Receptor Chemistry Towards the Third Millennium

PHARMACOCHEMISTRY L I B R A R Y - V O L U M E 31 RECEPTOR CHEMISTRY TOWARDS THE THIRD MILLENNIUM PHARMACOCHEMISTRY ...

Author: M. Gianella | G. Marucci | W. Quaglia | U. Gulini

39 downloads 1100 Views 23MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

PHARMACOCHEMISTRY

L I B R A R Y - V O L U M E 31

RECEPTOR CHEMISTRY TOWARDS THE THIRD MILLENNIUM

PHARMACOCHEMISTRY LIBRARY, edited by H. Timmerman Other titles in this series Volume 18

Trends in Receptor Research. Proceedings of the 8th Noordwijkerhout-Camerino Symposium, Camerino, Italy, 8-12 September, 1991 edited by P. Angeli, U. Gulini and W. Quaglia

Volume 19

Small Peptides. Chemistry, Biology and Clinical Studies edited by A.S. Dutta

Volume 20

Trends in Drug Research. Proceedings of the 9th Noordwijkerhout-Camerino Symposium, Noordwijkerhout (The Netherlands), 23-27 May, 1993 edited by V. Claassen

Volume 21

Medicinal Chemistry of the Renin-Angiotensin System edited by P.B.M.W.M. Timmermans and R.R. Wexler

Volume 22

The Chemistry and Pharmacology of Taxol| and its Derivatives edited by V. Farina

Volume 23

Qsar and Drug Design: New Developments and Applications edited by T. Fujita

Volume 24

Perspectives in Receptor Research edited by D. Giardin&, A. Piergentili and M. Pigini

Volume 25

Approaches to Design and Synthesis of Antiparasitic Drugs edited by Nitya Anand

Volume 26

Stable Isotopes in Pharmaceutical Research edited by Thomas R. Browne

Volume 27

Serotonin Receptors and their Ligands edited by B. Olivier et al.

Volume 28

Proceedings XlVth International Symposium on Medicinal Chemistry edited by F. Awouters

Volume 29

Trends in Drug Research II. Proceedings of the 1lth Noordwijkerhout-Camerino Symposium, Noordwijkerhout (The Netherlands), 11-15 May, 1997 edited by H. Van der Goot

Volume 30

The Histamine H3 Receptor. A Target for New Drugs edited by Rob Leurs and Henk Timmerman

http ://www. elsevier, nI/locate/is b n/0444504249

PHARMACOCHEMISTRY LIBRARY Editor: H. Timmerman

Volume 31

RECEPTOR CHEMISTRY TOWARDS THE THIRD MILLENNIUM

Proceedings of the 12th Camerino-Noordwijkerhout Symposium Camerino, Italy, 5-9 September 1999

Edited by

Ugo Gulini, Mario Gianella, Wilma Quaglia and Gabriella Marucci Department of Chemical Sciences, University of Camerino 1 Via S a n A g o s t i n o , 6 2 0 3 2 C a m e r i n o (MC), Italy

2000 ELSEVIER Amsterdam - Lausanne - New York- Oxford - Shannon - Singapore - Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands 92000 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier Science Rights & Permissions Department, PO Box 800, Oxford OX5 1DX, UK; phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also contact Rights & Permissions directly through Elsevier=s home page (http://www.elsevier.nl), selecting first >Customer Support=, then >General Information=, then >Permissions Query Form=. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (978) 7508400, fax: (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London W1P 0LP, UK; phone: (+44) 171 631 5555; fax: (+44) 171 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Science Rights & Permissions Department, at the mail, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made.

This volume is reprinted from: Pharnaceutica Acta Helvetiae, 74/2-3 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for.

ISBN: 0-444-50424-9

e The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Printed in The Netherlands.

PHARMACEUTICA ACTAHELVETIAE ELSEVIER

Pharmaceutica Acta Helvetiae 74 (2000) v www.elsevier.com/locate/pharmactahelv

Preface Despite Langley's brilliant intuition way back in 1878, it was still a long time before the receptor could be called a reality; in fact, even in the early 1960s De Jongh described this molecule as a woman whose lovely seductive image could be deduced only from the type of answer received to the many "messages" sent to her. Thus, when we met together for the first time in Camerino back in 1978, we were rightly inspired by that enthusiasm typical of pioneers attracted by the fascination of a discipline still all to be discovered. Over the last twenty years our joints may well have begun to creak due to increasing age, but our enthusiasm has by no whit been dulled; indeed, even if we now know almost all there is to be known about the way ligands " m a t e " with receptors, which have by now been isolated, characterized, and cloned, many other secrets still remain to tease our curiosity. In particular, differentiation into distinct subpopulations and the multiplicity of transduction processes seem to offer us unhoped for, and even more specific targets in our search for new drugs. And so, that

dream of Ehrlich in 1908 to design for each individual pathology a highly selective "charmed bullet", and thus one with reduced toxicity, now appears increasingly less utopistic. It is with these ideas that the 12th Camerino-Noordwijkerhout Symposium has seen us into the third millennium with an awareness that the only success for converting our hopes into reality is a multidisciplinary study based on a wakeful and critical comparison between experiences that have been reaching maturity through different approaches to the problematic - as has always been the case in our Symposia. Ugo Gulini Mario Giannella Gabriella Marucci Wilma Quaglia Guest Editors

PHARMACOCHEMISTRY LIBRARY

ADVISORY BOARD T. Fujita E. Mutschler N.J. de Souza F.J. Zeelen

Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan Department of Pharmacology, Universityof Frankfurt, Frankfurt, Germany Research Centre, Wookhardt Centre, Bombay, India Heesch, The Netherlands

Vol. 74 Nos. 2-3

PHA1LMACEUTICA ACTAHELVETIAE

March 2000

CONTENTS Contents list~Abstracts published in: Chemical Abstracts, Excerpta Medica, Intemational Pharmaceutical Abstracts Special Issue: Receptor Chemistry Towards the Third Millenium, Proceedings of the 12 th Camerino-Noordwijkerhout Symposium, Camerino, Italy, 5-9 September 1999 Preface Acknowledgements Pharmacological receptors: a century of discovery- and more D.J. Triggle Cholinergic receptors and neurodegenerative diseases F. Gualtieri Nicotinic systems in central nervous systems disease: degenerative disorders and beyond P.A. Newhouse and M. Kelton Central nicotinic receptor ligands and pharmacophores R.A. Glennon and M. Dukat Structural aspects of high affinity ligands for the 0~4132neuronal nicotinic receptor M.J. Dart, J.T. Wasicak, K.B. Ryther, M.R. Schrimpf, K.H. Kim, D.J. Anderson, J.P. Sullivan, M.D. Meyer Recombinant human receptors and functional assays in the discovery of altinicline (SIB-1508Y), a novel acetylcholine-gated ion channel (nAChR) agonist N.D.P. Cosford, L. Bleicher, J.-M. Vernier, L. Chavez-Noriega, T.S. Rao, R.S. Siegel, C. Suto, M. Washburn, G.K. Lloyd, I.A. McDonald Receptors in neurodegenerative diseases, muscarinic cholinergic receptors P. Angeli Design and development of selective muscarinic agonists for the treatment of Alzheimer's disease: characterization of tetrahydropyrimidine derivatives and development of new approaches for improved affinity and selectivity for M 1 receptors W.S. MesserJr., W.G. Rajeswaran, Y. Cao, H.-J. Zhang, A.A. EI-Assadi, C. Dockery, J. Liske, J. O'Brien, F.E. Williams, X.-P. Huang, M.E. Wroblewski, P.I. Nagy, S.M. Peseckis C1-1017, a functionally Ml-selective muscarinic agonist: design, synthesis, and preclinical pharmacology H. Tecle, R.D. Schwarz, S.D. Barrett, M.J. Cailahan, B.W. Caprathe, R.E. Davis, P. Doyle, M. Emmerling, D.J. Lauffer, T. Mirzadegan, D.W. Moreland, W. Lipiniski, C. Nelson, C. Raby, C. Spencer, K. Spiegel, A.J. Thomas, J.C. Jaen Ligands for the common allosteric site of acetylcholine M2-receptors: development and application U. Holzgrabe, W. Bender, H.M. Botero Cid, M. Staudt, R. Pick, C. Pfletschinger, E. Balatkov~, C. Tr&nkle, K. Mohr Receptors in cardiovascular disease: review and introduction A. Leonardi, G. Sironi, G. Motta Adrenoceptor subclassification: an approach to improved cardiovascular therapeutics J.P. Hieble The oLla and O~b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action S. Cotecchia, O. Rossier, F. Fanelli, A. Leonardi, P.G. De Benedetti oL~-Adrenoreceptor antagonists bearing a quinazoline or a benzodioxane moiety C. Melchiorre, P. Angeli, M.L. Bolognesi, A. ChiarinL D. Giardin&, U. GulinL A. Leonardi, G. Marucci, A. Minarini, M. Pigini, W. Quaglia, M. Rosini, V. Tumiatti Selection, design and evaluation of new radioligands for PET studies of cardiac adrenoceptors V.W. Pike, M.P. Law, S. Osman, R.J. Davenport, O. Rimoldi, D. Giardin~, P.G. Camici Enigmatic receptors L. Brasili Imidazoline receptors: a challenge P. Bousquet, V. Bruban, S. Schann, J. Feldman

v vi 79 85 91 103 115

125 131

135

141 149 157 163 173

181 191 201 205

Sigma receptors: recent advances and new clinical potentials W.D. Bowen Excitatory amino acid receptors G. Gaviraghi Excitatory amino acid agonists and antagonists: pharmacology and therapeutic applications D. G. Trist Metabotropic glutamate receptors: a structural view point R. Pellicciari, G. Costantino, A. Macchiarulo Synthesis and pharmacological properties of novel glycine antagonists D. Donati and R. Di Fabio Receptors in neurodegenerative diseases W. Froestl Neurotrophin receptor structure and interactions H. Yano and M.V. Chao The RET receptor tyrosine kinase: activation, signalling and significance in neural development and disease L Mason The ciliary neurotrophic factor and its receptor, CNTFRo~ M.W. Sleeman, K.D. Anderson, P.D. Lambert, G.D. Yancopoulos, S.J. Wiegand Rediscovering good old friend IGF-I in the new millenium: possible usefulness in Alzheimer's disease and stroke S. Dor#, S. Kar, W.-H. Zheng, R. Quirion Apoptosis induced by death receptors P. Schneider and J. Tschopp Hijacked receptors D.J. Triggle ICAM-1 receptors and cold viruses J. Bella and M.G. Rossmann Viral-encoded G-protein coupled receptors: new targets for drug research? M.J. Smit, H. Timmerman, D. Verzijl, R. Leurs Chemokine receptors: interaction with HIV-1 and viral-encoded chemokines S. SozzanL P. Allavena, A. Vecchi, J. Van Damme, A. Mantovani General topics and perspectives H. Timmerman Pharmacological evidence of muscarinic receptor heterodimerization S. Chiacchio, M. Scarselli, M. Armogida, R. Maggio Constitutive activity of G protein coupled receptors and drug action R. Leurs, M.S.R. Pena, R.A. Bakker, A.E. Aiewijnse, H. Timmerman New dimensions in G protein signalling: G135 and the RGS proteins W.F. Simonds and J.-H. Zhang Kappa opioid agonists as targets for pharmacotherapies in cocaine abuse J.L. Neumeyer, N.K. Mello, S. Stevens Negus, J.M. Bidlack Author Index Keyword Index

211 219 221 231 239 247 253 261 265 273 281 287 291 299 305 313 315 327 333 337 345 347

PHARMAGEUTIGA ACTAHELVETIAE ELSEVIER

Pharmaceutica Acta Helvetiae 74 (2000) 79-84 www.elsevier.com/locate/pharmactahelv

Pharmacological receptors" a century of discovery

and more

D a v i d J. Triggle * The Graduate School, 562 Capen Hall, State Universityof New York,Buffalo,NY14260, USA

Abstract

A brief survey of the history of the development of the concept of the pharmacological receptor is presented. From the pioneering concepts of Paul Ehrlich, John Langley and others, receptors are described in terms of their recognition properties, their structures, transducing abilities and the impact of genomics and their role in contributing to genetic diseases. The receptor concept has firmly underpinned our advances in drug development and molecular medicine of the latter half of this century and it is clear that it will continue to drive pharmaceutical developments in the 21st century. 9 2000 Elsevier Science B.V. All rights reserved.

Keywords: Receptors; Receptor history; Paul Ehrlich; John Newton Langley; Emil Fischer; Louis Pasteur; Drug-receptor interactions; Stereoselectivity; Receptor diseases; Receptor regulation; Receptor structure

1. Introduction

Even prior to these speculations, Langley (1878) had observed:

Separate, but complementary, lines of evidence led in the late 19th century to the establishment of the concept of "the receptor" as the cellular site at which drugs, toxins and antibodies mediated their physiological or pathological effects. These lines of evidence are particularly associated with Paul Ehrlich in Germany and John Newton Langley in England. However, their work built upon many centuries of work that attempted to define the action of naturally occurring materials on the body. These earlier contributions have been expertly summarized in the books, "Murder, Magic and Medicine" by John Mann (1992) and " I n Search of a Cure" by M. Weatherall (1990). From his extensive work on immunology and the chemotherapy of parasitic infections, Ehrlich argued that cells must possess specific and defined protoplasmic side chains that, because of their unique chemistry and steric architecture, could interact specifically with the complementary groups of a chemotherapeutic agent, toxin or antibody (Parascondola, 1981; Ehrlich, 1900):

" F o r the sake of brevity in what follows we shall in general designate as receptor that binding group of the protoplasmic molecule to which a foreign, newly introduced group binds." P. Ehrlich, 1900

* Tel.: +1-716-645-7315; [email protected]

fax: +1-716-645-2941;

e-mail:

"We may, I think, without much rashness assume that there is some substance or substances in the nerve endings or gland cells with which both atropine and pilocarpine are capable of forming compounds. On this assumption, then, the atropine or pilocarpine compounds are formed according to some law of which their relative mass and chemical affinity for the substance are factors." J.N. Langley, 1878 But Langley (1906) also recognized the receptor as a transducing engine that: " . . . receives the stimulus and, by transmitting it causes contraction." J.N. Langley, 1906 Langley, contemporaneously with the work of Ehrlich, used the term "receptive substance" for these specific entities and speculated that specific receptors must exist for curare, atropine, pilocarpine and the other autonomic drugs with which his research had been principally concerned. Certainly, the specificity of such drug-receptor interactions had been anticipated by Emil Fischer who wrote: " . . . I will say that enzyme and glucoside must fit together like lock and key in order to be able to exercise a chemical action on each other." Emil Fischer, 1894

0031-6865/00/$ - see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S0031-6865(99)00046-1

80

D.J. Triggle/ Pharmaceutica Acta Helvetiae 74 (2000) 79-84

Thus, by the beginning of this century, the conceptual foundation had been laid for the existence of pharmacological receptors, albeit as "black boxes", that received input and translated it into a physiological, pharmacological or pathological output. The present century has been largely devoted to opening this box and defining its contents. It has been a spectacularly successful century that has culminated with the classification, isolation, characterization and cloning of pharmacological receptors, with the identification of receptors - - "orphan receptors" - - for which ligands may not have been identified and with the determination of the detailed three-dimensional structure of a membrane receptor - - a bacterial potassium channel.

2. Receptors as recognition entities

The specificity of the drug-receptor recognition process has long been regarded as a critical feature of the receptor concept, even when the nature of receptors was entirely unknown. Indeed, the absence of such specificity, including stereoselectivity, is often a component of arguments that a receptor event is not involved in the action of a particular drug. These structure-activity relationships were originally qualitative in character, but were transformed first by the application of regression techniques that permitted the elucidation of one-dimensional quantitative structure-activity relationships (QSARs) and then by protein sequence determination and the determination of three-dimensional protein structures and the mapping of receptor sites (Greer et al., 1994). With these approaches, it is increasingly possible to interpret the actions of drugs at their receptors and to facilitate the design of drugs for new receptor sites. Thus, the design of the HIV-protease inhibitors, a critically available class of drugs for the treatment of this lethal disease, was greatly facilitated by the resolution of the structure of the enzyme. The dimeric, essentially symmetric, structure composed of two identical aspartate protease-like domains, was critical to the development of the first protease inhibitors. Stereochemistry of interaction has long been recognized in drug-receptor interactions and Pasteur very explicitly recognized that different stereoisomers could have very different physiological properties: "There cannot be the slightest doubt that the only and exclusive cause of this difference in the fermentation of the two tartaric acids is caused by the opposite molecular arrangements of the tartaric acids. In this way, the idea of the influence of the molecular asymmetry of natural organic products is introduced into physiological studies, this important characteristic being perhaps the only distinct line or demarcation which we can draw today between dead and living matter. I have in fact set up a theory of molecular asymmetry, one of the

most important and wholly surprising chapters of the science, which opens up a new, distant but definite horizon for physiology." Louis Pasteur, 1860 The stereochemical basis of drug actions was early investigated by Arthur Cushny at the beginning of this century (Cushny, 1926). These pioneering investigations on atropine and related compounds revealed the quantitative differences that can occur between drug enantiomers. Today, the issue of the chirality of drug-receptor interactions has assumed both scientific and regulatory significance. Scientific and clinical significance derives from consideration of the efficacy of a single enantiomer versus a racemate, from considerations of stereoselective metabolism and disposition, and from the impact of the route of administration and patient variability. Regulatory issues derive from considerations that racemic drugs may represent separate agents in fixed combinations: development issues derive from considerations of the costs, including those for chemical synthesis, of pursuing a single enantiomer or a racemic mixture. Recent developments in stereochemistry have focused upon the gaseous general anesthetics, long a topic of discussion concerning their potential interactions with receptors. The long-standing assumption that these agents interacted non-specifically through partitioning into lipid membranes has been challenged from a variety of sources, including the stereospecificity of interaction of isoflurane and halothane (Moody et al., 1994; Sedensky et al., 1994). The differences, though small, are potentially significant from the perspective of clinical safety.

3. Receptors as transduction machines

As realized by Langley, recognition is a necessary, but not sufficient, characteristic of a receptor. Receptors are also biological machines translating the information of the interaction with the ligand into the cellular response. And with this translation comes the necessary amplification of the input information by several orders of magnitude. Several types of physical and biochemical process are involved in this biological transduction. Of particular significance, because of their widespread occurrence are the transduction events mediated by G protein-coupled receptors and by ion channels. The G proteins are a large group or superfamily of GTP hydrolases and the interaction of an activated receptor with the heteromeric G protein releases bound GDP and replaces it with GTP with concomitant liberation of the activated GTP-associated GoL subunit. This activated subunit then interacts with a number of effector systems, including phospholipase C, adenylyl cyclase and ion channels. Similar amplification events occur during the opening or closing of ion channels mediated by chemical (ligandgated channels) or physical (potential-dependent channels).

D.J. Triggle / Pharmaceutica Acta Helvetiae 74 (2000) 79-84

To a first approximation, ion channel opening is an all-ornone stochastic event and the effect of a stimulus, chemical or physical, is to alter the probability of channel opening. Patch clamp techniques make it possible to observe the opening or closing of single channels and thus to measure single molecular events. This ability, coupled with recent structural information on the K + channel, makes possible to a first approximation a molecular description of ion channel function.

4. Receptor classification Receptor structure, the linear and ultimately the threedimensional representation of the sequence, provides a definitive classification and basis for the classification of receptors. This permits the identification of "families" and of "super-families" of receptors and, in recent years, has made possible the isolation and characterization of so-called "orphan receptors", for which physiological ligands or physiological function may not have been identified. Earlier classification schemes that used the identity of the physiological ligand that interacts with the receptor, the nature of the physiological or pharmacological response induced by receptor activation or the nature of the antagonist drug all have significant limitations. Multiple systems and receptors control blood pressure and similarly many receptors share a common biochemical cascade adenylyl cyclase or phospholipase C activation or the opening and closing of K + channels. To further complicate matters, many receptors are pleiotropic, initiating multiple consequences that may differ according to cell type and even agonist quality. Similarly, many physiological ligands may interact with multiple receptors that are of fundamentally different classes: acetylcholine interacts with both muscarinic and nicotinic receptors, the former being members of the G protein-coupled family (Bikker et al., 1998) and the latter a member of the ligand-gated ion channel family (Holladay et al., 1997). Finally, many receptors are heteromeric assemblies of multiple types of subunits: the

Table 1 Classification of pharmacological receptors Class Type 1

2 3

4

Ion channels

Characteristics

Integral membrane; subunit composition; each subunit has two or more membrane inserts as a pore region and four or more form the central pore of the channel G protein-coupled Seven-transmembrane integral proteins that couple to the G protein family of proteins Enzyme-associated One-transmembrane integral proteins that have kinase activity; may dimerize during receptor activation Nuclear receptors Non-membrane, cytosolic proteins with DNA binding domains; transcriptional regulators

81

pharmacological specificity and the actions induced can be very dependent upon subunit composition. Despite this complexity, it is convenient to recognize four principal families of chemically sensitive pharmacological receptors (Table 1).

5. Receptor structure The majority of receptors under discussion are integral membrane proteins and have not, until recently, yielded to three-dimensional structural determination. However, progress is now being made in three principal areas the structure of rhodopsin as a model for the very large G protein-coupled receptor family, the role of the nicotinic acetylcholine receptor as a model for ligand-gated ion channels and a bacterial K + channel from Streptomyces lividans that will materially define the ionic conductivity, selectivity and gating processes of ion channels. Additionally, powerful molecular biological approaches including selective mutagenesis and the use of chimeric constructs have served to define the roles of particular sequences or residues in receptor recognition and activation processes. The very large G protein-coupled receptor family has provided many examples of the definition of residue roles in drug interactions. Thus, for the beta-adrenoceptor, critical interacting residues have been determined to be aspartate-113 on helix III, serine-204 and -207 on helix V and phenylalanine-290 on helix VI. Such studies have defined a "homologous" binding pocket on this receptor family that is shared by the cationic neurotransmitters, acetylcholine, histamine, norepinephrine etc., and related small ligands. Perhaps the most recent dramatic advance has been the determination of the three-dimensional structure of a bacterial K + channel from S. lividans (Doyle et al., 1998). This channel is composed of four identical subunits, each with two trans-membrane sequences and a "pore" region, that associate in "tepee" shape to form the functional ion channel containing within it the selectivity filter that discriminates K + from other ions. The selectivity filter contains a so-called "signature" sequence, highly conserved residues that characterize K + ion channels and the GlyTyr-Gly components of this sequence in the four subunits bind K + through their carbonyl residues and are responsible for the ionic selectivity of the channel. These structural studies have also revealed the importance that very minor changes, frequently a single residue, can have on the drug-receptor interaction. Thus, the 5HT~B receptor in the rodent and man is pharmacologically quite distinct, a differentiation that is provided by residue 355, threonine in the human and asparagine in the rat (Oksenberg et al., 1992). Similarly, the interaction of barbiturates and other anesthetics with the GAB A p l receptor depends upon the presence of a single isoleucine residue: replacement of this residue by serine confers

82


anesthetic sensitivity (Belelli et al., 1999). Such changes are of extreme importance in the determination of individual human sensitivity to drugs where single nucleotide polymorphisms (SNPs) may determine clinically significant drug responses and interactions (Kleyn and Vessell, 1998). Thus, there are a number of polymorphisms in the human beta-adrenoceptors and these have been associated with bronchodilation in response to beta z-agonists and with the development of hypertension (Buscher et al., 1999). The P450-mediated drug metabolism process is highly polymorphic leading to extensive inter-individual variation in drug metabolism (Ingelman-Sundberg et al., 1999). Exploitation of this knowledge, now possible through gene-array technologies, will increasingly alter both drug development and drug prescribing.

6. Orphan receptors The classical route to the receptor concept has always been the existence first of a drug and an associated family structure with defined physiological and pharmacological effects. A classic example is morphine and the opiates and the subsequent discovery of the endogenous ligands and subsequently the G protein-coupled opiate receptor. The isolated and expressed receptor could then be used as a screen for novel structures that might have more desirable therapeutic properties. Advances in molecular biology now permit the reverse of this process. DNA sequences are identified that are analogs of known receptors. These sequences can be expressed to yield novel or "orphan" receptors for which the endogenous ligand can now be hunted (Soontjens et al., 1996; Robertson and Willy, 1997; Civelli et al., 1998; Wilson et al., 1998). Both the G protein family and the steroid hormone family have yielded many orphan receptors. At least 140 G protein receptors have been identified from the human genome and since this class has generated major drugs for many therapeutic targets, the status of orphan receptors here has attracted much attention. The identification of the opiod receptor ORL1 is but one example, interacting with a specific endogenous ligand nociceptin that appears to have widespread roles in the mediation of nociception and stress reduction. Over 70 orphan receptors have thus far been identified in the steroid receptor family and for most of these, neither endogenous ligand nor physiological action has yet been defined.

7. Receptors and genetic diseases Defects in the structure and expression of receptor proteins are increasingly known to be associated with specific disease states. As one of the largest families of receptors, the G protein-coupled receptors exhibit a variety of mutations and associated functional changes, including both "loss of function" and "gain of function" (Spiegel,

1995; Farfel et al., 1999). These defects can lie in the actual receptors or in the associated G proteins. Similarly, mutations in ion channels are being associated with a variety of diseases from cardiac abnormalities to cystic fibrosis. Loss-of-function mutations in G protein-coupled receptors are quite common, with approximately 100 having been described, and include nephrogenic diabetes insipidus (V2 vasopressin receptor), familial hypothyroidism (TSH receptor), Hirschprung disease (endothelin B receptor) hypercalcemia and neonatal hyperparathyroidism (Ca 2+ sensing receptor). These loss of function mutations may prevent protein expression, folding or insertion in the membrane or may impair agonist binding or interaction of the receptor with G proteins. Loss-of-function defects may also arise in the associated G proteins. Pseudohypoparathyroidism, resistance to parathyroid hormone with a subnormal urinary cAMP response to the hormone, provides one example. Individuals with the type l a form of the disease also show resistance to a variety of other hormones that stimulate cAMP formation and this is associated with a defect in the Gs-oLsubunit. A number of gain-of-function mutations have also been described in which there is constitutive receptor activation. These include McCuneAlbright syndrome characterized by excessive cell proliferation, including hyperpigmented skin, precocious puberty, hyperthyroidism, acromegaly and polyostotic fibrous dysplasia and results from persistent activation of the G protein from an inability to hydrolyze GTP and thus to terminate the receptor-G protein cycle. A defect in the beta-subunit of a G protein has been shown to be associated with an increased incidence of hypertension, being found in 53% of patients with essential hypertension and 44% of normotensive patients (Siffert et al., 1998). Defects associated with ion channels underlie a variety of diseases, including cystic fibrosis, cardiac arrhythmias, episodic ataxia, heritable myasthenia and nocturnal frontal lobe epilepsy (Keating and Sanguinetti, 1996; Ackerman and Clapham, 1997; Cooper and Jan, 1999). Cystic fibrosis arises from a defect in a chloride channel the cystic fibrosis transmembrane regulator - - that blocks chloride transport in epithelial cells. It is a remarkably common defect amongst Caucasians some 3.5% of the population carrying a defective gene. The most common defect is the deletion of phenylalanine 508, which results in a protein that does not insert properly into the membrane. In long QT syndrome, there is a lengthening of the QT interval of the electrocardiogram, a delay that may initiate cardiac arrhythmias, fibrillation and death. The defect has several origins associated primarily with K + channels: one of these channels associated with the HERG gene and accounting for some 30% of LQTS cases is of particular importance since it is a target for a number of clinically available drugs, including some antibiotics, antihistamines and antifungal agents that increase the chance of arrhythmias and sudden death.


8. Genetically modified animals Techniques, targeted mutations and transgenes, that permit the creation of animals - - transgenic animals that have been genetically modified in their receptor function provide a powerful tool with which to dissect the specific sites of action of drugs (Rudolph and Mohler, 1999). Gene inactivation - - receptor knockout - - provides a valuable technique for determining the action of subtype-selective drugs. Thus, mice lacking the A 2A receptor fail to show a stimulant effect to caffeine, both confirming the receptor subtype at which adenosine acts and suggesting that antagonists at this receptor may be cognition enhancers. Receptor knockouts may also yield unanticipated targets: mice lacking the GoLq subunit had a prolonged bleeding time and were not responsive to platelet activators, thus identifying this subunit as a potential target for antithrombotic therapy. Finally, animals with receptors bearing specific mutations can be very useful to dissect the spectrum of pharmacological actions that a given drug may produce. An interesting example is provided by the GABA a receptor, which, like many other ligand-gated channel receptors, is a heteromeric collection of subunits making many distinct subtypes. In a system where the oL1 subunit has been mutated to produce a diazepam-insensitive oLl-receptor, the major actions of diazepam are unaltered mediated by the oL2, oL3 and e~5 subunits while other actions are lost (Rudolph et al., 1998).

9. Conclusions It has been a remarkable century for receptors! Still a hypothetical entity in 1900, receptors come to the year 2000 as known and defined entities and more. We can now synthesize receptors of defined character and properties and we can produce genetically modified animals that display our own human receptors. And our concept of receptors has expanded enormously: from their beginning as sites for neurotransmitters and chemotherapeutic agents they are now recognized to be critical cellular communication routes for both chemical and physical stimuli, for ligands small and large and whose aberrant expression is associated with a multiplicity of disease states. Langley and Ehrlich might today be strangers in a strange land were they to return, but they would surely recognize the magnificent fruits of their toil in the vineyards.

References Ackerman, M.J., Clapham, D.E., 1997. Ion channels - - basic science and clinical disease. N. Engl. J. Med. 336, 1575-1586. Belelli, D., Pau, D., Cabras, G., Peters, J.A., Lambert, J.J., 1999. A single amino acid confers barbiturate sensitivity upon the GABA p l receptor. Br. J. Pharmacol. 127, 601-604. Bikker, J.A., Trumpp-Kallmeyer, S., Humblet, C., 1998. G-Protein c o u -

83

pled receptors: models, mutagenesis and drug design. J. Med. Chem. 41, 2911-2927. Buscher, R., Herrmann, V., Inel, P.A., I999. Human adrenoceptor polymorphisms: evolving recognition of clinical importance. Trends Pharmacol. Sci. 20, 94-99. Civelli, O., Nothacker, H.-P., Reinscheid, R., 1998. Reverse physiology: discovery of the novel neuropeptide, orphanin FQ/nociceptin. Crit. Rev. Neurobiol. 12, 163-176. Cooper, E.C., Jan, L.Y., 1999. Ion channel genes and human neurological disease: recent progress, prospects, and challenges. Proc. Natl. Acad. Sci. U S A 96, 4759-4766. Cushny, A.R., 1926. Biological Relations of Optically Isomeric Substances. Balliere, Tindall and Cox, London. Doyle, D.A., Cabral, J.M., Pfeutzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., McKinnon, R., 1998. The structure of the potassium channel: molecular basis of K + conduction and selectivity. Science 280, 69-74. Ehrlich, P., 1900. On immunity with special reference to cell life. Proc. R. Soc. London 125, 424-449. Farfel, Z., Bourne, H.R., Iiri, T., 1999. Mechanisms of disease: the expanding spectrum of G protein diseases. N. Engl. J. Med. 340, 1012-1020. Fischer, E., 1894. Einfliuss der konfiguration auf die wirkung der enzyme. Ber. Dtsch. Chem. Ges. 27, 2985-2993. Greer, J., Erickson, J.W., Baldwin, J.J., Varney, M.D., 1994. Application of the three-dimensional structures of protein target molecules structure-based drug design. J. Med. Chem. 37, 1035-1054. Holladay, M.W., Dart, M.J., Lynch, J.K., 1997. Neuronal nicotinic receptors as targets for drug discovery. J. Med. Chem. 40, 4169-4194. Ingelman-Sundberg, M., Oscarson, M., McLellan, R.A., 1999. Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment. Trends Pharmacol. Sci. 20, 342-349. Keating, M.T., Sanguinetti, M.C., 1996. Pathophysiology of ion channel mutations. Current Biology 6, 326-333. Kleyn, P.W., Vessell, E.S., 1998. Genetic variation as a guide to drug development. Science 281, 1820-1821. Langley, J.N., 1906. On nerve endings and on special excitable substances in cells. Proc. R. Soc. London, Ser. B 78, 170-184. Mann, J., 1992. Murder Magic and Medicines. Oxford Univ. Press, Oxford. Moody, E.J., Harris, B.D., Skolnick, P., 1994. The potential for safer anesthesia using stereoselective anesthetics. Trends Pharmacol. Sci. 15, 387-390. Oksenberg, D., Marsters, S.A., O'Dowd, B.F., Jin, H., Havlik, S., Peroutka, S.J., Askenazi, A., 1992. A single amino-acid difference confers pharmacological variation between human and rodent 5-HT1B receptors. Nature 360, 161-163. Parascondola, J., 1981. The theoretical basis of Paul Ehrlich's chemotherapy. J. Hist. Med. 36, 19-43. Pasteur, L., 1860. On the asymmetry of naturally occurring compounds (Two lectures delivered before the Chemical Society of Paris, 20th of January and 3rd of February, 1860). In: Richardson, G.M. (Ed.), Memoirs of Stereochemistry. American Book, London, 1901, pp. 1--33. Robertson, D.W., Willy, P.J., 1997. Nuclear orphan receptors: scientific progress and therapeutic opportunities. Annu. Rep. Med. Chem. 32, 251-260. Rudolph, U., Mohler, H., 1999. Genetically modified animals in pharmacological research: future trends. Eur. J. Pharmacol. 375, 327-337. Rudolph, U., Crestani, F., Benke, D., Martin, J.R., Benson, J.A., Keist, R., Fritschy, J.M., Low, K., Bluthmann, H., Mohler, H., 1998. Function of GABAA-receptor subtypes: mice with point-mutated diazepam insensitive c~l-subunits. Soc. Neurosci. Abstr. 24, 1990, cited in Rudolph and Mohler (1999). Sedensky, M.M., Cascorbi, H.F., Meinwald, J., Radford, P., Morgan, P.G., 1994. Genetic differences affecting the potency of stereoisomers of halothane. Proc. Natl. Acad. Sci. U S A 91, 10054-10058.

84


Siffert, W., Rosskopf, D., Siffert, G. et al., 1998. Association of a human G-protein (beta)3 subunit variant with hypertension. Nature Genetics 18, 45-48. Soontjens, C.D., Rafter, J.J., Gustafsson, J.-A., 1996. Ligands for orphan receptors?. J. Endocrinol. 150, $241-$257. Spiegel, A.M., 1995. Defects in G protein-coupled signal transduction in human disease. Annu. Rev. Physiol. 58, 143-170.

Weatherall, M., 1990. In Search of a Cure. Oxford Univ. Press, Oxford, UK. Wilson, S., Bergsma, D.K., Chambers, J.K., Muir, A.I., Fantom, K.G.M., Ellis, C., Murdock, P.R., Herrity, N.C., Stadel, J.M., 1998. Orphan G-protein-coupled receptors: the next generation of drug targets?. Br. J. Pharmacol. 125, 1387-1392.

PHARMACEL ICA ACTAHELVETIAE ELSEVIER


Cholinergic receptors and neurodegenerative diseases Fulvio Gualtieri

*

Department of Scienze Farmaceutiche, University of Florence Via G. Capponi, 9-5012I Florence, Italy

Keywords: Neurodegenerative diseases; Alzheimer's disease; Parkinson's disease; Cholinergic hypothesis; Muscarinic agonists; Muscarinic antagonists; Nicotinic agonists; Acetylcholine releasers; Acetylcholinesteraseinhibitors; High affinity choline uptake enhancers

1. Introduction The severe disturbance of cholinergic function in dementias and particularly in Alzheimer's disease (AD) was discovered nearly simultaneously by several researchers in the mid-seventies (Bowen et al., 1976; Davies and Maloney, 1976; Perry et al., 1977). As a consequence, some 20 years ago several scientists (McGeeer, 1981; Coyle et al., 1983), among whom Bartus et al. (1982) are the most cited, suggested that at least some of the symptoms of dementias were due to impaired cholinergic transmission in the brain and proposed that drugs able to restore CNS cholinergic tone would be able to revert the symptoms of the disease and to slow down its progress. Known as the Cholinergic Hypothesis of Alzheimer's disease, this hypothesis has served as the main rationale for the development of anti-AD drugs, even if alternative approaches, such as the use of neurotrophic agents, nootropics, glutamate antagonists, benzodiazepine receptor ligands, calcium antagonists, anti-inflammatory agents, radical scavengers and compounds interfering with amyloid precursor protein (APP) processing have been proposed and evaluated (Gualtieri et al., 1995). In the past 20 years, the popularity of the cholinergic hypothesis has experienced some ups and downs, mainly due to the fact that central cholinergic impairment appears as a consequence more than the cause of AD. However, the finding that cholinergic drugs can control correct processing of APP has boosted a recent revival of the approach.

* Tel.: +0039-055-2757295; fax: +0039-055-240776; e-mail: [email protected]

Familial AD is indeed associated with increased formation of a specific APP derivative, the 42 residue variant (A[~1_42) of the amyloid [3-peptides. Amyloid [3 peptides derive from abnormal proteolytic processing of APP (referred to as [3 and y secretase cleavage) and represent highly hydrophobic and self-aggregating molecules suspected of being the main determinant of the disease. It is becoming clearer and clearer that correct processing of APP (referred to as oL-secretase cleavage) can be accelerated by the stimulation of muscarinic M~ receptors within minutes of receptor activation, while the formation of A[3 peptides is decreased by approximately 50% (Schenk et al., 1995). Despite the drawbacks of the cholinergic hypothesis, this idea has guided most of the researchers involved with AD and enormous resources have been invested in developing compounds that would directly (nicotinic and M I selective muscarinic agonists) or indirectly (acetylcholinesterase inhibitors, M 2 selective muscarinic antagonists, acetylcholine releasers, high affinity choline uptake inhibitors) increase the level of cholinergic transmission in the brain.

2. Cholinesterase inhibitors Tacrine, donepezil and rivastigmine (Francis et al., 1999) (Fig. 1), all belonging to the acetylcholinesterase inhibitors class, represent, at the moment, the result of these intensive efforts. They produce a measurable, albeit modest improvement in cognition, with effects equivalent to a 6- to 12-month delay in the symptomatic progression of the disease. A few other compounds are presently in

0031-6865/00/$- see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S0031-6865(99)00021-7

86

F. Gualtieri / Pharmaceutica Acta Helvetiae 74 (2000) 85-89

.•oc.3 CH3

O tacrine

donepezil Aricept (Eisai)

Cognex (Park-Davis)

~H3

CH3

~H3

~

0

rivastigmine Exelon (Novartis)

Fig. 1. Launched anti-Alzheimerdrugs.

clinical trials or await approval; in Fig. 2, some selected compounds that have reached advanced developing phases are shown.

3. Muscarinic agonists In addition to acetylcholinesterase inhibitors, M I selective muscarinic agonists are among the most studied com-

pounds (Fig. 3). At the moment, their future does not appear particularly brilliant as their efficacy is low and the usually modest selectivity is causing side effect problems. The uncertainty in the efficacy and usefulness of M 1 muscarinic agonists is hampering the development of most of the compounds of this class. Forest Laboratories are reported to have abandoned their development of the M1 muscarinic agonist M 3 antagonist LU25109 and the same seems the fate of milameline, discovered by Hoechst Mar-

H3C%/~~)H

.,co'" "ccl Metrifonate (awating approval)

Eptastigmine (phase III)

~H3 H3CX /CH3 ~) H~e'Si~cF3

H

,~

H

O Zifrosilone (phase II)

Huperzine A (phase II)

Fig. 2. Selected acetylcholinesteraseinhibitors in development for AD.

87


4. Muscarinic antagonists

~

A

Talsaclidine (phase III)

The approach based o n M 2 antagonists has not produced, so far, compounds of practical interest, mainly because of poor brain penetration of known selective M 2 antagonists. The prototype BIBN-99 (Doods et al., 1993), which is M 2 selective and seems able to cross the blood brain barrier (BBB), is still in an early stage of development while, as regards the recently disclosed SCH 57790 (Lachowicz et al., 1999), available pharmacological data are insufficient to evaluate its interest (Fig. 4).

Cevimeline (phase III)

5. Acetylcholine releasers

C6H13

'~

CHa Xanomeline (phase III)

CHa Milameline (phase III)

..N,

N

N,~O"-,CH8 CHa LU-25109 (phase I)

Acetylcholine releasers, which can be viewed as muscarino-mimetics, are experiencing similar difficulties. Linopirdine, that blocks a variety of K + channels including I M, has been withdrawn from clinical trials (Kelly, 1999), while SM21 (Ghelardini et al., 1997) is still at the pre-clinical stage (Fig. 5).

Sabcomeline (phase III)

Fig. 3. Selected muscarinic agonists in developmentfor AD.

ion Russel. (Kelly, 1999). Very recently, when the compound had reached phase III clinical development as a cognition enhancer, Smith Kline Beecham put development of sabcomeline hydrochloride on hold, pending further decisions, because of its inconsistent effect (Anonymous, 1999). Nevertheless, it has been observed that although the effects of muscarinic agonists on cognition are modest at tolerable doses, in several cases, they seem to improve the psychiatric symptoms usually present in AD patients.

6. High affinity choline uptake enhancers Increasing the uptake of choline is another possible way to improve central cholinergic tone. Two compounds of this class are in preclinical evaluation: MKC-231 from Mitsubishi (Chaki et al., 1995) and Z-4105 from Zambon (Anonymous, 1995)(Fig. 6).

7. Nicotinic agonists Apparently, the expectations deriving from the cholinergic hypothesis, based on muscarinic agonists, have been transferred to nicotinic agonists, as one can hardly pick up a pre-clinical journal without reading about nicotinic ago-

C1

CH3

0

CH3 BIBN-99

SCH 57790

Fig. 4. M2 selective muscarinic antagonists.

Linopirdine (phase III)

SM-21 (preclinical)

Fig. 5. Selected acetylcholinereleasers.

CH 3

88


~9 N-"-O

H

0

H

Z-4105

LY297802 muscarinic antinociceptive

MKC-231

ABT-594 nicotinic antinociceptive

Fig. 6. Selected HACU enhancers.

Fig. 8. Anti-nociceptive cholinergic agents.

nists and their highly selective presynaptic action on a variety of neurotransmitters release in the brain. Although nicotine has been reported to be beneficial for m e m o r y in h u m a n and animal tests, the cardiovascular, gastrointestinal and endocrine side effects of nicotine, as well as the negative connotation associated with tobacco smoking, have apparently delayed intensive research efforts to develop nicotinic agonists which would restore central cholinergic tone. In the past few years, however, thanks also to progress in molecular biology, physiology and p h a r m a c o l o g y of central nicotinic receptors (Boyd, 1997; Chavez-Noriega et al., 1997), the potential of nicotinic agonists for the treatment of neurodegenerative disorders has been recognised. Indeed, besides their utility in AD, centrally acting nicotinic agonists have potentials for the treatment of Parkinson's disease (PD) due to their ability to release other neurotransmitters like dopamine. As a consequence, intensive research has been performed on the synthesis and pharmacological evaluation of several classes of drugs that possess nicotinic properties without the undesirable side effects of nicotine (Decker et al., 1994; Holladay et al., 1995, 1997; Brioni et al., 1996; Glennon and Dukat, 1996). Most of the work in this field has been performed in the Abbott and SIBIA research laboratories; in Fig. 7 are reported some of the molecules that seem promising and are in clinical trial. It is interesting that in addition to potential anti-A1zheimer and anti-Parkinson drugs, the research on cholin-

ergic agonists has identified c o m p o u n d s (both nicotinic and muscarinic) that are potent analgesics and that could be developed as non-classical painkillers (Fig. 8).

CH 3

CH3

CH 3 (S)-ABT-418

(R)-ABT 089

(S)-SIB-1508Y Fig. 7. Selected nicotinic agonists in development for AD/PD treatment.

References Anonymous, 1995. Z-4105. Drugs of the Future, 20, 584. Anonymous, 1999. Sabcomeline hydrochloride. Drugs of the Future, 24, 113-114. Bartus, R.T., Dean, R.L.I., Beer, A.S., Lippa, A.S., 1982. The cholinergic hypothesis of geriatric memory dysfunctions. Science 217, 408-417. Bowen, D.M., Smith, C.B., White, P., Davidson, A.N., 1976. Neurotransmitter related enzymes and indexes of hypoxia in senile dementia and other abiothrophies. Brain 99, 459-496. Boyd, R.T., 1997. The molecular biology of neuronal nicotinic acetylcholine receptors. Crit. Rev. Toxicol. 27, 299-318. Brioni, J.D., Morgan, S.J., Oneill, A.B., Sykora, T.M., Postl, S.P., Pan, J.B., Sullivan, J.P., Arneric, S.P., 1996. In vivo profile of novel nicotinic ligands with CNS selectivity. Med. Chem. Res. 6, 487-510. Chaki, H., Yamabe, H., Sugano, M., Morita, S., Bessho, T., Tabata, R., K.-I., S., Egawa, M., Tobe, A., Morinaka, Y., 1995. Design and synthesis of 4-acylaminopyridine derivatives: novel high affinity choline uptake enhancers II. Bioorg. Med. Chem. Lett. 5, 1495-1500. Chavez-Noriega, L.E., Crona, J.H., Washburn, M.S., Urrutia, A., Elliott, K.J., Johnson, E.C., 1997. Pharmacological characterization of recombinant human neuronal acetylcholine receptors h-oL-2-[3-2, h-e~-2-[3-4, h-e~-3-[3-2, h-oL-4-[3-2, h-oL-4-[3-4 and h-c~-7 expressed in Xenopus oocytes. J. Pharmacol. Exp. Ther. 280, 346-356. Coyle, J.T., Price, C.L., De Long, M.R., 1983. Alzheimer's disease: a disorder of cortical cholinergic innervations. Science 219, 1184-1190. Davies, P., Maloney, A.J.F., 1976. Selective loss of central cholinergic neurons in Alzheimer's disease. Lancet 2, 1403-1408. Decker, M.W., Brioni, J.D., Sullivan, J.P., Buckley, M.J., Radek, R.J., Raszkiewicz, J.L., Kang, C.H., Kim, D.J.B., Giardina, W.J., Wasicak, J.T., Garvey, D.S., Williams, M., Arneric, S.P., 1994. (S)-3-Methyl5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT 418): a novel cholinergic ligand with cognition-enhancing and anxiolytic activities: II. In vivo characterization. J. Pharmacol. Exp. Ther. 270, 319-328. Doods, H., Entzeroth, M., Ziegler, H., Schiavi, G., Engel, W., Mihm, G., Rudolf, K., Eberlein, W., 1993. Characterization of BIBN 99: a lipophilic and selective muscarinic M2 receptor antagonist. Eur. J. Pharmacol. 242, 23-30. Francis, P.T., Palmer, A.M., Snape, M., Wilcock, G.K., 1999. The cholinergic hypothesis of Alzheimer's disease: a review of progress. J. Neurol. Neurosurg. Psychiatry 66, 137-147. Ghelardini, C., Galeotti, N., Gualtieri, F., Scapecchi, S., Bartolini, A., 1997. 3-oL-Tropanyl 2-(4-Cl-phenoxy)butyrate (SM21): a review of the pharmacological profile of a novel enhancer of cholinergic transmission. CNS Drug Reviews 3, 346-362. Glennon, R.A., Dukat, M., 1996. Nicotine receptor ligands. Med. Chem. Res. 6, 465-486.


Gualtieri, F., Dei, S., Manetti, D., M.N., R., Scapecchi, S., Teodori, E., 1995. The medicinal chemistry of Alzheimer and Alzheimer-like diseases with emphasis on the cholinergic hypothesis. I1 Farmaco 50 (7-8), 489-503. Holladay, M.K., Lebold, S.A., Lin, N.-H., 1995. Structure-activity relationships of nicotinic acetylcholine receptor agonists as potential treatments for dementia. Drug Dev. Res. 35, 191-213. Holladay, M.W., Dart, M.J., Lynch, J.K., 1997. Neuronal nicotinic acetylcholine receptors as target for drug discovery. J. Med. Chem. 40, 4169-4194. Kelly, J.S., 1999. Alzheimer's disease: the tacrine legacy. TIPS 20, 127-129.

89

Lachowicz, J.E., Lowe, D., Duffy, R.A., Rupero, W., Taylor, L.A., Guzik, H., Brown, J., Berger, J.G., Tice, M., McQuade, R., Kozlowski, J., Clader, J., Strader, C.D., Murgolo, N., 1999. SCH 57790: a novel M2 receptor selective antagonist. Life Sciences 64, 535-539. McGeeer, E.G., 1981. Neurotransmitter system in aging and dementia. Prog. Neuropsychopharmacol. 5, 435-445. Perry, E.K., Perry, R.H., Blessed, G., Tomlinson, B.E., 1977. Necropsy evidence of central cholinergic deficits in senile dementia. Lancet 1, 189. Schenk, D.B., Rydel, R.E., May, P., Little, S., Panetta, J., Lieberburg, I., Sinha, S., 1995. Therapeutic approaches related to amyloid betapeptide and Alzheimer's disease. J. Med. Chem. 38, 4141-4154.

This Page Intentionally Left Blank

PHARMAGEUTICA ACTAHELVETIAE ELSEVIER


Nicotinic systems in central nervous systems disease" degenerative disorders and beyond Paul A. Newhouse *, Megan Kelton Clinical Neuroscience Research Unit, Department of Psychiatry University of Vermont College of Medicine, 1 South Prospect Street, Burlington, VT 05403, USA

Abstract Advances in the understanding of the structure, function, and distribution of central nervous system (CNS) nicotinic receptors has provided the impetus for new studies examining the role(s) that these receptors and associated processes may play in CNS functions. Further motivation has come from the realization that such receptors are changed in degenerative neurologic diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Ongoing investigations of the molecular substructure of CNS nicotinic receptors and their pharmacology have begun to open up new possibilities for novel CNS therapeutics with nicotinic agents. Exploiting these possibilities will require understanding of the role(s) that these receptor systems play in human cognitive, behavioral, motor, and sensory functioning. Clues from careful studies of human cognition and behavior are beginning to emerge and will provide direction for studies of potentially therapeutic novel nicotinic agents. Modulation of these receptors with the ultimate goal of producing therapeutic benefits is the goal of these investigations and drug development. This paper will review studies from our laboratory and others that point to the importance of CNS nicotinic mechanisms in normal human cognitive and behavioral functioning as well as their role in disease states. In addition, this paper will examine potential clinical applications of nicotine and/or nicotinic agonists in a variety of CNS disorders with particular emphasis on structural brain disease including: movement disorders such as Parkinson's disease and Tourette's syndrome, cognitive/behavioral disorders such as Alzheimer's disease, attention deficit/hyperactivity disorder, and schizophrenia, and other more speculative applications. Important results from early therapeutic studies of nicotine and/or nicotinic agonists in these disease states are presented. [:'or example, recent studies with nicotine and novel nicotinic agonists such as ABT-418 by our group in AD patients suggest that nicotinic stimulation can improve the acquisition and retention of verbal information and decrease errors. Preliminary results from a series of studies examining the acute and subchronic quantitative effects of nicotine on cognitive and motor functioning in Parkinson's disease suggest that acute nicotine administration and stimulation improves some aspects of cognitive and motor performance and may improve the processing speed of more complex tasks. The most likely near-term applications of novel nicotinic agonists in CNS disorders are likely to be in those disorders that are degenerative in nature, e.g. Parkinson's disease and Alzheimer's disease, or other movement disorders such as Tourette's syndrome. The most likely direct therapeutic role for nicotinic agonists is as augmentation therapy in combination with other agents rather than as monotherapy, except early in disease states or as a prophylactic or preventative treatment. @ 2000 Elsevier Science B.V. All rights reserved. Keywords: Nicotine; Alzheimer's disease; Parkinson's disease; Tourette's syndrome; Schizophrenia; Analgesia; Nicotinic agonists

1. Introduction: Interest in the possibility of utilizing agents that directly interact with nicotinic receptors for the treatment of central nervous system (CNS) disease has followed as understanding of the structure, function, and distribution of CNS nicotinic receptors has increased. Ongoing investigations of the molecular substructure of CNS nicotinic receptors and their pharmacology have begun to open up new possibilities for novel CNS therapeutics with nicotinic agents

* Corresponding author. Tel.: +1-802-847-4560; fax: +1-802-8477889; e-mail: [email protected]

(Arneric et al., 1995). There is considerable evidence from both animal and human studies for the involvement of CNS nicotinic cholinergic receptors in a variety of cognitive, motor and behavioral systems. Modulation of these receptors with the ultimate goal of producing therapeutic benefits is the goal of these investigations and drug development. This chapter will briefly examine potential clinical applications of nicotine a n d / o r nicotinic agonists in a variety of CNS disorders with particular emphasis on structural brain disease. For further details, the reader is referred to a more comprehensive review (Newhouse and Kelton, 1999). Clinical areas covered include: cognitive disorders, move-


92

P.A. Newhouse, M. Kelton / Pharmaceutica Acta Helvetiae 74 (2000) 91-101

ment disorders, analgesia, smoking cessation, cytoprotection, and other more speculative applications. Important results from early therapeutic studies of nicotine and/or nicotinic agonists in these disease states are presented. As clinical therapeutic research in this field is still in its infancy, few long-term controlled clinical trials have been conducted except for smoking cessation.

2. Cognitive disorders 2.1. Alzheimer's diseases

Nicotinic mechanisms may be important in explaining the pathophysiology and in designing treatments for AD (James and Nordberg, 1995). Patients suffering from AD have a marked reduction in cortical nicotinic cholinergic receptor binding compared to age-matched controls (Flynn and Mash, 1986; Whitehouse et al., 1986; Aubert et al., 1992). Normal aged subjects show an age-related decline in cortical nicotinic binding (Flynn and Mash, 1986). Warpman and Nordberg (1995) used epibatidine and ABT418 to show selective losses of o~4132 nicotinic receptors in the brains of patients with AD. Perry et al. (1996) showed that the entorhinal cortex (important in memory formation) rich in nicotinic binding, appears particularly vulnerable to amyloid plaque-induced loss of receptors. More generally, Perry et al. (1995) have shown that nicotine receptor loss seems tightly linked to the primary pathology in the dementias, e.g., linked to dopaminergic cell loss in PD and Lewy Body dementia, and linked to amyloid plaques and tangles in hippocampal and parahippocampal areas in AD. In humans, nicotine is reported to increase arousal and attention as well as decrease reaction time and prevent decline in efficiency over time (Wesnes and Warburton, 1983, 1985). In both animals and humans nicotine improves the subject's ability to withhold responses to inappropriate stimuli (Myrsten et al., 1972; Wesnes and Warburton, 1983; Newhouse et al., 1988). This may be relevant to AD because a cardinal feature of the cognitive disorder of AD and a possible marker of cholinergic dysfunction (Fuld et al., 1982) is the difficulty demented patients have in inhibiting inappropriate responses or in responding to inappropriate stimuli. This difficulty in response selection and/or suppression is one explanation of the liberal response bias seen in AD. Gray et al. (1996) have shown that nicotine enhances hippocampal synaptic transmission which may be critical for new learning to take place. AD is associated with a marked cerebral blood flow (CBF) perfusion deficit in parietotemporal cortex in addition to the global decrease in cerebral perfusion. It is of interest that the nicotinic antagonist mecamylamine reliably reproduces this abnormal CBF pattern in normal volunteers, (Gitelman and Prohovnik, 1992). Nicotine reli-

ably augments the enhancement in CBF produced by electrically stimulating basal forebrain cholinergic neurons (Arneric, 1989). As the basal forebrain cholinergic neurons are heavily damaged in AD, changes in observed CBF may be secondary to damage to nicotinic systems. Presumably, the inability to autoregulate CBF impairs cognitive functioning. Neuroimaging studies also support the involvement of nicotinic cholinergic systems in AD. Nordberg (1993) showed a significant correlation change between the change in temporal cortex labeling of ~1C-nicotine and cognitive function scores in AD patients using positron emission tomography (PET). This result was bolstered by further work from these investigators (Nordberg et al., 1995) in which a kinetic model was developed to quantify the loss of nicotinic receptor binding in vivo in AD patients. Significant correlations were shown between cognitive dysfunction and the loss of nicotinic receptor binding in temporal and frontal cortices and hippocampus in these patients using PET. Nordberg (1993) also examined the effects of treatment with the anticholinesterase tacrine on AD patients using PET and showed that brain nicotinic receptor binding of 1~C-nicotine increased along with CBF after 3 weeks of treatment. Epidemiologic studies of AD that assess risk factors show that, like Parkinson's disease, smokers are at a lower risk of developing AD than nonsmokers, even when other factors are controlled for (Tyas, 1996). Lee (1994) has done a meta-analysis of these studies and has calculated a relative risk of 0.64 for smokers to develop AD. In a retrospective case-control autopsy study, Ulrich et al. (1997) analyzed 72 age- and sex-matched smoker-nonsmoker pairs and showed that an apparent protective action against senile plaque formation could be demonstrated in 28 age matched pairs of smoking-nonsmoking women, although a positive correlation between the amount of smoking and neurofibrillary changes in smokers of both sexes was also seen. Whether potential protective effects of smoking are secondary to nicotine is unclear but in vitro data suggesting a neuroprotective effect of nicotine are consistent with this possibility (Arneric et al., 1995). 2.2. Studies of nicotinic antagonists and agonists in Alzheimer's disease 2.2.1. Antagonist studies Studies utilizing antagonists are useful for establishing the cognitive relevance of neuro-receptor changes in brain as they produce a temporary chemical "lesion". Newhouse et al. (1992; 1993; 1994) have studied the effects of the centrally-active non-competitive nicotinic antagonist and peripheral ganglionic blocker mecamylamine on cognitive functioning in young and elderly normals and Alzheimer's and Parkinson's disease patients. These studies attempted to establish that nicotinic blockade produced cognitive impairment in humans, and examined whether


there were age- or disease-related changes in sensitivity to nicotinic blockade, which would be indicated by shifts in dose-response curves between groups. Mecamylamine administration produced dose-related impairment of the acquisition of new information with group differences in sensitivity. Young normals showed significant cognitive impairment errors after the highest dose. By contrast, the elderly normals showed significant impairment after the middle and high doses, and the Alzheimer's disease subjects showed impairment after all three active doses. This pattern was seen in both verbal and nonverbal learning tasks. In the AD patients, the learning rate actually became negative at 10 and 20 mg of mecamylamine, i.e., they were actually getting worse with increasing trials. Interestingly, in the old normals, mecamylamine produced a dose-related change in response bias with a significant liberal shift after the high dose, which has been seen in AD patients. This did not occur with the young normals. Regarding psychomotor speed, mecamylamine produced dose-related slowing in a number of tasks that measured reaction time. These included increases in reaction time for the CRT and manikin tasks. Older subjects tended to show proportionately greater increases in reaction time than the younger subjects did. By contrast, there were minimal behavioral effects. Pickworth (1997) examined the effects of mecamylamine in smoking and non-smoking volunteers on electrophysiological and performance measures. In both groups, mecamylamine cause dose-related decreases in alpha EEG frequency and increases in delta frequency. In addition, response time slowed in both vigilance and distractibility tasks and delayed recall was impaired. These results confirm prior studies concerning the cognitive-impairing effects of blocking central nicotinic receptors and provide support for an important role for nicotinic receptor loss in the pathogenesis of the cognitive impairment in Alzheimer' s disease. These results suggest that the deficits produced by mecamylamine resemble in several respects those seen in AD. Deficits in short- and long-term memory, impaired attention, liberal response bias, and decreases in reaction time are hallmarks of the dementing picture seen in these disorders. The age-related nature of some of the findings suggest that the decline in nicotinic receptors with age produces increased vulnerability to the effects of nicotinic blockade.

2.2.2. Agonist studies Newhouse et al. (1988; 1993; 1996)have examined the effects of intravenous nicotine in AD with particular attention on tasks that are affected by mecamylamine. Analysis of the cognitive effects of nicotine in the AD group showed that there was a significant dose-related decrease in verbal learning errors, with a "U"-shaped dose-response curve. A similar improvement pattern was seen in

93

long-term verbal recall. Neuroendocrine measures (Newhouse et al., 1990) tended to confirm that the doses used were active at CNS nicotinic receptors. Results show that nicotine produces improvements in attentionally driven tasks with improved reaction time, hits and false alarms on a continuous performance task. Throughput (speed-accuracy product) was improved as well. These findings of the beneficial results of acute nicotinic stimulation in AD have been supported by the studies of Sahakian et al. (Jones et al., 1992; Sahakian and Coull, 1994) who have shown that subcutaneous nicotine administration in AD patients produced improvements in attentional functioning. This group found that nicotine produced a highly significant improvement in accuracy on a sustained visual attention task (which involved the detection of number sequences). Importantly, there was no speed-accuracy tradeoff, i.e., patients do not become slower, even though they become more accurate. Further, they showed that the AD subjects improved in a dose-dependant matter on attentional aspects of a visual short-term memory and attention task. Katayama et al. (1995) showed that nicotine improved performance in dementia patients using event-related potentials. More chronic administration of nicotine to AD has also shown promise. Wilson et al. (1995) administered nicotine by patch to six AD patients for 8 days. Compared to the placebo patch condition, there were significantly fewer errors on a non-verbal learning task while subjects were on nicotine. This effect persisted for at least a week after withdrawal. However, Snaedal et al. (1996) were unable to find a significant effect of 4 weeks of transdermal nicotine administration on memory in 18 AD patients, possibly due to a significant placebo effect as patients on both nicotine and placebo showed improvements in short-term memory. Potter et al. (1999) have recently examined the acute effects of the novel nicotinic agonist ABT-418 on cognitive functioning in Alzheimer's disease. Subjects showed significant linear dose-related improvements in verbal learning and memory on the Selective Reminding Task as reflected by improved total recall and a decline in recall failure. Qualitatively similar improvements were seen in nonverbal learning tasks such as spatial learning and memory and repeated acquisition. Positive dose-related effects on reaction time were also seen. Interestingly, subjects also showed a dose-related decline in anxiety and fear, confirming prior animal studies suggesting that this agent may also have anxiolytic effects. These positive results echo studies of this agent in aged monkeys by Buccafusco et al. (1995) who showed dose-related improvements in a delayed matching-to-sample task performance following administration of ABT-418. These studies represent significant evidence that stimulation of nicotinic receptors can improve the acquisition and retention of verbal (declarative) and non-verbal information in humans. The role of attentional effects of nicotinic stimulation has been stressed by Sahakian and Coull (1994). However, as has been suggested by Warburton and

94


Rusted (1993), nicotine's effects are most often seen in tasks that have a large attentional load. Preclinical studies of other novel nicotinic agonists also show promise. Aged rats show improved learning when treated with GTS-21 (Arendash et al., 1995). SIB-1553A is an oL4132 subtypeselective nicotinic agonist and appears to be efficacious in acute and chronically stimulating hippocampal acetylcholine release (Lloyd et al., 1998). This compound appears to produce enhanced performance in a variety of models of cognitive dysfunction (e.g., aged rats, rhesus monkeys, rats with cholinergic lesions) in areas such as spatial and non-spatial working and reference memory (Lloyd et al., 1998). A profile such as this suggests that this compound may have activity in disorders of cortical and subcortical cholinergic dysfunction such as AD. RJR2403 (Lipiello et al., 1996) appears to be a highly selective ligand for the oL4132 subtype of nicotinic receptor and may be a useful agent for investigating the clinical and cognitive effects of stimulating this receptor subtype in degenerative neurologic disease.

3. Schizophrenia The very high rates of cigarette smoking in schizophrenia (as high as 93% in male schizophrenic patients; Kirch, 1999) do not appear to be explained by gender, age or socioeconomic status (Dalack et al., 1998) and appear to reflect either disease- or treatment-related processes that encourage cigarette use. A major hypothesis for this high rate of use is that nicotine may have salutary effects on cognitive and/or behavioral functioning in this disorder (reviewed in detail in Dalack et al., 1998). Cigarette smoking appears to improve abnormal smooth pursuit eye movements that are commonly found in schizophrenic subjects (Olincy et al., 1998). The P50 auditory evoked response to repeated stimuli appears to be abnormal in many schizophrenic patients (Freedman et al., 1994) and their first-degree relatives (Waldo et al., 1991). Studies of this wave are designed to examine inhibitory control of sensory processing and involve examining the ratio of the two P50 waves evoked after auditory stimuli 500 ms apart. In schizophrenic subjects Adler et al. (1992; 1993) have shown that nicotine administration via smoking or nicotine gum appears to transiently normalize this impaired response and restore sensory gating. The neurobiological mechanism responsible for this response has been traced to pyramidal neurons of the hippocampus as a major source (Bickford-Wimer et al., 1990). The normal response is blocked by the oL7-nicotinic receptor antagonist oLbungarotoxin (Luntz-Leybman et al., 1992). Autoradiography has shown that these c~7 receptors appeared to occur on non-pyramidal hippocampal GAB A-containing inhibitory neurons (Freedman et al., 1993). An extension of this work (Freedman et al., 1997) has linked this abnormality in schizophrenics and their first-degree relatives to a

dinucleotide polymorphism at chromosome 15q13-14, the site of the oL7-nicotinic receptor. In addition other studies performed in schizophrenic patients support salutary effects of nicotine on cognitive performance and attention in this disorder. Levin et al. (1996a; b) found that nicotine administered via skin patch reversed some of the haloperidol-related impairments in a variety of cognitive tests assessing memory and reaction time. As with normal volunteers, nicotine also improved attentive performance during a continuous performance task in these subjects. Haloperidol administration has also been found to increase smoking behavior (McEvoy et al., 1995a), and the atypical neuroleptic clozapine appears to decrease smoking in schizophrenic subjects (McEvoy et al., 1995b). Clozapine, unlike most neuroleptics, appears to improve P50 gating in schizophrenic patients (Nagamoto et al., 1996) in a similar manner to nicotine. Dalack et al. (1998) have suggested that the high rate of nicotine use in schizophrenia may be associated with a partial correction of a putative cortical-subcortical dissociation of dopamine activity and that nicotine use is associated with increased glutamatergic activity in limbic regions implicated in schizophrenia, particularly the frontal cortex and hippocampus. An optimal nicotinic agonist for use in this patient population may have to have mixed properties to improve both dopaminergic and glutamatergic functioning as this may require stimulation of several different subtypes, including oL7 homomeric receptors as well as oL5- or oL6containing receptors. An important concern in any clinical trial of a potential nicotinic agonist for schizophrenia would be the identification of appropriate clinical endpoints. In addition to the highly specific attentional/ sensory abnormalities heretofore demonstrated in schizophrenic patients, negative and deficit symptomatology may well be an appropriate target for such trials as there is little convincing evidence thus far that the florid psychotic symptoms are likely to be responsive to nicotinic stimulation. Nonetheless, improvements in cognitive symptoms may produce significant long-term clinical benefit. The development of effective nicotinic agonists could be an important contribution to progress in this challenging disorder.

4. Attention deficit hyperactivity disorder Given the attentional improvement that has been demonstrated with nicotinic receptor stimulation, there are implications for nicotine or novel nicotinic agonists as a possible treatment strategy in attention deficit-hyperactivity disorder (ADHD). This is a disorder primarily of children, but affects adults as well. ADHD afflicts as many as 3-5% of American children (American Psychological Association, 1994) and is characterized by inattention, restlessness, impulsiveness and hyperactivity. There is sig-


nificant co-morbidity of smoking in teens and adults with ADHD. It has been reported that 40% of adults with ADHD smoke cigarettes compared to 26% of the general population (Pomerleau et al., 1995). As the symptomatology of ADHD often leads to difficulty in school and other behavioral problems, there is some debate as to whether cigarette smoking is an act of self-medication, or a manifestation of the behavioral problems related to the symptomatology. Several studies have examined the effects of nicotine administered via patch on the attentional processes in adults with ADHD. Conners et al. (1996) administered placebo or low and high dose nicotine patches to nonsmoking and smoking adults with ADHD, respectively. Nicotine significantly improved attentional performance on the Continuous Performance Task and increased attentional self-ratings of smokers on the Profile of Mood States. All subjects were rated as having a decrease in ADHD symptoms in the Clinical Global Impressions scale. These finding were replicated in a separate study by Levin et al. (1996a; b). In this study, nicotine administration also reduced the standard error of reaction time over blocks of trials, suggesting improved consistency in attentional performance. Novel nicotinic agonists such as ABT-418 have also been found to improve some aspects of attention in aged primates (Prendergrast et al., 1998). In the primate study, Prendergrast et al. found that ABT-418 and ABT-089 prevented distractibility and increased delayed recall accuracy in trials where a distracter was present. It has been suggested that the central disorder of ADHD is an impairment of behavioral inhibition of responding to inappropriate external stimuli or distracters (Barkley, 1997). Children with ADHD showed excessive errors of commission and a stronger tendency to respond correctly and in error during a Continuous Performance Task (Iaboni et al., 1995). If nicotinic stimulation improves attention and allows for more effective inhibition of attention to inappropriate stimuli, then perhaps ADHD may be treatable by stimulating the nicotinic system both in adults and children. This may also potentially prevent cigarette smoking in some subjects. As the abuse liability of nicotine separate from tobacco products is extremely low (Hughes, 1998), such treatment may be acceptable for adolescents.

5. Movement disorders

5.1. Parkinson's disease

A number of studies have shown that smokers have a lower than expected incidence of PD, suggesting a protective effect of nicotine (Baumann et al., 1980; Baron, 1986, 1994). These studies have been carefully reviewed by Morens et al. (1995) who conclude that the association is not artifactual. While epidemiologic studies do not confirm that nicotine is the protective agent, the only other possible

95

protective aspect of cigarette smoke identified thus far is a reduction in monoamine oxidase-B (MAO-B) activity after long-term smoking (Fowler et al., 1996). Nicotine has also been shown to counteract the locomotor effects of MPTPinduced lesions in mice, a putative model for PD (Sershen et al., 1987). A similar loss of cholinergic cells in the basal forebrain nuclei as occurs in AD has been described in PD (Whitehouse et al., 1983). The loss of cholinergic markers in the cortex (Perry et al., 1995) that occur in PD may be related to lesions in these nuclei and other cholinergic projections to the cortex (Whitehouse et al., 1988). In demented PD patients, the loss of cortical cholinergic markers has been shown to be of greater magnitude and more extensive than that of nondemented PD patients (Perry et al., 1985). Studies have shown a marked reduction in cortical nicotinic receptor binding that parallels the degree of dementia in PD and increasing age (Whitehouse et al., 1988; Aubert et al., 1992). There is similarity between the cortical nicotinic binding site loss in PD and AD as well as similar changes in other cholinergic markers. Nicotine was examined as a treatment for PD as early as the 1920s (Moll, 1926) in patients with a form of secondary parkinsonism due to encephalitis lethargica. Marshall and Schniden (1966) examined the effects of nicotine on tremor, including that secondary to Parkinson's disease and showed mildly positive effects in several patients. More recently FagerstriSm et al. (1994) reported a detailed study of two patients who had nicotine gum and patch added to their Parkinson's disease therapy. Using a single subject, placebo-control reversal design, improvement was associated with nicotine dosing and involved diminished tremor and disorganized thinking in one patient and lessened bradykinesia and increased energy in the other. Newhouse et al. (1998) have preliminarily examined the quantitative effects of nicotine in PD patients. Subjects with mild to moderate PD received dose-ranging infusions of intravenous nicotine up to 1.25 Ixg/kg/min, followed by chronic administration of nicotine by transdermal patch with doses ranging up to 14 m g / d a y for 2 weeks. Testing occurred both during drug administration and up to 2 weeks after drug cessation to look for prolonged effects. Nicotine appeared to acutely improve attention/arousal in PD patients as measured by the Critical Flicker Fusion (CFF) and the Choice Reaction Time test. Mecamylamine pre-administration antagonized the improvement, suggesting a specific effect on nicotinic receptors. During the chronic phase of administration by transdermal patch, nicotine appeared to improve performance speed in standard clinical motor performance tasks. In most cases, improvement appeared to persist after drug withdrawal, although there was some evidence for the beginning of a return towards baseline values at the session 2 weeks after drug withdrawal. For the computerized performance tasks, subjects showed improvement on the motor portions of certain

96


tasks at day 14, but by 2 weeks post drug, they had returned almost to baseline values. These effects are consistent with the possibility that a sustained evoked increase in the release of dopamine in nigrostriatal pathways may be occurring as a result of presynaptic nicotinic receptor stimulation. Studies such as these provide optimism that nicotinic stimulation may be a fruitful strategy for PD treatment, either by utilizing nicotinic agonists as monotherapy in early cases or as a dopa-augmenter or dopa-sparing drug in later stage disease. In addition to nicotine, other novel nicotinic agonists are being developed specifically focused on Parkinson's disease. SIB-1508 and its racemate SIB-1765F are subtype selective nicotinic agonists particularly for oL4[32-containing nicotinic receptors (Sacaan et al., 1997). These compounds appear to have greater efficacy than nicotine at releasing dopamine from striatal slices. SIB-1765F potentiated the positive locomotor effects of L-dopa in a reserpine model of PD in rats (Menzaghi et al., 1997) with a rapid onset of action. The compound produced a small improvement in locomotion when administered alone, however the effect was much greater when combined with L-dopa. SIB-1508Y, an isomer of SIB-1765F, is even more potent in this model and has also shown positive activity in the MPTP-treated monkey model of PD (Schneider et al., 1998).

6. Tourette's syndrome Tourette's syndrome (TS) is a hyperkinetic movement disorder with symptoms of sudden, rapid and brief, recurrent, stereotyped motor movements or sounds (tics) and can range from mild to severe. TS is commonly treated with dopamine antagonists such as haloperidol, which may be effective but has significant adverse side effects and is ineffective in up to 30% of cases. While the etiology is not known it is proposed that, unlike PD, TS represents a disorder of excess dopamine transmission in the striatum (Shapiro et al., 1989; Wolf et al., 1996), either through dopamine excess or receptor hypersensitivity. Following laboratory studies showing that acute nicotine administration could potentiate haloperidol-induced catalepsy and locomotor activity in rodents (Emerich et al., 1991), an intuitive leap was made and clinical trials were begun in TS patients (Sanberg et al., 1989). Initial open trials with nicotine gum showed reductions in both tic severity and frequency particularly when used to augment the effects of haloperidol (McConville et al., 1992). More recent studies (Silver and Sanberg, 1995) using transdermal nicotine have confirmed the positive effects of nicotine when added to haloperidol therapy in TS patients and have also shown that in some patients the positive effects persist for several weeks after patch removal. Long-term positive effects of transdermal nicotine were also found by Dursan et al. (1994) who found that two consecutive

nicotine patches reduced tic severity scores for up to 4 weeks after patch removal. The potential efficacy of nicotine and/or nicotinic agonists in both hypodopaminergic (PD) and hyperdopaminergic disorders (TS) may reflect the mixed agonist/antagonist activities of nicotine. In the case of TS, available evidence from animal studies suggests that a prolonged desensitization and inactivation of nicotinic receptors following exposure to nicotine may be producing the therapeutic response (Shytle et al., 1999). This theory has been tested by the use of the nicotinic antagonist mecamylamine to augment the effect of antidopaminergic agents in TS with positive results (Sanberg et al., 1998). Whether novel agonists which do not produce as rapid or as long-term a desensitization of nicotinic receptors would be helpful in TS remains untested.

7. Other potential clinical applications 7.1. Analgesia The possible analgesic effects of nicotine have been a subject of dispute. Decreased sensitivity to pain has been demonstrated in studies involving men but the effect has been difficult demonstrate in women (Jamner et al., 1998). The discovery of the potent antinociceptive effects of the frog-derived nicotinic neurotoxin epibatidine has activated the search for analogs that might provide significant analgesia without unacceptable toxicity. Epibatidine itself appears to have antinociceptive activity 200-fold more potent than that of morphine (Brioni et al., 1997), but has significant toxicity due to potent activity at the ganglionic and neuromuscular junction. There is evidence for both central and peripheral sources of nicotine-induced analgesia (Caggiula et al., 1995), as well as activity at the level of the primary sensory neuron (Puttfarcken et al., 1997) and dorsal root ganglia (Roberts et al., 1995). A major goal, therefore, is to develop novel nicotinic analogs with both a large therapeutic index and significant analgesic activity. The potent novel nicotinic agonist ABT594 (a potent oL4132 agonist) appears to exhibit antinociceptive properties equal in efficacy to those of morphine across a series of animal models of acute thermal, chemical and neuropathic pain (Bannon et al., 1998; DonnellyRoberts et al., 1998). The analgesic effects are blockable by mecamylamine and repeated treatment did not appear to elicit opiod-like withdrawal or physical dependence. Such a compound appears to be a promising agent for clinical development as a non-opiate analgesic agent if toxicity is low.

8. Cytoprotection Intriguing evidence has been developed that suggests that nicotine and nicotinic drugs may have cytoprotective


effects. In vitro studies have shown that nicotine can protect against the excitotoxic effects of glutamate when it was administered before, but not during exposure to glutamate or NMDA (Shimohama et al., 1996), perhaps mediated through the inhibition of nitric oxide formation. Nicotine also appears to offer protection against systemic kainic acid-induced excitotoxic effects (Borlongan et al., 1995). Protection against excitotoxic neuronal death by nicotine appears to involve oL7 receptor mechanisms as the effect can be antagonized in vitro by oL7 antagonists such as oL-bungarotoxin (Kaneko et al., 1997; Carlson et al., 1998). Nicotine also prevents the neuronal degeneration that occurs after destruction of the basal forebrain by neurotoxins (Owman et al., 1989). Other nicotinic agonists such as ABT-418 and GTS-21 have also shown neuroprotective effects in cell culture studies (Marin et al., 1994; Donnelly-Roberts et al., 1996). Yamashita and Nakamura (1996) have shown that nicotine prevents cell death in PC-12 cells after withdrawal of growth factors. In the case of Alzheimer's disease, disease modifying therapy may involve therapeutic approaches designed to modify the basic pathogenetic mechanisms of AD. In particular, [3-amyloid appears to produce dysfunction of nicotine-stimulated release of acetylcholine and dopamine (Itoh et al., 1996) and other cholinergic dysfunction which can to some extent be ameliorated by nicotine administration (Maurice et al., 1996). It has been shown that nicotine may act to inhibit the deposition of [3-amyloid in vitro (Salomon et al., 1996) by impairing the aggregation of ~1-42 peptide into [3-pleated sheets. It appears that nicotine may bind to and stabilize the a-helical structure of the [3-peptide. Nicotine appears to inhibit [3-amyloid toxicity in rat cortical neurons (Kihara et al., 1997; Zamani et al., 1997). In addition, Kihara et al. (1997) showed that the novel oL7 nicotinic agonist anabaseine derivative DMXB produced a similar effect. Thus, the potential cytoprotective effects of nicotine appear to be receptor-mediated, suggesting the possibility that novel nicotinic agonists might provide more potent effects. These studies, in conjunction with the epidemiologic data, suggest the possibility that chronic treatment with nicotine or other nicotinic agonists might delay or prevent the clinical onset of AD. The evidence for the neuroprotective effect of nicotine is at least as strong as for some of the other classes of agents under investigation. The use of nicotine as a chronic protective agent may raise more complex questions than other compounds such as antioxidants, namely the potential for other chronic adverse effects (e.g., cardiovascular) or dependence. However, even after patients become symptomatic, chronic nicotine (and/or muscarinic) stimulation may slow disease progression and prolong a period of less impaired functioning. Such a strategy is worth considering, particularly for high risk groups, such as individuals with a strong family history of AD and/or who are positive for the APOE4 allele, or individuals who appear, based on cognitive or

97

clinical assessment, to be either questionably impaired or in very earliest stages of dementia.

9. Smoking cessation Smoking cessation is the only currently approved indication for nicotinic therapy. Several meta-analyses of randomized controlled trials using nicotine for smoking cessation have concluded that nicotine replacement therapy is an effective treatment for smoking cessation, although 6-month quit rates do not appear to be much better than approximately 20% for any type of nicotine replacement device (Fiore et al., 1996; Westman and Rose, 1999), especially if given in the absence of a behavioral program. A more promising approach may be to utilize a combined agonist/antagonist approach. In a series of studies, Rose et al. (1994; 1996) have shown that combining low-dose mecamylamine administration with nicotine in smokers produces dramatic improvements in smoking 6-month quit rates over nicotine alone (40% versus 20%). In addition to this novel approach, the development of orally available novel nicotinic agonists may provide additional therapeutic options with reduced side effects and improved patient acceptability. Lobeline, a mixed nicotinic agonist/antagonist, is being examined for efficacy in smoking cessation clinical trials, for further information on the use of nicotine in smoking cessation, the reader is referred to several recent reviews for coverage of this topic (Balfour and Fagerstrom, 1996; Westman and Rose, 1999).

10. Anxiety / depression The anxiety relieving effect of nicotine appears to be independent of the subject's smoking status (Gilbert, 1979), but may be quite dependent on the baseline anxiety state of the individual. Novel nicotinic agonists such as ABT-418 show a non-benzodiazepine anxiolytic profile in animal tests (Brioni et al., 1994) and studies in AD patients show fear and anxiety reducing characteristics without cognitive impairment (Potter et al., 1999). The possibility therefore exists that nicotinic agonists could be developed specifically for their anti-anxiety properties which would offer the potential benefit of decreasing anxiety without cognitive impairment, a combination that is hard to achieve with today's agents. Studies have shown that individuals who smoke heavily are at high risk of depressive illness, either first onset or recurrent, if they cease smoking, especially without nicotine replacement therapy (Glassman et al., 1990). Although direct antidepressive effects of nicotine remain to be demonstrated, there is no question that nicotine can have significant salutary effects on mood, at least in deprived smokers (Foulds et al., 1997). As nicotine has significant effects on the release of monoamine neurotransmitters

98


which are important in depression, such as serotonin, the possibility exists that novel nicotinic agonists without the adverse side effects seen with nicotine could be developed specifically for their antidepressive effects, either as monotherapy or as an augmentative therapy.

11. Epilepsy Autosomal dominant nocturnal frontal-lobe epilepsy (ADNFLE) is a form of partial epilepsy which is characterized by frontal-lobe motor seizures occurring during sleep (Philips et al., 1998). This disorder has been linked to a mutation in the channel-lining domain (M2) of the oL4 nicotinic receptor subunit with "use-dependent potentiation" of the electrophysiological response to nicotinic agonists (Lena and Changeux, 1998). If the symptoms of ADNFLE are secondary to overactivity of this receptor or unusual potentiation, then nicotinic antagonists such as mecamylamine may be helpful.

12. Further directions The most likely near-term applications of novel nicotinic agonists in CNS disorders are likely to be in those disorders that are degenerative in nature, e.g., Parkinson's disease and Alzheimer's disease, or other movement disorders such as Tourette's syndrome. The most likely direct therapeutic role for nicotinic agonists is as augmentation therapy in combination with other agents rather than as monotherapy, except early in disease states or as a prophylactic or preventative treatment. A major problem remains as to whether compounds can be developed which are selective in producing improvement in cognition, motoric behavior, attention or pain without significant side-effects. Therapeutic trials of nicotine and novel nicotinic agonists will be important to assess the realistic likelihood of long-term improvements in functioning as heretofore virtually all studies have been short-term or acute. Future clinical studies should carefully focus on cognitive and behavioral measures that are likely to be positively affected by nicotinic stimulation based on preliminary acute studies.

Acknowledgements Some of the work described in this chapter was supported in part by NIMH R29-46625, GCRC M01-00109, Abbott Laboratories, and Japan Tobacco.

References Adler, L.E., Hoffer, L.J., Griffith, J., Waldo, M.C., Freedman, R., 1992. Normalization by nicotine of deficient auditory sensory gating in the relatives of schizophrenics. Biol. Psychiatry 32, 607-616.

Adler, L.E., Hoffer, L.D., Wiser, A., Freedman, R., 1993. Normalization of auditory physiology by cigarette smoking in schizophrenic patients. Am. J. Psychiatry 150, 1856-1861. Arendash, G.W., Sengstock, G.J., Sanberg, P.R., Kern, W.R., 1995. Improved learning and memory in aged rats with chronic administration of the nicotinic receptor agonist GTS-21. Brain Res. 674, 252259. Arneric, S.P., 1989. Basal forebrain neurons modulate cortical cerebral blood flow: increases by nicotinic cholinergic mechanisms. J. Cereb. Blood Flow Metab. 9, $502, Suppl. 1. Arneric, S.P., Anderson, D., Bannon, A., Briggs, A.W., Clark, A., Brion, J. et al., 1995. Preclinical pharmacology of ABT-418: a prototypical cholinergic channel activator for the potential treatment of Alzheimer' s disease. CNS Drug Rev. 1, 1-26. Aubert, I., Araujo, D.M., C~cyre, D., Robitaille, Y., Gauthier, S., Quirion, R., 1992. Comparative alterations of nicotinic and muscarinic binding sites in Alzheimer's and Parkinson's diseases. J. Neurochem. 58, 529-541. Balfour, D.J., Fagerstrom, K.O., 1996. Pharmacology of nicotine and its therapeutic use in smoking cessation and neurodegenerative disorders. Pharmacol. Ther. 72, 51-81. Bannon, A.W., Decker, M.W., Holladay, M.W., 1998. Broad-spectrum, non-opioid analgesic activity by selective modulation of neuronal nicotinic acetylcholine receptors. Science 2, 77-81. Barkley, R.A., 1997. Attention-deficit/hyperactivity disorder, self-regulation, and time: towards a more comprehensive theory. J. Dev. Behav. Pediatr. 18, 271-279. Baron, J.A., 1986. Cigarette smoking and Parkinson's disease. Neurology 36, 1490-1496. Baron, J.A., 1994. Epidemiology of smoking and Parkinson's disease. In: Clarke, Quik, Thurau, Adlkofer (Eds.), Effects of Nicotine on Biological Systems II, Birkh~iuser, Boston, $42. Baumann, R.J., Jameson, H.D., McKean, H.D., Maack, D.G., Weisberg, L.M., 1980. Cigarette smoking and Parkinson's disease: 1. Comparison of cases with matched neighbors. Neurology 30, 839-843. Bickford-Wimer, P.C., Nagamoto, H., Johnson, R., Adler, L.E., Egan, M., Rose, G.M. et al., 1990. Auditory sensory gating in hippocampal neurons: a model system in the rat. Biol. Psychiatry 27, 183-192. Borlongan, C.V., Shytle, R.D., Ross, S.D., Shimizu, T., Freeman, T.B., Cahill, D.W., 1995. Nicotinic protects against systemic kainic acid-induced excitotoxic effects. Exp. Neurol. 136, 261-265. Brioni, J.D., O'Neill, A.B., Kim, D.J.B., Buckley, M.J., Decker, M.W., Arneric, S.P., 1994. Anxiolytic-like effects of the novel cholinergic channel activator ABT 418. J. Pharmacol. Exp. Ther. 271, 353-361. Brioni, J.D., Decker, M.W., Sullivan, J.P., Arneric, S.P., 1997. The pharmacology of (-)-nicotine and novel cholinergic modulators. Adv. Pharmacol. 37, 153-214. Buccafusco, J.J., Jackson, W.J., Terry, A.V. Jr., Marsh, K.C., Decker, M.W., Arneric, S.P., 1995. Improvement in performance of a delayed matching-to-sample task by monkeys following ABT-418: a novel cholinergic channel activator for memory enhancement. Psychopharmacology 120, 256-266. Caggiula, A.R., Epstein, L.H., Perkins, K.A., Saylor, S., 1995. Different methods of assessing nicotine-induced antinociception may engage different neural mechanisms. Psychopharmacology 122, 301-306. Carlson, N.G., Bacchi, A., Rogers, S.W., Gahring, L.C., 1998. Nicotine blocks TNF-alpha-mediated neuroprotection to NMDA by an alphabungarotoxin-sensitive pathway. Neurobiology 35, 29-36. Conners, C.K., Levin, E.D., Sparrow, E., Hinton, S., Ernhardt, D., Meck, W.H., Rose, J.E., March, J., 1996. Nicotine and attention deficit in adult ADHD. Psychopharmacol. Bull. 32, 67-73. Dalack, G., Healy, D., Meador-Woodruff, J., 1998. Nicotine dependence in schizophrenia: clinical phenomena and laboratory findings. Am. J. Psychiatry 155, 1490-1501. Donnelly-Roberts, D.L., Xue, I.C., Arneric, S.P., Sullivan, J.P., 1996. In vitro neuroprotective properties of the novel cholinergic channel activator (ChCA), ABT-418. Brain Res. 719, 36-44.


Donnelly-Roberts, D.L., Puttfarcken, P.S., Kuntzweiler, T.A., Briggs, C.A., Anderson, D.J., Campbell, J.E. et al., 1998. ABT-594 [(R)-5(2-azetidinylmethoxy)-2-chloropyridine]: a novel, orally effective analgesic acting via neuronal nicotine acetylcholine receptors: I. In vitro characterization. J. Pharmacol. Exp. Ther. 285, 777-786. Dursan, S.M., Revely, M.A., Bird, R., Stirton, F., 1994. Long lasting improvement of Tourette's syndrome with transdermal nicotine. Lancet 344, 1577. Emerich, D.F., Zanol, M.D., Norman, A.B., McConville, B.J., Sanberg, P.R., Fiore, M.C. et al., 1991. Nicotine potentiates haloperidol-induced catalepsy and hyperactivity. Pharmacol. Biochem. Behav. 38, 875-880. Fagerstr~Sm, K.O., Pomerleau, O., Giordani, B., Stelson, F., 1994. Nicotine may relieve symptoms of Parkinson's disease. Psychopharmacology 116, 117-119. Fiore, M.C., Bailey, W.C., Cohen, S.J. et al., 1996. Smoking cessation. Clinical Practice Guideline No 18. Rockville, MD: US Department of Health and Human Services, Public Health Service, Agency for Health Care Policy and Research. AHCPR Publication No. 96-0692. Flynn, D., Mash, D., 1986. Characterization of 1-[3H]nicotine binding in human cerebral cortex: comparison between Alzheimer's disease and the normal. J. Neurochem. 47, 1948-1954. Foulds, J., Stapleton, J.A., Bell, N., Swedenham, J., Jarvis, M.J., Russell, M.A., 1997. Mood and physiological effects of subcutaneous nicotine in smokers and never-smokers. Drug Alcohol Depend. 44, 105-115. Fowler, J.S., Volkow, N.D., Wang, G.J., Pappas, N., Logan, J., MacGregor, R. et al., 1996. Inhibition of monoamine oxidase B in the brains of smokers. Nature 379, 733-736. Freedman, R., Wetmore, C., Stromberg, I., Leonaard, S., Olson, L., 1993. c~-Bungarotoxin binding to hippocampal interneurons: immunocytochemical characterization and effects on growth-factor expression. J. Neurosci. 13, 1965-1975. Freedman, R., Adler, L.E., Bickford, P., Byerley, W., Coon, M., Cullum, C.N. et al., 1994. Schizophrenia and nicotine receptors. Harvard Rev. Psychiatry 2, 179-192. Freedman, R., Coon, H., Mytes-Worsley, M., Orr-urteger, A., Otincy, A., Davis, A. et al., 1997. Linkage of a neurophysiological deficit in schizophrenia to a chromosome 15 locus. Proc. Natl. Acad. Sci. 94, 587-592. Fuld, P., Katzman, R., Davies, P., Terry, R.D., 1982. Intrusions as a sign of Alzheimer dementia: chemical and pathological verification. Ann. Neurol. I1, 155-159. Gilbert, D.G., 1979. Paradoxical tranquilizing and emotion-reducing effect of nicotine. Psychol. Bull. 86, 643-661. Gitelman, D.R., Prohovnik, I., 1992. Muscarinic and nicotinic contributions to cognitive function and cortical blood flow. Neurobiol. Aging 13, 313-318. Glassman, A.H., Helzer, J.E., Covey, L.S., Cuttler, L.B., Stetner, F., Tipp, J.E. et al., 1990. Smoking, smoking cessation, and major depression. JAMA 264, 1546-1549. Gray, J., Rajan, A.S., Radcliffe, K.A., Yakehiro, M., Dani, J.A., 1996. Hippocampal synaptic transmission enhanced by low concentrations of nicotine. Nature 383, 713-716. Hughes, J.R., 1998. Dependence on the abuse of nicotine replacement medications: an update. In: Benowitz, N. (Ed.), Nicotine Safety and Toxicity. Oxford, New York, pp. 147-160. Iaboni, F., Douglas, V.I., Baker, A.G., 1995. Effects of reward and response costs on inhibition in ADHD children. J. Abnorm. Psychol. 104 (1), 232-240. Itoh, A., Nitta, A., Nadai, M., Nishimura, K., Hirose, M., Hasegawa, T. et al., 1996. Dysfunction of cholinergic and dopaminergic neuronal systems in [3-amyloid protein-infused rats. J. Neurochem. 66, 11131117. James, J.R., Nordberg, A., 1995. Genetic and environmental aspects of the role of nicotinic receptors in neurodegenerative disorders: empha-

99

sis on Alzheimer's disease and Parkinson's disease. Behav. Genet. 25, 149-159. Jamner, L.D., Girdler, S.S., Shapiro, D., Jarvik, M.E., 1998. Pain inhibition, nicotine and gender. Exp. Clin. Psychopharmacol. 6, 96-106. Jones, G.M.M., Sahakian, B.J., Levy, R., Warburton, D.M., Gray, J.A., 1992. Effects of acute subcutaneous nicotine on attention, information processing and short term memory in Alzheimer's disease. Psychopharmacology 108, 485-494. Kaneko, S., Maeda, T., Kume, T., Kochiyama, H., Akaike, A., Shimohama, S. et al., 1997. Nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via alpha 7-neuronal receptors and neuronal CNS receptors. Brain Res. 765, 135-140. Katayama, S., Hirata, K., Tanaka, H. et al., 1995. Efficacy of transdermal nicotine in dementia: a study using event-related potentials and a middle latency response. In: Brain Imaging of Nicotine and Tobacco Smoking. NPP Books, Ann Arbor, pp. 289-302. Kihara, T., Shimohama, S., Sawada, H., Kimura, J., Kume, T., Kochiyama, H. et al., 1997. Nicotinic receptor stimulation protects neurons against beta-amyloid toxicity. Ann. Neurol. 42, 159-163. Kirch, D.G., 1999. Nicotine and major mental disorders. In: Piasecki, M., Newhouse, P. (Eds.), Nicotine in Psychiatry: Emerging Trends in Psychopathology and Novel Therapeutics. American Psychiatric Press, Washington, in press. Lee, P.N., 1994. Smoking and Alzheimer's disease: a review of the epidemiologic evidence. Neuroepidemiology 13, 131-144. Lena, C., Changeux, J.P., 1998. Allosteric nicotinic receptors, human pathologies. J. Physiology (Paris) 92, 63-74. Levin, E.D., Conners, C.K., Sparrow, E., Hinton, S.C., Erhardt, D., Meck, W.M. et al., 1996a. Nicotine effects on adults with attention deficit/hyperactivity disorder. Psychopharmacology 123, 55-63. Levin, E.D., Wilson, W., Rose, J.E., McEvoy, J., 1996b. Nicotine-haloperidol interactions and cognitive performance in schizophrenics. Neuropsychopharmacology 15, 429-436. Lipiello, P.M., Bencherif, M., Gray, J.A., Peters, S., Grigoryan, G., Hodges, H. et al., 1996. RJR-2403: a nicotinic agonist with CNS selectivity: II. In vivo characterization. J. Pharmacol. Exp. Ther. 279, 1422-1429. Lloyd, G.K., Menzaghi, F., Bontempi, B., Suto, C., Siegel, R., Akong, M. et al., 1998. The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents. Life Sci. 62, 1601-1606. Luntz-Leybman, V., Bickford, P., Freedman, R., 1992. Cholinergic gating of response to auditory stimuli in rat hippocampus. Brain Res. 587, 130-136. Marin, P., Maus, M., Deagher, S., Glowinski, J., Premont, J., 1994. Nicotine protects cultured striatal neurons against N-methyl-~aspartate receptor-mediated neurotoxicity. NeuroReport 5, 1977-1980. Marshall, J., Schniden, H., 1966. Effect of adrenaline, noradrenaline, atropine and nicotine on some types of human tremor. J. Neurol. Neurosurg. Psychiatry 29, 214-218. Maurice, T., Lockhart, B.P., Privat, A., 1996. Amnesia induced in mice by centrally administered [3-amyloid peptides involves cholinergic dysfunction. Brain Res. 706, 181-193. McConville, B.J., Sanberg, P.R., Fogelson, H.M., King, J., Cirino, P., Parker, K.W. et al., 1992. The effects of nicotine plus haloperidol compared to nicotine only and placebo nicotine only in reducing tic severity and frequency in Tourette's disorder. Biol. Psychiatry 31, 832-840. McEvoy, J.P., Freudenreich, O., Levin, E.D., Rose, J.E., 1995a. Haloperidol increases smoking in patients with schizophrenia. Psychopharmacology 119, 124-126. McEvoy, J.P., Freudenreich, O., McGee, M., Vander Zwaag, C., Levin, E., Rose, J., 1995b. Clozapine decreases smoking in patients with chronic schizophrenia. Biol. Psychiatry 37, 550-552. Menzaghi, F., Whelan, K.T., Risbrough, V.B., Rao, T.S., Lloyd, G.K.,

100


1997. Interactions between a novel cholinergic ion channel agonist, SIB-1765F and L-DOPA in the reserpine model of Parkinson's disease in rats. J. Pharmacol. Exp. Ther. 280, 393-401. Moll, H., 1926. The treatment of postencephalitic Parkinsonism by nicotine. Br. Med. J. 1, 1079-1081. Morens, D.M., Grandinetti, A., Reed, D., White, L.R., Ross, G.W., 1995. Cigarette smoking and protection from Parkinson's disease: false association or etiologic clue?. Neurology 45, 1041-1051. Myrsten, A., Post, B., Frankenhaeuser, M., Johansson, G., 1972. Changes in behavioral and phsysiological activation induced by cigarette smoking in habitual smokers. Psychopharmacology (Berlin) 76, 232-235. Nagamoto, H.T., Adler, L.E., Hea, R.A., Griffith, J.M., McRae, K.A., Freedman, R., 1996. Gating of auditory P50 in schizophrenics: unique effects of clozapine. Biol. Psychiatry 40, 181-188. Newhouse, P.A., Kelton, M., 1999. Clinical aspects of nicotinic agents: therapeutic applications in central nervous system disorders. In: Clementi, F., Gotti, C., Fornasari, D. (Eds.), Handbook of Experimental Pharmacology: Neuronal Nicotinic Receptors. Springer, Heidelberg, in press. Newhouse, P.A., Sunderland, T., Tariot, P.N., Blumhardt, C.L., Weingartner, H., Mellow, A. et al., 1988. Intravenous nicotine in Alzheimer' s disease: a pilot study. Psychopharmacology 95, 171-175. Newhouse, P.A., Sunderland, T., Narang, P.K., Mellow, A., Fertig, J.B., Lawlor, B.A. et al., 1990. Neuroendocrine, physiologic and behavioral responses following intravenous nicotine in nonsmoking healthy volunteers and in patients with Alzheimer's disease. Psychoneuroendocrinology 15, 471-484. Newhouse, P.A., Potter, A., Corwin, J., Lenox, R., 1992. Acute nicotinic blockade produces cognitive impairment in normal humans. Psychopharmacology 10, 480-484. Newhouse, P.A., Potter, A., Lenox, R.H., 1993. Effects of nicotinic agents on human cognition: possible therapeutic applications in Alzheimer's and Parkinson's diseases. Med. Chem. Res. 2, 628-642. Newhouse, P.A., Potter, A., Corwin, J. et al., 1994. Age-related effects of the nicotinic antagonist mecamylamine on cognition and behavior. Neuropsychopharmacology 10, 93-107. Newhouse, P.A., Potter, A., Corwin, J. et al., 1996. Effects of nicotinic cholinergic agents on cognitive functioning in Alzheimer's and Parkinson's disease. Drug Dev. Res. 38, 278-289. Newhouse, P.A., Singh, J., Conrath, C., Kelton M.C., 1998. The effects of nicotine in Parkinson's disease. Presented at Annual Meeting, American Psychiatric Association, New York, NY; New Research Proceedings: NR 356, 165. Nordberg, A., 1993. Effect of long-term treatment with tacrine (THA) in Alzheimer's disease as visualized with PET. Acta Neurol. Scand., Suppl. 149, 62-65. Nordberg, A., Lundqvist, H., Hartvig, P., Lilja, A., Langstrom, B., 1995. Kinetic analysis of regional (S) ( - ) llC-nicotine binding in normal and Alzheimer brains - - in vivo assessment using positron emission tomography. Alzheimer Dis. Assoc. Disord. 9, 21-27. Olincy, A., Ross, R.G., Young, D.A., Roath, M., Freedman, R., 1998. Improvement in smooth pursuit eye movements after cigarette smoking in schizophrenic patients. Neuropsychopharmacology 18, 175185. Owman, C., Fuxe, K., Jason, A., Kahrstrom, J., 1989. Studies of the protective actions of nicotine on neuronal and vascular functions in rats: comparison between sympathetic noradrenergic and mesostriatal dopaminergic fiber system and the effect of a dopamine agonist. Prog. Brain Res. 79, 267-276. Perry, E.K., Curtis, M., Dick, D.J., Candy, J.M., Atack, J.R., Bloxham, C.A. et al., 1985. Cholinergic correlates of cognitive impairment in Parkinson's disease: comparisons with Alzheimer' s disease. J. Neurol. Neurosurg. Psychiatry 48, 413-421. Perry, E.K., Morris, C.M., Court, J.A., Cheng, A., Fairbairn, A.E., McKeth, I.G. et al., 1995. Alteration in nicotinic binding sites in Parkinson's disease, Lewy body dementia and Alzheimer's disease: possible index of early neuropathology. Neuroscience 64, 385-395.

Perry, E.K., Court, J.A., Lloyd, S., Johnson, M., Griffiths, M.H., Spurden, D. et al., 1996. [3-amyloidosis in normal aging and transmitter signaling in the human temporal lobe. Ann. N. Y. Acad. Sci. 777, 388-392. Philips, H.A., Shaffer, I.E., Crossland, K.M., Bhatia, K.P., Fish, D.R., Marsden, C.D. et al., 1998. Autosomal dominant nocturnal frontal-lobe epilepsy: genetic heterogeneity and evidence for a second locus at 15q24. Am. J. Hum. Genet. 63, 1108-1116. Pickworth, W.B., 1997. Effects of mecamylamine on spontaneous EEG and performance in smokers and non-smokers. Pharmacol., Biochem. Behav. 56, 181-187. Pomerleau, O.F., Downey, K.K., Stelson, F.W., Pomerleau, C.S., 1995. Cigarette smoking in adult patients diagnosed with attention deficit hyperactivity disorder. J. Subst. Abuse 7, 373-378. Potter, A., Corwin, J., Lang, J., Lenox, R., Newhouse, P.A., 1999. Acute effects of the selective cholinergic channel activator (nicotinic agonist) ABT-418 improved learning in Alzheimer's disease. Psychopharmacology 142, 334-342. Prendergrast, M.A., Jackson, W.J., Terry, A.V. Jr., Decker, M.W., Arneric, S.P., Buccafusco, J.J., 1998. Central nicotine receptor agonists ABT-418, ABT-089 and (-)-nicotine reduce distractibility in adult monkeys. Psychopharmacology 136, 50-58. Puttfarcken, P.S., Manelli, A.M., Arneric, S.P., Donnelly-Roberts, D.L., 1997. Evidence for nicotinic receptors potentially modulating nociceptive transmission at the level of the primary sensory neuron: studies with F11 cells. J. Neurochem. 69, 930-938. Roberts, R.G., Stevenson, J.E., Westerman, R.A., Pennefather, J., 1995. Nicotinic acetylcholine receptors on capsaicin-sensitive nerves. NeuroReport 6, 1578-1582. Rose, J.E., Behm, F.M., Westman, E.C., Levin, E.D., Stein, R.M., Ripka, G.V., 1994. Mecamylamine combined with nicotine skin patch facilitates smoking cessation beyond nicotine patch treatment alone. Clin. Pharmacol. Ther. 56, 86-99. Rose, J.E., Westman, E.C., Behm, F.M., 1996. Nicotine/mecamylamine combination treatment for smoking cessation. Drug Dev. Res. 38, 243-256. Sacaan, A.I., Reid, R.T., Santori, E.M., Adams, P., Correa, L.D., Mahaffy, L.S. et al., 1997. Pharmacological characterization of SIB1765F: a novel cholinergic ion channel agonist. J. PharmacoI. Exp. Ther. 280, 373-383. Sahakian, B.J., Coull, J.T., 1994. Nicotine and THA: evidence for improved attention in patients with Dementia of the Alzheimer type. Drug Dev. Res. 31, 80-88. Salomon, A.R., Marcinowski, K.J., Zagorski, M., 1996. Nicotine inhibits amyloid formation by the [3-peptide. Biochemistry 35, 13568-13578. Sanberg, P.R., McConville, B.J., Fogelson, H.M., Manderscheid, P.Z., Parker, K.W., Blythe, M.M. et al., 1989. Nicotine potentiates the effects of haloperidol in animals and patients with Tourette syndrome. Biomed. Pharmacother. 43, 19-23. Sanberg, P.R., Shytle, R.D., Silver, A.A., 1998. Treatment of Tourette's syndrome with mecamylamine. Lancet 352 (9129), 705-706. Schneider, J.S., Van Velson, M., Menzaghi, F., Lloyd, G.K., 1998. Effects of the nicotinic acetylcholine receptor agonist SIB-1508Y on object retrieval performance in MPTP-treated monkeys: comparison with levodopa treatment. Ann. Neurol. 43, 311-317. Sershen, H., Hashim, A., Lajtha, A., 1987. Behavioral and biochemical effects of nicotine in an MPTP-induced mouse model of Parkinson's disease. Pharmacol., Biochem. Behav. 28, 299-303. Shapiro, E.S., Shapiro, A.K., Fulop, G., Hubbard, M., Mandeli, J., Nordlie, J. et al., 1989. Controlled study of haloperidol, pimozide and placebo for the treatment of Gilles de la Tourette's syndrome. Arch. Gen. Psychiatry 46, 722-730. Shimohama, S., Akaike, A., Kimura, J., 1996. Nicotine-induced protection against glutamate cytotoxicity: nicotinic cholinergic receptormediated inhibition of nitric oxide formation. Ann. N. Y. Acad. Sci. 777, 356-361. Shytle, R.D., Baker, M., Silver, A.A., Reid, B.M., Sanberg, P.R., 2000.


Smoking, nicotine and movement disorders. In: Piasecki, M., Newhouse, P. (Eds.), Nicotine in Psychiatry: Emerging Trends in Psychopathology and Novel Therapeutics. American Psychiatric Press, Washington, in press. Silver, A.A., Sanberg, P.R., 1995. Transdermal nicotine patch and potentiation of haloperidol in Tourette's syndrome. Lancet 342 (8864), 182. Snaedal, J., Johannesson, T., Jonsson, J.E., Gylfadottir, G., 1996. The effects of nicotine in dermal plaster on cognitive functions in patients with Alzheimer's disease. Dementia 7, 47-52. Tyas, S.L., 1996. Are tobacco and alcohol use related to Alzheimer's disease? A critical assessment of the evidence and its implications. Addict. Biol. 1,237-254. Ulrich, J., Johanson-Locher, G., Seiler, W.O., Stahelin, H.B., 1997. Does smoking protect from Alzheimer's disease? Alzheimer-type changes in 301 unselected brains from patients with known smoking history. Acta Neuropathol. 94, 450-454. Waldo, M.C., Carey, G., Myles-Worsley, M., Cawthra, E., Adler, L.E., Nagamoto, H.T. et al., 1991. Codistribution of a sensory gating deficit and schizophrenia in multi-affected families. Psychiatry Res. 39, 257-268. Warburton, D.M., Rusted, J.M., 1993. Cholinergic control of cognitive resources. Neuropsychobiology 28, 43-46. Warpman, U., Nordberg, A., 1995. Epibatidine and ABT 418 reveal selective losses of alpha 4 beta 2 nicotinic receptors in Alzheimer brains. NeuroReport 6, 2419-2423. Wesnes, K., Warburton, D., 1983. Smoking, nicotine and human performance. Pharmacol. Ther. 21, 189-208. Wesnes, K., Warburton, D., 1985. Effects of scopolamine and nicotine on human performance. Psychopharmacology 82, 147-150.

101

Westman, E.C., Rose, J.E., 1999. Nicotine replacement therapies and beyond. In: Piasecki, M., Newhouse, P. (Eds.), Nicotine in Psychiatry: Emerging Trends in Psychopathology and Novel Therapeutics. American Psychiatric Press, Washington, in press. Whitehouse, P.J., Hedreen, J.C., White, C.L., Price, D.L. et al., 1983. Basal forebrain neurons in dementia of Parkinson's disease. Ann. Neurol. 13, 243-248. Whitehouse, P., Martino, A., Antuono, P., Lowenstein, P.R., Cayle, J.T., Price, D.L. et al., 1986. Nicotinic acetylcholine binding sites in Alzheimer's disease. Brain Res. 371, 146-151. Whitehouse, P.J., Martino, A.M., Wagster, M.V., Price, D.L., Mayeux, R., Atack, J.R. et al., 1988. Reductions in [3H] nicotinic acetylcholine binding in Alzheimer's disease and Parkinson's disease: an autoradiographic study. Neurology 38, 720-723. Wilson, A.L., Langley, L.K., Monley, J., Bauer, T., Rotfunda, S., McFalls, E. et al., 1995. Nicotine patches in Alzheimer's disease: pilot study on learning, memory and safety. Pharmacol. Biochem. Behav. 51,509-514. Wolf, S.S., Jones, D.W., Knable, M.B., Gorey, J.G., Lee, K.S., Hyde, T.M. et al., 1996. Tourette syndrome: prediction of phenotypic variation in monozygotic twins by caudate nucleus D2 receptor binding. Science 273, 1225-1227. Yamashita, H., Nakamura, S., 1996. Nicotine rescues PC12 cells from death induced by nerve growth factor deprivation. Neurosci. Lett. 213, 145-147. Zamani, M.R., Allen, Y.S., Owen, G.P., Gray, J.A., 1997. Nicotinic modulates the neurotoxic effect of beta-amyloid protein (25-35) in hippocampal cultures. NeuroReport 8, 513-517.




Central nicotinic receptor ligands and pharmacophores Richard A. Glennon *, Matgorzata Dukat Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Box 581, MCV Station, Richmond, VA 23298-0540, USA

Abstract

Multiple populations of pentameric nicotinic acetylcholinergic (nACh) receptors exist and several may be classified as central or neuronal. Neuronal nACh receptors, however, are primarily of the oL4[~2 and o~7 types, and these have been the focus of most recent investigations aimed at the development of novel agents and identification of pharmacophores. Selectivity data are limited. Furthermore, because several populations of nACh receptors might indirectly influence a given functional effect, it is difficult to discuss structure-activity relationships (SAR) in terms of differential SAR, or to formulate SAR on the basis of functional studies. For the most part, studies are limited to the formulation of structure-affinity relationships (SAFIR) for the binding of agents at nACh receptors, and for these the O~4[~2 population has been the most extensively investigated. SAFIR and newer agents are reviewed here with reference to earlier studies. Novel agents now have been identified that bind with up to 30 times higher affinity than nicotine and these are providing new insight into the understanding of nACh receptors. 9 2000 Elsevier Science B.V. All rights reserved.

Keywords: A-84543; A-85380; ABT-089; ABT-418; ABT-594; Aminoethoxypyridines;Anabaseine; Anabasine; Chloronicotine, 6-; Cotinine; Cytisine; Dihydro-[3-erythroidine; DMXB; Epibatidine; GTS-21 ; Isonicotine; Lobelanidine; Lobelanine; Lobeline; Metanicotine; Myosmine; Nicotine; Nicotinoids; Nornicotine; SIB-1508Y; SIB-1553A; SIB-1765F; Tetrahydropyrido[3,4-d]azepines

1. Introduction Structure-activity studies on nicotinic acetylcholinergic (nACh) agents may be thought of as belonging to two different eras; the first era involved investigations prior to the 1990s when most studies were focused on the agonist or antagonist actions of nACh agents, whereas the second era began with the realization that multiple populations of nACh receptors exist, and with the introduction of radioligands and techniques to investigate neuronal receptor populations. Studies conducted during the first era were devoted primarily, although not exclusively, to peripheral nACh receptors and receptor function; the second era involves investigations targeting mainly, although not exclusively, neuronal or central nACh receptors and receptor function. It would be a mistake to refer to the two eras as


the peripheral era and the central era; however, significantly more attention has been accorded to central nACh receptors in recent years than ever before. Due to its rich history, and to the extensive literature associated with nACh receptor research emanating from the early studies, it was only natural to use this early information as a starting point for the more recent investigations. Indeed, a considerable effort has been made to determine the binding of many older nACh receptor ligands and their derivatives at newly identified populations of sites so as to identify binding differences between peripheral and central nicotine receptors, and to identify potentially selective agents. Relatively few nACh agents or general structure-types are truly novel; most of these were introduced to the literature in the 1950s and 1960s and, for some, even earlier. Early studies with nicotinic agents also identified very few that were more potent than nicotine ( l a ) as nACh agonists. Over the past decade there have been several revelations that have shaped subsequent research direction. One of the most important of these is the

0031-6865/00/$ - see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S003 1-6865(99)00022-9

104

R.A. Glennon, M. Dukat / Pharmaceutica Acta Helvetiae 74 (2000) 103-114

discovery of epibatidine (2), an alkaloid isolated from South American frogs by Daly et al. (reviewed: Badio et al., 1994; Dukat, 1994). Epibatidine (2) might not have been expected to bind with high affinity at nACh receptors on the basis of published first-era structure-activity relationships (SAR) or pharmacophore models; however, epibatidine has been demonstrated to be a nACh agonist that binds with more than 30 times the affinity of nicotine at O~4 ~2-typ e nicotinic receptors. The present overview will focus on the SAR formulated for the binding of nicotinic agents at, primarily, oL4132-type nicotinic receptors. The review will also target those compounds that might be considered as nicotinoids. The term nicotinoids was introduced by Yamamoto nearly 35 years ago and refers to "substances that are analogous to the nicotine structure in some respects" (for review see Yamamoto, 1965). It may be appreciated that the definition is rather vague and open to interpretation. In an attempt to be somewhat more restrictive, we have previously employed the term to refer to compounds "displaying a structural similarity to nicotine in that they contain a basic nitrogen atom and a pyridine (or equivalent) aromatic ring" (Glennon and Dukat, 1996). At the very least, these definitions exclude nACh neurotoxins which, for the most part, are structurally different from nicotine (and which, in most instances, bind in a noncompetitive manner, or display low affinity, for ot4[32-type nACh receptors). Certain other agents have been termed nicotinoid pharmacophoromimetics because, although they might not possess a pyridine (or equivalent) ring, they bind with high affinity at oL4~2 receptors; examples of such agents are dihydro-[3-erythroidine (3) and cytisine (4). For the purpose of the present review, we will simply classify agents either as nicotinoids or as miscellaneous agents. Several reviews on nicotinic agents have recently appeared (McDonald et al., 1995; Glennon and Dukat, 1996, 1999a,b; Rosecrans and Brioni, 1996; Brioni et al., 1997; Holladay et al., 1997, 1999; Arneric and Brioni, 1999; Yamamoto and Casida, 1999); hence, the present overview will focus on more current findings. These reviews can be consulted for further information on individual agents, on agents not discussed herein, and for additional references. Multiple populations of pentameric nACh receptors exist and several may be classified as neuronal (Sargent, 1993; Holladay et al., 1997; Arneric and Brioni, 1999). The major population of nACh receptors in mammalian brain for which nicotine displays high affinity are the oL4[3z-type. Because several populations of nACh receptors might indirectly influence a given functional effect, or because the specific population(s) of nACh receptors involved in producing some of nicotine's effects have not yet been elucidated (Shacka and Robinson, 1996), it is difficult to discuss SAR in terms of differential SAR, or to formulate SAR on the basis of functional studies. For the most part, studies are limited to the formulation of structure-affinity relationships (SAFIR) for the binding of

agents at nACh receptors, and the bulk of the existing data are for oL4[~2 binding. Over the past decade several different radioligands have been used to label these receptors in brain homogenates and the most prevalent are tritiated nicotine (la), cytisine (4), and methylcarbamylcholine (5). Anderson and Arneric (1994) compared all three and concluded that they label the same nACh receptor subtype in rat brain and are unable to distinguish subtypes of nACh receptors. Several radioligands are based on an epibatidine template (e.g., Houghtling et al., 1994; Holladay et al., 1997); these radioligands appear to label multiple populations of nACh receptors including the oL4132-type receptors.

2. Nicotinoids

With the advent of tritiated nicotine (and other radioligands) to label neuronal nACh receptors came fairly simple questions such as (a) which is the higher affinity optical isomer of nicotine, (-)nicotine or (+)nicotine, (b) what is the effect of N-demethylation, to afford nornicotine, and what is the effect of N-methyl homologation, (c) is the pyridine ring of nicotine necessary for high affinity, (d) is the pyridine-ring nitrogen atom required for binding, (e) is an intact pyrrolidine ring required, (f) what is the influence of pyridine-ring and pyrrolidine-ring substitution on receptor affinity, (g) what is the effect of quaternization, and (h) what is the preferred conformation of nicotine for binding at the receptors. Many of these questions have now been answered; they are summarized in Fig. 1 and will be discussed below; several reviews are available and were cited in Section 1. Another question was whether or not earlier nicotinic pharmacophore models were applicable to the binding of nicotinic agents at neuronal nACh receptors. A nicotinic pharmacophore model was formulated in 1970 and subsequently elaborated upon. The primary tenets of the "Beers-Reich/Sheridan" nicotinic pharmacophore (hereafter referred to simply as the B RS pharmacophore) were that a molecule must possess a basic amine situated a certain distance from a hydrogen bond acceptor (see Beers and Reich, 1970; Sheridan et al., 1986). The hydrogen bonding feature in nicotine is the

D

3'

4'/ c

Fig. 1. General structure and numbering system of (-)nicotine. The bolded letters refer to portions of the molecule for which structure-activity informationis discussed in the text.

R.A. Glennon, M. Dukat/ Pharmaceutica Acta Helvetiae 74 (2000) 103-114

pyridine nitrogen atom, and the optimal internitrogen distance was proposed to be 4.8 A. Other details of this and other models have been recently reviewed (Glennon and Dukat, 1999a). The introduction of epibatidine, at about the same time that many of the above-questions were being addressed, had a significant impact. As already mentioned, epibatidine, although bearing some structural similarity to nicotine, did not seem to fit the optimal dimensions of the B RS pharmacophore. The calculated internitrogen distance for the lowest energy conformation of epibatidine, 5.5 A, was about 0.7 A greater than seemingly optimal (Dukat et al., 1994). Nevertheless, several groups of investigators independently found that (-)epibatidine (K i ca. 0.05 nM) binds with considerably higher affinity than (-)nicotine (K i ca. 1-5 nM) (e.g., Qian et al., 1993; Dukat et al., 1994). To determine if it was the greater internitrogen distance, or the presence of the pyridyl chloro function, that accounted for the enhanced affinity of epibatidine, several related derivatives were prepared for examination including isonicotine (6) and 6-chloronicotine (Dukat et al., 1994; Glennon et al., 1994). Isonicotine (6) (K i = 12.5 nM) displayed reduced affinity relative to epibatidine, and 6-chloronicotine displayed only several-fold greater affinity than nicotine; it was suggested that although the chloro group might contribute to the binding of nicotinoids, the greater internitrogen distance, as found in epibatidine, might play a key role. This distance issue has recently become controversial and will be further discussed below. On the other hand, the pyridine 6-chloro substituent is becoming a fixture in newer nicotinic ligands (e.g., see Holladay et al., 1998; Latli et al., 1999).

2.1. Nico#noid binding: general SAFIR for OL4 [32 binding What structural features are required for the high-affinity binding of nicotine-related agents? The nicotine structure is arbitrarily divided into several regions for purpose of discussion: A, the chiral center; B, the pyridine ring; C, the pyrrolidine amine and its substituents; D, the pyrrolidine ring (see Fig. 1).

2.1.1. Chiral center (A) S ( - )Nicotine (K i c a . 1-5 nM) displays modest stereoselectivity and binds with about 20- to 50-fold higher affinity than its R(+)enantiomer. Nornicotine shows little stereoselectivity suggesting that the N-methyl group of nicotine might play a role in enantiomeric binding. Certain nicotine-related agents with extended internitrogen distances also show little stereoselectivity for binding; this will be further discussed in the appropriate section below (i.e., see Section 2.2). 2.1.2. Pyridine ring (B) Although this has not been extensively investigated, the pyridine ring of nicotine can be replaced with certain other

105

heterocycles with little effect on affinity; ABT-418 (7) (K i ca. 2 nM) is an example of such an agent. Fusion of nicotine to a benzene ring provides several different derivatives with varying results. Fusion at the d-face, affording benzo[ d]nicotine, dramatically decreases affinity whereas fusion at the e-face, affording benzo[e]nicotine (8) (K i --- 0.9 nM), is tolerated (Flammia et al., 1999). The presence of the pyridine-ring nitrogen atom seems to contribute to binding and its replacement with an sp2-hy bridized carbon atom results in about a 500-fold decrease in affinity. Quaternization of the pyridine nitrogen atom with a methyl group (i.e., nicotine isomethiodide, 9), abolishes affinity. Interestingly, however, homologation of the N-methyl group of 9 to longer groups such, as nonyl to dodecyl, results in restoration of affinity (Dwoskin, 1999). Pyridyl-ring substitution provides some interesting results. Introduction of small substituents at either the 2- or 5-position decreases affinity for a 4[32-type receptors, and substitution at the 4-position has not been systematically investigated. The best studied position is the 6-position; introduction of substituents can either increase or decrease the affinity of the resulting nicotinoid depending upon the nature of the substituent (Dukat et al., 1996, 1999a). Small alkyl groups are tolerated (e.g., 6-methyl, 6-ethyl) with little reduction in affinity (K i ca. 2-6 nM), but 6halogenated derivatives of nicotine all bind with affinities at least comparable to that of nicotine itself and are at least as potent as, or are more potent than, nicotine in functional assays.

2.1.3. Pyrrolidine amine (C) Replacement of the N-methyl group by hydrogen (i.e., nornicotine, lb) reduces affinity by approximately 30-fold. At this time it is not known if the N-methyl group directly participates in a ligand-receptor interaction or whether its major role is to influence the conformation of the molecule. To date, there have been no attempts to correlate the basicity of the pyrrolidine amine with oL4[~2 affinity; however, agents with reduced basicity typically bind with lower affinity than nicotine. For example, the basicity of the low-affinity agents myosmine and cotinine (see next section; p K a = 5.5 and 4.5, respectively) are well below that of racemic nicotine (pK a = 8.05) and nornicotine (pK a = 9.12) (Yamamoto, 1965; Fujita et al., 1971). Quaternization of nicotine (i.e., nicotine methiodide, 10) doubles its affinity whereas concurrent quaternization of both amines (i.e., nicotine dimethiodide) abolishes affinity. Homologation of the N-methyl group results in a dramatic decrease in affinity. 2.1.4. Pyrrolidine ring (D) Contraction of the pyrrolidine ring of nicotine to an azetidine, as with MPA (11), has little effect on affinity (Abood et al., 1987), whereas increasing the size of the pyrrolidine ring to a piperidine ring reduces receptor affinity. This can be seen in comparing, for example, nomico-


106

Table 1 Affinities of chain-extended nicotinoids at

oL4132

n A C h receptors a R

Isomer

Designation

Ki, nM

-H -CH 3 - C 2H 5 -H -CH 3 - C 2H 5 -H -CH 3 - C 2H 5 -H -H -CH 3 - C 2H 5

-

N-Methyl A M P N, N-Dimethyl A M P N-Ethyl-N-methyl A M P N-Methyl A E P N, N-Dimethyl AEP N-Ethyl-N-methyl A E P N-Methyl A P P N , N - D i m e t h y l APP N-Ethyl-N-methyl A P P N-Methyl A B P N-Methyl A X P N, N-Dimethyl A X P N-Ethyl-N-methyl A X P

> 10,000 540 28 289 47 18 > 10,000 > 10,000 > 10,000 900 35 21 22

26a 26b 26c 26d

-CH 3 -CH 3 -H -H

S( - ) R( + ) S(-) R(+)

A-84543

0.15 19.70 0.16 0.14

27a 27b 27c 27d

-CH 3 -CH 3 -H -H

S(-) R( + ) S(-) R(+)

-CH 3 -CH 3

S(--) R( + )

-H

S(-)

-H

R( + )

27.7 3000 16.7 39.0

32a 32b

-H -H

S(-) R(+)

0.09 0.45

33a 33b

-CH 3 -n

S(-) s(-)

0.04

33c

-n

R(+)

34a 34b

-CH 3 -H

~

X~R~/CHs

16a ]6b 16c 20a 20b 20c 21a 2]b 2]c 22a 25a 25b 25c

X: - C H 2 - -

-CH2CH 2-

-CH2CH2CH2

--

-CHzCHzCHzCH 2-O-CH2CH 2-

"N"

--

-

A-85380

0.45 3.50 0.05 0.05

"CH 3

3]a 3]b 31c 3]d

a See text for references.

ABT-089

1.60 ABT-594

0.04

57 1400


O

HaCO" V

Nicotine: R = -CH 3 (la) Nornicotine: R = -H (lb)

pN

~"

H3%3C[

Epibatidine (2)

~

"CH3

Benzo[e]nicotine (8)

0 Cytisine (4)

Dihydro-[3-erythroidine (3)

U

Methylcarbamylcholine (5)

V

H3C

9 N-'O

Isonicotine (6)

ABT-418 (7)

(-)Nicotine Methiodide (10)

CHa (-)Nicotine Isomethiodide (9)

C 3

MPA (11)

~ Anabasine (12)

Methylanabasine (13)

AMP (16) Derivatives

t

2-Methyl THN (17)

Cotinine (15)

8-Methyl PcA (18)

R

~

(CH2)n-NRR'

AEPs: n = 2 (20) APPs: n = 3 (21) ABPs: n = 4 (22)

PdA (19) Derivatives

'

Myosmine (14)

~ N H . C H

24

3

~

NHCH3

Metanicotine (R JR-2043) (23)

~o/~NH-CH3 AXP (25) Derivatives

28

R~

R N--'R'

29

Furopyridines (30)

.3\

35

O

36

37

107

108

R.A. Glennon, M. Dukat/Pharmaceutica Acta Helvetiae 74 (2000) 103-114

(-)Lobeline (38)

Lobelanine (39)

L o b d ~ d m e (411)

L o b e l ~ (41)

l~oxolobdine (42)

Deshydroxylobeline (43)

44

(-)Lobeline-Q (45)

6oc. Anabaseine (46)

oc.,

Y

DMXB (GTS-21) (47)

DMAC (48)

2MeCA (49)

51 H

H

-. H3CO"

.\

-N"

H3CO

52

53

CH3 55

.,

H3CO

54

3 56

tine (K i = 30 nM) with anabasine (12) (K i = 210 nM) or nicotine (K i = 2 nM) with N-methylanabasine (13) (K i -180 nM) (Dukat, Damaj, Martin, Glennon; unpublished data). Other modifications of the pyrrolidine ring that result in reduced affinity are found in the nicotine metabo-

~CH3 SIB-1553A (57)

lites myosmine (14) (K i > 3000 nM) and cotinine (15) (K i = 1500 nM); these compounds bind with affinities of about 1000-fold lower than that of nicotine. Although the intact pyrrolidine ring is not required for binding, its presence seems optimal (Dukat et al., 1996). That is,


aminomethylpyridine (AMP, 16) derivatives (see Table 1 for several examples) bind with modest affinity when the amine is tertiary, but none binds with as high an affinity as nicotine itself. One of the higher affinity agents is N-ethylN-methyl AMP 16c (K i = 28 nM). Introduction of substituents into the pyrrolidine ring typically reduces, and quite frequently significantly reduces, nACh receptor affinity (Lin et al., 1994).

2.2. Chain-extended nicotinoids

Problems associated with the definition of nicotinoids were alluded to above. For example, if a methylene group is inserted into the side chain of the above-mentioned AMP (16) derivatives, do the resulting agents still represent nicotinoids? Notwithstanding this potential nomenclature or classification problem, we will refer to the following compounds simply as chain-extended nicotinoids. The simple AMP (16) derivatives bind with modest to high affinity at (x4~2-type nACh receptors depending upon the nature of their amine substituents (i.e., R and R'). Conformational restriction of N-ethyl-N-methyl AMP (16e, K i = 28 nM; Table 1) to a tetrahydronaphthyridine derivative (i.e., 2-methyl THN (17), K i - 18 nM) results in retention of affinity. However, the corresponding ring-expanded tetrahydropyrido[3,4-c]azepine, 8-methyl PcA (18) (K i = ca. 1000 nM), binds with reduced affinity. Relocation of the more basic nitrogen atom of PcA derivatives provides structurally similar tetrahydropyrido[3,4-d]azepines (PdA derivatives); the PdA derivative where R = - C H 3 (i.e., 19, K i = 48 nM) binds with enhanced affinity relative to 8methyl PcA (Cheng et al., 1995, 1999). Ring-opening of the latter ring system provides a series of aminoethylpyridines (AEP derivatives 20) that retain the affinity of the PdA derivatives for nACh receptors, and that bind with higher affinity than the shorter AMPs. Further extension of the alkyl chain abolishes nACh receptor affinity (amino-npropylpyridines; APP derivatives 21; K i > 10,000 nM, see Table 1) (Cheng et al., 1999), whereas introduction of yet an additional methylene group (i.e., amino-n-butylpyridines; ABP derivatives 22) restores some affinity (Caldwell et al., 1997). These chain-extended derivatives are not wholly novel but represent compounds that were studied 35 years ago as nicotinic insecticidal agents (Yamamoto, 1965). Of particular interest are ABP derivatives where unsaturation has been introduced. For example, metanicotine or RJR-2403 (23) (K i = 26 nM), which may be viewed as an unsaturated ABP derivative, binds with respectable affinity at nACh receptors (Bencherif et al., 1996; Caldwell et al., 1997), whereas a related chain-shortened unsaturated analog (E-N-methyl-3-(3-pyridinyl)-2propene-l-amine, K i ca. 6000 nM) binds with significantly reduced affinity (Cheng et al., 1999). Another chain unsaturated derivative is alkyne 24 (K i - 5 8 nM) (Caldwell et al., 1997).

109

Replacement of the benzylic methylene of the APPs by an oxygen atom (to afford aminoethoxypyridines, AXPs; pyridyl ethers, 25) has been independently shown by two groups of investigators to result in retention or enhancement of nACh receptor affinity (reviewed: Glennon and Dukat, 1999a). The simple N-ethyl-N-methyl AXP (2ge, R = - C H 3, R' = - C 2H 5; Ki - 22 nM, Table 1) binds with an affinity comparable to that of the corresponding AMP (16) derivative bearing the same amine substituents. Interestingly, however, parallel alterations of the terminal amine substituents in the AMP, AEP, and AXP series compounds did not result in parallel shifts in receptor affinity suggesting that the different series, or members within each series, of compounds might be binding in a different manner (Glennon and Dukat, 1999a). For example, both the Nmonomethyl and N,N-dimethyl AMP 16a and 16b bind with low affinity (K i > 10,000 and 540 nM, respectively), whereas the corresponding AXP derivatives 25a and 25b bind with affinities in the 20- to 35-nM range (Cheng et al., 1995, 1999). The Abbott group has developed a series of AXP derivatives that rivals epibatidine in affinity (Abreo et al., 1996). For example, pyridyl ether 26a (Table 1), which may be viewed as a chain-extended analog of nicotine, binds with at least 10 times the affinity of nicotine (la). As with nicotine, the S ( - ) isomer binds with higher affinity than its R ( + ) enantiomer. Likewise, as with nornicotine (lb), the two isomers of the desmethyl derivative (i.e., 26e and 26d) show no stereoselectivity. Unlike what is seen with nicotine and nornicotine, however, is that the desmethyl derivative 26e binds with an affinity comparable to that of 26a. In the nicotine series, the pyrrolidine ring can be replaced with an azetidine with relatively little effect on affinity (Abood et al., 1987). Similar results are seen with 27. The S ( - )azetidine analog A-85380 (27e) (K i = 0.05 nM) binds with very high affinity. Similar N-desmethyl and stereochemical trends are seen as with derivatives of 26. A fluorinated analog of 27e, i.e., 2-[lSF]F-A-85380, is being explored as a new PET imaging agent (Dolle et al., 1999). Relocation of the side chain to the pyridine 2-position (e.g., 28; K i > 10,000 nM) and conformational restriction, as with 29 (e.g., R = R' = - C H 3, Ki = 290 nM), result in decreased affinity (Cheng et al., 1999); nevertheless, with the appropriate amine substituents and stereochemistry (i.e., 30; R ( + ) N - H , K i = 5.11 nM; S ( - ) N - C H 3 , K i = 2.73 nM) the furo[3,2-b]pyridines can bind with affinities comparable to that of nicotine (Elliott et al., 1997). Interesting, though, is that both the N - H and N-methyl derivatives of 30 bind in a stereoselective fashion and display opposite enantioselectivity; that is, S ( - ) 3 0 with an N - H and R ( + ) 3 0 with an N-methyl bind with reduced affinity (K i = 75.8 and 97.9 nM, respectively) (Elliott et al., 1997). There are other curious binding similarities and differences between the pyridyl ethers or AXP analogs and derivatives of nicotine. For example, substitution at the pyridine 2-position typically decreases the affinity of nico-

110


tine, whereas substitution at the 6-position is generally tolerated (depending upon the nature of the specific substituent). In the pyridyl ether series, 2-methylation of 26 decreases affinity (Lin et al., 1997); for example, compare 31a-d with 26a-d (Table 1). In contrast, substitution at the 6-position is seemingly tolerated (Holladay et al., 1998) (see 32 and 33; Table 1). Pyridyl substitution has been examined in detail both in the pyrrolidine series (Lin et al., 1998) and in the azetidine series (Koren et al., 1998). The general conclusions reached are somewhat similar between the two series; that is, substituents are better tolerated at the pyridyl 6-position than at the 2-position. Bromo and methyl substitution at the pyridyl 4-position decreases the affinity of the pyrrolidine analogs by more than 20-fold (Lin et al., 1998); substituents at the 5-position have little effect in either the pyrrolidine (Lin et al., 1998) or azetidine (Koren et al., 1998) series. However, introduction of a chloro group at the 5-position of 25c (i.e., 5-chloro-N-ethyl-N-methyl AXP; K i = 244 nM) reduces affinity by an order of magnitude. Introduction of a second nitrogen atom into the pyrrolidine ring of AXP derivatives affords imidazoline derivatives 34. The Nmethyl derivative 34a (K i --57 nM; Table 1) binds with an affinity comparable to the simple AXP derivatives 25a-c; N-demethylation to 34b decreases affinity to a similar extent seen upon demethylation of nicotine (Giannella, Ferretti, Dukat, Martin, Glennon; unpublished data). There is no question that the chain-extended analogs have re-opened a new and exciting chapter of nACh receptor research. Complicating the issue, however, is the varying efficacies of these agents (see Lin et al., 1998).

2.3. Epibatidine analogs Epibatidine (2) burst on to the nACh receptor scene in the early 1990s and stirred considerable interest with its high ( K i = 0 . 0 3 - 0 . 0 6 nM) affinity (reviewed: Dukat, 1994). Almost immediately after ( - ) - and (+)epibatidine were shown to bind at e~4~2 receptors with nearly identical affinity, there was evidence that tritiated epibatidine can label multiple populations of nACh receptors (for review see Holladay et al., 1997). Since then, several radiolabeled analogs of epibatidine have been explored as pharmacological tools and imaging agents (for example, see Davila-Garcia et al., 1997; Horti et al., 1998). Several different total syntheses of epibatidine have been reported (Broka, 1994), and numerous epibatidine analogs have now been prepared and evaluated; the nACh receptor affinities of these compounds span a broad range. More recently, attempts have been made to escape some of the toxic effects associated with epibatidine by modifying its structure in a somewhat more dramatic fashion. This has led to analogs such as 35 and 36. The Z-isomer of compound 35 (IC50 = 1.4 nM) binds with an affinity comparable to that of nicotine, and with 200-fold higher affinity than its E-isomer; this compound, like nicotine itself,

also binds at 0~3~2 nACh receptors with high (IC50 = 1.9 nM) affinity (Olesen et al., 1997). 3,8-Diazabicyclooctane derivatives display nicotine-like antinociceptive properties and one, 36, was reported to bind with high (K i = 4.1 nM) affinity at nACh receptors (Barlocco et al., 1998). Compound 37 (RJR-2429; K i = 1 nM) binds with an affinity comparable to nicotine (Bencherif et al., 1998).

2.4. The nicotinoid pharmacophore As previously mentioned, the BRS pharmacophore is the currently accepted model used to identify important nicotinic geometries. The high affinity~ of epibatidine (calculated internitrogen distance = 5.5 A) initially questioned the importance of the 4.8-A internitrogen distance as being optimal (Dukat et al., 1994); however, altemative binding models now suggest that distances of 4.6 or 6.3 ,~ might explain the common binding of epibatidine and related agents (Abreo et al., 1996). Upon reexamination of the epibatidine structure, Koren et al. (1998) identified two stable conformations of el~ibatidine with internitrogen distances of 5.5 and 4.5 A; although the lowest energy conformation possesses the longer distance, the shorter distance conformer is only slightly (0.18 kcal/mol) less preferred. Furthermore, upon examination of a series of pyridine-modified derivatives of A-85380 (27c), these same investigators found that the agents could achieve internitrogen distances of as little as 4.33 to 4.39 A if they used conformers with calculated energies not exceeding the minimum energy conformations by more than 0.42 kcal/mol (Koren et al., 1998). These investigators favored the shorter distance as being more important because although most analogs could achieve both shorter- and longer-distance conformations, no stable conformers with intemitrogen distances of < 5.1 /k could be identified for derivatives with low affinity (e.g., the 2-bromo and 2-iodo analogs of 27c; K i = 240 nM and > 1000 nM). The optimal intemitrogen distance question remains unanswered. At this time, the question of how these agents bind in a common manner, and indeed if they bind in a common manner, has yet to be resolved. There is some general agreement that a basic amine and a hydrogen bonding site are probably required for binding. But, even here, there is a lack of consensus; for example, Barlow and Johnson (1989) have suggested an "amine plus flat surface" concept that avoids the issue of a hydrogen bonding site. In any event, assuming that a hydrogen bonding site is an important feature, most nACh agents are conformationally flexible and can assume several different low-energy conformations of widely varying internitrogen distance. Agents prevented from assuming short-distance conformations, such as for example 19 and 24, bind with affinities (K i values ca. 50 nM) significantly lower than those of epibatidine and the high-affinity pyridyl ethers (K i values ca. 0.03-0.2 nM). Unless, and until, conformationally-restricted high-affinity agents can be identified with intemitrogen distances significantly longer than 4.5 to


111

o

4.8 A, there is little reason to assume that the longer distances are favored for high affinity. However, because parallel structural changes do not always result in parallel shifts in affinity, and because agents with internitrogen distances exceeding 5.5 A bind at nACh receptors and produce nicotine-like pharmacological effects, the possibility exists that either the amine functions (or amine substituents) of these nACh receptor ligands are not all oriented in the same manner, and/or that different receptor features are being employed to bind these ligands.

3. Miscellaneous agents 3.1. Lobeline analogs

One nACh agent that has recently attracted some attention is lobeline. (-)Lobeline (38) is being studied in smoking cessation therapy and is capable of mimicking some, but not all, effects produced by nicotine (e.g., Damaj et al., 1997). The agent is also interesting from a theoretical perspective. (-)Lobeline is a high-affinity nACh receptor ligand and was initially considered by Beers and Reich (1970) to fit their pharmacophore model; that is, lobeline possesses the required pharmacophoric features, a basic amine and a hydrogen-bonding site. However, Barlow and Johnson (1989) were the first to point out that lobeline possesses two hydrogen bonding features: a carbonyl group and a hydroxyl group. On the basis of X-ray crystallographic studies on (-)lobeline HC1, the latter investigators suggested that the carbonyl oxygen atom better fits the BRS pharmacophore model than does the hydroxyl group. Glaser et al. (1992) obtained a different solution to the crystal structure using lobeline HBr, and on the basis of the X-ray structure and on conformational studies using NMR spectrometry, they too agreed that the carbonyl oxygen atom was the more likely hydrogen bonding site. However, Abood et al. (1988) had earlier reported that lobelanine (39) and lobelanidine (40) (analogs of lobeline where the hydroxyl group is oxidized to the corresponding ketone, and where carbonyl group is reduced to the corresponding alcohol, respectively) bind with similar affinity and with affinities comparable to that of (-)lobeline (38). These findings would argue that either the carbonyl group or the hydroxyl group could serve as a hydrogen bond acceptor to account for the binding of lobeline. Interestingly, neither the des-oxo nor the des-hydroxy analogs of lobeline had ever been examined in binding studies. A recent structure-affinity investigation has provided some new insight (Dukat et al., 1999b). Lobelan (41) ( K i > 10,000 nM) lacks affinity for OL4~ztype nACh receptors indicating that at least one oxygen function is required for binding. Interestingly, lobelanine (39) (K i = 7800 nM) and lobelanidine (40) (K i = 300 nM) bind with significantly lower affinity than previously reported. Others have also recently reported low affinities for

39 and 40 (Crooks et al., 1999). Furthermore, deso x o ( - ) l o b e l i n e (42) ( K i - 2 3 5 nM) and deshydroxy(-)lobeline (43) ( K i - - 1 1 0 nM) also bind with lower affinity than (-)lobeline (K i = 4 nM) (Flammia et al., 1999). It would appear that the presence of either oxygen function alone is not a sufficient condition for high-affinity binding and that the second oxygen atom plays a role. It has also been shown that 44 (K i = 5 nM), an analog of (-)lobeline where the hydroxyl group has been replaced by a chloro function, retains the affinity of (-)lobeline (Dukat et al., 1999b). Due to the possibility that the chloro analog 44 might undergo cyclization to the quaternary amine under the conditions of the binding assay, the simple N-methyl quaternary amine derivative of (-)lobeline (i.e., (-)lobeline methiodide or (-)lobelineQ, 45; K~ > 2000 nM) was examined and found to bind with reduced affinity. It is unlikely that a cyclized form of 44 accounts for its affinity. In any event, the presence of both oxygen functions is not specifically required for binding. The specific role of the carbonyl group and the hydroxy group of lobeline, or the chloro group of 44, is currently unknown. The presence of these polar functions seem to result in optimal nACh receptor affinity; they may participate in the formation of internal hydrogen bonds with the (protonated) amine to influence the conformation of the lobeline-type structures. Nevertheless, it is not yet known if (-)lobeline (38) conforms to the BRS pharmacophore, or indeed, whether lobeline analogs bind in a manner analogous to that of nicotine.

4. Non-~413 2 nACh receptors The vast majority of published structure-affinity studies have involved oL4~2 nACh receptors. Although there exist other populations of nACh receptors, much less has been done with these. There are a few agents that bind with high affinity at certain of these sites (or subunits) (for example, see Holladay et al., 1997 for a recent review), but for the most part, SAFIR have yet to be formulated. One population of receptors that has received some attention are the o~7 nACh receptors. The oL7 receptors are characterized by their high affinity for ot-bungarotoxin (BTX; ot-Bgt) and reduced affinity for nicotine; [~25I]BTX is commonly used to label this homo-oligomeric receptor. Anabaseine (46) is a partially unsaturated analog of anabasine (12); structurally, anabaseine is to anabasine what myosmine (14) is to nornicotine (lb). The finding that anabaseine might possess cytoprotective and memory-enhancing actions prompted an investigation of its SAFIR (De Fiebre et al., 1995). Anabaseine (46) binds with relatively high affinity at o~4~2 sites (K i ~ 75 nM) but with several-fold lower affinity at oL7 sites (K i --- 374 nM). Incorporation of a benzylidine group at the tetrahydropyridine 3-position and either ring substitution or chain elongation resulted in compounds such as 47 (DMXB, GTS-21;

112


OL4~2 Ki ~ 85 nM, c~7 Ki ~ 212 nM) and 48 (DMAC; OL4~2 gi ~ 347 nM, a7 gi ~ 34 nM); in effect, it was

5. Summary

demonstrated that the selectivity of anabaseine for these two receptor populations could be altered and even reversed. Subsequent investigations further examined the SAFIR and SAR of these agents (Meyer et al., 1998a,b and references therein). Removal of the N,N-dimethylamino group of 48 resulted in a compound that displayed good affinity of oL7 receptors (IC50 = 80 nM); the role of hydroxyl and methoxy substitution was explored and compound 49 (2MeCA; oL7 IC50 = 8 nM) was identified. For the most part these agents behave as a7 partial agonists. On the basis of studies by Abbott laboratories on pyridyl ethers and on an old observation that oxystilbene derivatives are active at ganglionic nACh receptors, an Italian group prepared a series of stilbenes 50 (Gotti et al., 1998). MG624 (i.e., 50 where Y = H and R = Et; K i = 27 nM) binds at ot 7 sites with high affinity. Related compounds include 50 where R = Y = Me; the S (i.e., F3A) and R (i.e., F3B) isomers bind with comparable affinity ( K i = 70 and 81 nM, respectively). Glassco et al. (1993) reported that the bridged nicotine analog 51 where R = H, and its isomers, possess low affinity for O~4[~2 nACh receptors but that they nevertheless produced analgesic activity in animals. A series of related compounds has been explored by SIBIA Neurosciences that differ primarily with the introduction of an aromatic methoxy group; that is, 51 where R = - O M e (Vernier et al., 1998). Compound 52 possesses greater structural similarity than 53 to (-)nicotine; compound 52 was also the more efficacious of the two as a nicotinic agonist at [3a-Containing (e.g., OL4~4, O~2[~4) nACh receptors. Ring-expansion to the anabasine analog 54 was detrimental to activity. Some of the agents are active in animal models of Parkinson's disease and pain (Vernier et al., 1998). Certain 5-substituted nicotine analogs are being explored for their utility as cognition enhancers and for their ability to modulate dopaminergic function. For example, SIB-1765F (55) (S-isomer = SIB-1508Y) is being studied for use in Parkinson's disease (Sacaan et al., 1997). SIB1765F binds at oL4[~2 receptors with an affinity comparable to that of ( - ) n i c o t i n e but was far less potent at oL7 receptors ( K i > 100,000 nM); in addition, this agent displayed a different pharmacology than (-)nicotine. Replacement of the ethynyl group of 55 with a phenyl group shifted the binding pattern of 56 away from a4~2 receptors to receptors possessing ~4 subunits. Modification of 56 resulted in 57 (SIB-1553A). SIB-1553A stimulated the release of ACh and dopamine in brain regions associated with learning and memory and, when tested in cells stably expressing human nACh receptor subunits, was more efficacious at oL4~4 receptors and had weak actions on oL3~4, oL7, and other receptors (Vernier et al., 1999). These agents are being explored for possible use in Alzheimer's disease.

It should be apparent that nACh receptors, and particularly central nACh receptors, are enjoying a new-found popularity. Of the two major populations of nACh receptors found in the brain, oL4[~2 and oL7, much needs to be learned about the former, and even less is known about the latter. Only very recently is attention turning to the " m i n o r " subpopulations. Numerous agents now have been developed and examined but, for the most part, very little is known about selectivity. Indeed, at this time, the vast majority of studies being published are more concerned with the binding requirements or functional properties of the receptor populations of interest to individual investigators. Probably of peak interest are new compounds that bind with high affinity at oL4~2 nACh receptors but fail to produce (or antagonize) nicotine-like functional actions, and agents that lack affinity for these receptors but that do produce (or antagonize) nicotine-like activity. It is likely that these types of agents will provide us with clues necessary to develop subtype-selective nicotinic agents. The exceptionally broad potential therapeutic utility of nACh receptors promises to make these receptor populations some that will witness significant scientific exploration at the beginning of the new millennium.

Acknowledgements Work from the authors' laboratories was supported in part by PHS grant DA05274.

References Abood, L.G., Lu, X., Banerjee, S., 1987. Receptor binding characteristics of a 3H-labelled azetidine analogue of nicotine. Biochem. Pharmacol. 36, 2337-2341. Abood, L.G., Shahid, K.S., Maiti, A., 1988. Structure-activity studies of carbamate and other esters: agonists and antagonists to nicotine. Pharmacol. Biochem. Behav. 30, 403-408. Abreo, M.A., Lin, N.-H., Garvey, D.S., Gunn, D.E., Hettinger, A.-M., Wasicak, J.T., Pavlik, P.A., Martin, Y.C., Donnelly-Roberts, D.L., Anderson, D.J., Sullivan, J.P., Williams, M., Arneric, S.P., Holladay, M.W., 1996. Novel 3-pyridyl ethers with subnanomolar affinity for central neuronal nicotinic acetylcholine receptors. J. Med. Chem. 39, 817-825. Anderson, D.J., Arneric, S.P., 1994. Nicotine receptor binding of [3H]cytisine, [3H]nicotineand [3H]methylcarbamylcholinein rat brain. Eur. J. Pharmacol. 252, 261-267. Arneric, S., Brioni, J.D. (Eds.), 1999. Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities. Wiley-Liss, New York. Badio, B., Garraffo, H.M., Spande, T.F., Daly, J.W., 1994. Epibatidine: discovery and definition as a potent analgesic and nicotinic agonist. Med. Chem. Res. 4, 440-448. Barlocco, D., Cignarella, G., Tondi, D., Vianello, P., Villa, S., Bartolini, A., Ghelardini, C., Galeotti, N., Anderson, D.J., Kuntzweiler, T.A., Colombo, D., Toma, L., 1998. Mono- and disubstituted 3,8-diazabicyclo[3.2.1]octane derivatives as analgesic structurally related to epibatidine: synthesis, activityand modeling.J. Med. Chem. 41,674-681.


Barlow, R.B., Johnson, O., 1989. Relations between structure and nicotine-like activity: X-ray crystal structure analysis of (-)-cytisine and (-)-lobeline hydrochloride and a comparison with (-)-nicotine and other nicotine-like compounds. Br. J. Pharmacol. 98, 799-808. Beers, W.H., Reich, E., 1970. Structure and activity of acetylcholine. Nature 225, 917-922. Bencherif, M., Lovette, M.E., Fowler, K.W., Arrington, S., Reeves, L., Caldwell, W.S., Lippiello, P.M., 1996. RJR-2403: a nicotinic agonist with CNS selectivity: I. In vitro characterization. J. Pharmacol. Exp. Ther. 279, 1413-1421. Bencherif, M., Schmitt, J.D., Bhatti, B.S., Crooks, P., Caldwell, W.S., Lovette, M.E., Fowler, K., Reeves, L., Lippiello, P.M., 1998. The heterocyclic substituted pyridine derivative(_+)2-(3-pyridinyl)-lazabicyclo[2.2.2]octane (RJR-2429): a selective ligand at nicotinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 284, 886-894. Brioni, J.D., Decker, M.W., Sullivan, J.P., Arneric, S.P., 1997. The pharmacology of (-)nicotine and novel cholinergic channel modulators. Adv. Pharmacol. 37, 153-214. Broka, C.A., 1994. Synthetic approaches to epibatidine. Med. Chem. Res. 4, 449-460. Caldwell, W.S., Benchenif, M., Bhatti, B.S., Deo, N.M., Dobson, G.P., Dull, G.M., Lipiello, P.M., Lovette, M.E., Miller, C.H., Ravard, A., Schmitt, J.D., Crooks, P.A., 1997. Synthesis and structure-activity relationships of analogs of RJR-2403, a CNS-selective nicotinic agonist. Abstracts of International Business Communications Symposium on Nicotinic Acetylcholine Receptors as Pharmaceutical Targets, Washington, DC, July 24-25. Cheng, Y.X., Fiedler, W., Dukat, M., Damaj, I., Martin, B., Glennon, R.A., 1995. Conformationally-restricted aminomethylpyridine derivatives as novel nicotinic receptor ligands. Va. J. Sci. 46, 135. Cheng, Y.X., Dukat, M., Dowd, M., Fiedler, W., Martin, B., Damaj, M.I., Glennon, R.A., 1999. Synthesis and binding of 6,7,8,9-tetrahydro-5H-pyrido[3,4-d]azepine and related ring-open analogs at central nicotinic receptors. Eur. J. Med. Chem. 34, 177-190. Crooks, P.A., Jones, M.D., Chesnut, M.D., Jaromczyk, A.M., Dwoskin, L.P., 1999. Stereochemically-defined lobeline analogues: inhibition of [3H]dopamine uptake and [3H]nicotine binding in rat striatum. Abstracts, College on Problems of Drug Dependence meeting, Acapulco, Mexico, June 12-17, 1999, p. 29. Damaj, M.I., Patrick, G.S., Creasy, K.R., Martin, B.R., 1997. Pharmacology of lobeline, a nicotinic receptor ligand. J. Pharmacol. Exp. Ther. 282, 410-419. Davila-Garcia, M.I., Musachio, J.L., Perry, D.C., Xiao, Y.X., Horti, A., London, E.D., Dannals, R.F., Kellar, K.J., 1997. [125I]IPH, an epibatidine analog, binds with high affinity to neuronal nicotinic cholinergic receptors. J. Pharmacol. Exp. Ther. 282, 445-451. De Fiebre, C.M., Meyer, E.M., Henry, J.C., Muraskin, S.I., Kem, W.R., Papke, R.L., 1995. Characterization of a series of anabaseine-derived compounds reveals that the 3-(4)-dimethylamino-cinnamylidine derivative is a selective agonist at neuronal nicotinic OL7/125I-oLbungarotoxin receptor subtypes. Mol. Pharmacol. 47, 164-171. Dolle, F., Dolci, L., Valette, H., Hinnen, F., Vaufrey, F., Guenther, I., Fuseau, C., Coulon, C., Bottaender, M., Crouzel, C., 1999. Synthesis and nicotinic acetylcholinergic receptor in vivo binding properties of 2-fluoro-3-[2(s)-2-azetidinylmethoxy]pyridine: a new positron emission tomography ligand for nicotinic receptors. J. Med. Chem. 42, 2251-2259. Dukat, M., 1994. 208/210 a.k.a. Epibatidine. Med. Chem. Res. 4, 433-439, and following articles. Dukat, M., Damaj, M.I., Glassco, W., Dumas, D., May, E.L., Martin, B.R., Glennon, R.A., 1994. Epibatidine: a very high affinity nicotinereceptor ligand. Med. Chem. Res. 4, 131-139. Dukat, M., Fiedler, W., Dumas, D., Damaj, I., Martin, B.R., Rosecrans, J.A., James, J.R., Glennon, R.A., 1996. Pyrrolidine-modified and 6-substituted analogs of nicotine: a structure-affinity investigation. Eur. J. Med. Chem. 31,875-888. Dukat, M., Dowd, M., Damaj, I.M., Martin, B., E1-Zahabi, M., Glennon,

113

R.A., 1999a. Synthesis, receptor binding and QSAR studies on 6-substituted nicotine derivatives as cholinergic ligands. Eur. J. Med. Chem. 34, 31-40. Dukat, M., Flammia, D.D., Damaj, M.I., Martin, B., Glennon, R.A., 1999b. Lobeline: a nicotine receptor structure-affinity study. Abstracts, College on Problems of Drug Dependence meeting, Acapulco, Mexico, June 12-17, 1999, p. 37. Dwoskin, L., 1999. Novel subtype selective nicotinic receptor antagonists block the neurochemical effects of nicotine. Presented at College on Problems of Drug Dependence meeting, Acapulco, Mexico, June 12-17, 1999. Elliott, R.L., Ryther, K.B., Anderson, D.J., Piattoni-Kaplan, M., Kuntzweiler, T., Donnelly-Roberts, D., Arneric, S.P., Holladay, M.W., 1997. Novel 2-(2'-furo[3,2-b]pyridinylpyrrolidines: potent neuronal nicotinic acetylcholine receptor ligands. Bioorg. Med. Chem. Lett. 7, 2703-2708. Flammia, D.D., Dukat, M., Damaj, M.I., Martin, B.R., Glennon, R.A., 1999. Lobeline: failure to meet Sheridan pharmacophore requirements. Virginia Academy of Sciences meeting, Norfork, VA, May 25-28, 1999. Fujita, T., Nakajima, M., Soeda, Y., Yamamoto, I., 1971. Physicochemical properties of biological interest of nicotine and its related compounds. Pestic. Biochem. Physiol. 1, 151-162. Glaser, R., Hug, P., Drouin, M., Michel, A., 1992. Solution- and solid-state stereochemistry of (-)ot-lobeline hydrochloride and hydrobromide, a respiratory stimulant. J. Chem. Soc. Perkin Trans., pp. 1071-1079. Glassco, W., Suchocki, J., George, C., Martin, B.R., May, E.L., 1993. Synthesis, optical resolution, absolute configuration and preliminary pharmacology of ( + ) - and (-)cis-2,3,3a,4,5,9b-hexahydro-1methyl-1H-pyrrolo[3,2-h]isoquinoline, a structural analog of nicotine. J. Med. Chem. 36, 3381-3385. Glennon, R.A., Dukat, M., 1996. Nicotinic receptor ligands. Med. Chem. Res. 6, 465-486. Glennon, R.A., Dukat, M., 1999a. Nicotinic cholinergic receptor pharmacophores. In: Arneric, S.P., Brioni, J.D. (Eds.), Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities. Wiley-Liss, New York, pp. 271-284. Glennon, R.A., Dukat, M., 1999b. Nicotine analogs: structure-affinity relationships for central acetyl-cholinergic receptor binding. In: Yamamoto, I., Casida, J.E. (Eds.), Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor. Spinger-Verlag, Tokyo, 237-252. Glennon, R.A., Herndon, J.L., Dukat, M., 1994. Epibatidine-aided studies toward definition of a nicotinic receptor pharmacophore. Med. Chem. Res. 4, 461-473. Gotti, C., Balestra, B., Moretti, M., Rovati, G.E., Maggi, L., Rossoni, G., Berti, F., Villa, L., Pallavicini, M., Clementi, F., 1998.4-Oxystilbene compounds are selective ligands for neuronal nicotinic ot-bungarotoxin receptors. Br. J. Pharmacol. 124, 1197-1206. Holladay, M.W., Dart, M.J., Lynch, J.K., 1997. Neuronal nicotinic acetylcholine receptors as targets for drug discovery. J. Med. Chem. 40, 4169-4194. Holladay, M.W., Wasicak, J.T., Lin, N.-H., Ryther, K.B., Bannon, A.W., Buckley, M.J., Kim, D.J.B., Decker, M.W., Anderson, D.J., Campbell, J.E., Kuntzweiler, T.A., Donnelly-Roberts, D.L., Piattoni-Kaplan, M., Briggs, C.A., Williams, M., Arneric, S.P., 1998. Identification and initial structure-activity relationships of (R)-5-(2-Azetidinylmethoxy)-2-chlorpyridine (ABT-594), a potent, orally active, nonopiate analgesic agent acting via neuronal nicotinic acetylcholine receptors. J. Med. Chem. 41,407-412. Holladay, M.W., Cosford, N.D.P., McDonald, I.A., 1999. Natural products as a source of nicotinic acetylcholine receptor modulators and leads for drug discovery. In: Arneric, S.P., Brioni, J.D. (Eds.), Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities. Wiley-Liss, New York, pp. 253-270. Horti, A.G., Scheffel, U., Kimes, A.S., Musachio, J.L., Ravert, H.T., Mathews, W.B., Zhan, Y., Finley, P.A., London, E.D., Dannals, R.F., 1998. Synthesis and evaluation of N-[11C]methylated analogues of

114


epibatidine as tracers for positron emission tomographic studies of nicotinic acetylcholine receptors. J. Med. Chem. 41, 4199-4206. Houghtling, R.A., Davila-Garcia, M.I., Hurt, S.D., Kellar, K.J., 1994. [3H]Epibatidine binding to nicotinic cholinergic receptors in brain. Med. Chem. Res. 4, 538-546. Koren, A.O., Horti, A.G., Mukhin, A.G., Gundisch, D., Kimes, A.S., Dannals, R.F., London, E.D., 1998. 2-, 5- and 5-Halo-3(2(S)azetidinylmethoxy)pyridines: synthesis, affinity for nicotinic acetylcholine receptors and molecular modeling. J. Med. Chem. 41, 3690-3698. Latli, B., D'Amour, K., Casida, J.E., 1999. Novel and potent 6-chloro-3pyridinyl ligands for the oL4[~2 neuronal nicotinic acetylcholine receptor. J. Med. Chem. 42, 2227-2234. Lin, N.-H., Carrera, G.M., Anderson, D.J., 1994. Synthesis and evaluation of nicotine analogs as neuronal nicotinic acetylcholine receptor ligands. J. Med. Chem. 37, 3542-3553. Lin, N.-H., Gunn, D.E., Ryther, K.B., Garvey, D.S., Donnely-Roberts, D.L., Decker, M.W., Brioni, J.D., Buckley, M.J., Rodrigues, A.D., Marsh, K.G., Anderson, D.J., Buccafusco, J.J., Prendergast, M.A., Sullivan, J.P., Williams, M., Arneric, S.P., Holladay, M.W., 1997. Structure-activity studies on 2-methyl-3-(2(S)-pyrrolidinylmethoxy)pyridine (ABT-089): an orally bioavailable 3-pyridyl ether nicotinic acetylcholine receptor ligand with cognition-enhancing properties. J. Med. Chem. 40, 385-390. Lin, N.-H., Gunn, D.E., Li, Y., He, Y., Bai, H., Ryther, K.B., Kuntzweiler, T., Donnelly-Roberts, D.L., Anderson, D.J., Campbell, J.E., Sulivan, J.P., Arneric, S.P., Holladay, M.W., 1998. Synthesis and structureactivity relationships of pyridine-modified analogues of 3-[2-(S)-pyrrolidinyl)methoxy]pyridine, A-84543, a potent nicotinic acetylcholine receptor agonist. Bioorg. Med. Chem. Lett. 8, 249-254. McDonald, I.A., Cosford, N., Vemier, J.-M., 1995. Nicotinic acetylcholine receptors: molecular biology, chemistry and pharmacology. Annu. Rep. Med. Chem. 30, 41-50. Meyer, E.M., Tay, E.T., Zoltewicz, J.A., King, M.A., Papke, R.L., de Fiebre, C.M., 1998a. Neuroprotective and memory-related actions of novel alpha-7 nicotinic agents with different mixed agonist/antagonist properties. J. Pharmacol. Exp. Ther. 284, 1026-1032. Meyer, E.M., Juryatov, A., Gerzanich, V., Lindstrom, J., Papke, R.L., 1998b. Analysis of 3-(4-hydroxy, 2-methoxybenzylidine)anabaseine selectivity and activity at human and rat alpha-7 nicotinic receptors. J. Pharmacol. Exp. Ther. 287, 918-925.

Olesen, P.H., Swedberg, M.D.B., Eskesen, K., Judge, M.E., Egebjerg, J., Tonder, J.E., Rasmussen, T., Sheardown, M.J., Rimvall, K., 1997. Identification of novel (isoxazole)methylene-1-azabicyclic compounds with high affinity for the central nicotinic cholinergic receptor. Bioorg. Med. Chem. Lett. 7, 1963-1968. Qian, C., Li, T., Shen, T.Y., Libertine-Garahan, L., Eckman, J., Biftu, T., Ip, S., 1993. Epibatidine is a nicotinic analgesic. Eur. J. Pharmacol. 250, R13-R14. Rosecrans, J.A., Brioni, J.D., 1996. Medicinal chemistry and pharmacology of neuronal nicotinic receptors. Med. Chem. Res. 6, 439-443, and following articles. Sacaan, A.I., Reid, R.T., Santori, E.M., Adams, P., Correa, L.D., Mahaffy, L.S., Bleicher, L., Cosford, N.D.P., Stauderman, K.A., McDonald, I.A., Rao, T.S., Lloyd, G.K., 1997. Pharmacological characterization of SIB-1765F: a novel cholinergic ion channel agonist. J. Pharmacol. Exp. Ther. 280, 373-383. Sargent, P.B., 1993. The diversity of neuronal nicotinic acetylcholine receptors. Annu. Rev. Neurosci. 16, 403-443. Shacka, J.J., Robinson, S.E., 1996. Central and peripheral anatomy of nicotinic sites. Med. Chem. Res. 6, 444-464. Sheridan, R.P., Nilakantan, R., Dixon, J.S., Venkataraghavan, R., 1986. The ensemble approach to distance geometry: application to the nicotinic pharmacophore. J. Med. Chem. 29, 899-906. Vernier, J.-M., Holsenback, H., Cosford, N.D.P., Whitten, J.P., Menzaghi, F., Reid, R., Rao, T.S., Sacaan, A.I., Lloyd, G.K., Suto, C.M., Chavez-Noriega, L.E., Washburn, M.S., Urrutia, A., McDonald, I.A., 1998. Conformationally restricted analogues of nicotine and anabasine. Bioorg. Med. Chem. Lett. 8, 2173-2178. Vernier, J.-M., E1-Abdellaouri, H., Holsenback, H., Cosford, N.D.P., Bleicher, L., Barker, G., Bontempi, B., Chavez-Noriega, L., Menzaghi, F., Rao, T.S., Teid, R., Sacaan, A.I., Suto, C., Washburn, M., Lloyd, G.K., McDonald, I.A., 1999. 4-[[2-(1-Methyl-2r. pyrrolidinyl)ethyl]thio]phenol hydrochloride (SIB-1553A): a novel cognitive enhancer with selectivity for neuronal nicotinic acetylcholine receptors. J. Med. Chem. 42, 1684-1686. Yamamoto, I., 1965. Nicotinoids as insecticides. In: Metcalf, R.L. (Ed.), Advances in Pest Control Research. Wiley-Interscience, New York, pp. 231-260. Yamamoto, I., Casida, J.E. (Eds.), 1999. Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor. Spinger-Verlag, Tokyo.



Structural aspects of high affinity ligands for the oL4132 neuronal nicotinic receptor Michael J. Dart *, James T. Wasicak, Keith B. Ryther, Michael R. Schrimpf, Ki H. Kim, David J. Anderson, James P. Sullivan, Michael D. Meyer Neurological and Urological Diseases Research and Department of Structural Biology, Abbott Laboratories, Abbott Park IL, 60064, USA

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are a heterogeneous family of related ion channels that are widely distributed throughout the central and peripheral nervous systems. They all share a common architecture of five subunit proteins that combine at the cell surface to create a ligand-gated cation permeable pore. Significant effort is currently being expended by medicinal chemistry teams to synthesize ligands that exhibit selectivity for central over peripheral nAChR subtypes. Within the CNS, multiple nAChR subtypes are recognized, and the discovery of ligands exhibiting selectivity among these subtypes offers an opportunity for the development of novel therapeutic agents. The a4[~2 subtype is one of the most abundant nAChR subtypes within the CNS, and has been the primary focus of high affinity ligand design. Nicotine (1), and more recently, epibatidine (2) have served as structural templates for the design of the majority of active compounds. Although the diversity of nAChR ligands is growing, the structural requirements necessary for high affinity binding with the e~4132 receptor remain poorly understood. The putative pharmacophoric elements common to all potent oL4132 ligands include (1) a basic or quaternized nitrogen atom, and (2) a less basic nitrogen or a carbonyl oxygen that presumably interact with electron rich and electron deficient sites on the receptor, respectively. The family of currently known high affinity analogs consists of a diverse array of azacycles containing a basic amine. Several additional basic amine fragments have been identified, including the pyrrolizidine nucleus (exemplified by 8) and the 2-azabicyclo[2.2.1 ]heptane skeleton (exemplified by 9). In addition, we have found that the furo[2,3-b]pyridine heterocycle (compound 10) serves as useful bioisosteric replacement for the pyridyl substituent of nicotine. A preliminary pharmacophore model is proposed in which a reasonable superposition of the putative pharmacophoric elements of the diverse array of high affinity ligands for the oL4~2 nAChR reported herein may be accommodated. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: Nicotinic acetylcholine receptor (nAChR); oLc4132; Nicotine; Epibatidine; Pyrrolizidine; 2-azabicyclo[2.2.1]heptane; Furo[2,3-b]pyridine; Furo[3,2-b]pyridine; Pharmacophore; Pyridyl ether; Furo[2,3-c]pyridine; Furo[3,2-c]pyridine; Molecular modelling

1. Introduction

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels that are found in abundance at the neuromuscular junction, and are widely distributed throughout the central and peripheral nervous systems. They comprise five subunit proteins that combine to form a cation permeable pore at the cell surface. In neuronal tissues, multiple nAChR subtypes may be assembled from a diverse array of subunits (OL2-OL9; [32-[34) and their * Corresponding author. Neuroscience Research, D-47W, Abbott Laboratories, Abbot Park, IL 60064-6117, USA. Tel.: +1-847-938-6996; fax: +1-847-937-9195; e-mail: [email protected]

specific pharmacology is directly influenced by their particular subunit composition. Among the numerous neuronal nAChR subtypes, the e~4132 receptor is generally acknowledged to be the most predominant CNS receptor subtype exhibiting high affinity for nicotine (see Ref. in Holladay et al., 1998). This is supported by the observation that high affinity nicotine binding is absent in the brains of mutant mice lacking the [32 nAChR subunit (Picciotto et al., 1995; Zoli et al., 1998). In addition, several reports have suggested that [32-containing nAChRs (likely oL4132) may play a fundamental role in mediating several important physiological processes including cognition (Nordberg, 1994; Perry et al., 1995; Picciotto et al., 1995; Zoli et al., 1999), addic-


116

M.J. Dart et al. / Pharmaceutica Acta Helvetiae 74 (2000) 115-123

tion (Picciotto et al., 1998; Lena and Changeux, 1999), neurotransmitter release (Wonnacott et al., 1996; Grady et al., 1997; Lu et al., 1998; Tani et al., 1998; Alkondon et al., 1999), and antinociception (Bitner et al., 1998; Marubio et al., 1999). Therefore, ligands that selectively bind and activate oL4132 receptors could potentially provide novel therapeutics for the treatment of a variety of debilitating CNS disorders such as Alzheimer's disease and the management of pain (Williams and Arneric, 1996; Brioni et al., 1997). Radioligand binding assays using [3HI-nicotine or [3H]cytisine as high-affinity binding probes are frequently employed as an initial screen to assess ligand interactions with et4132 nAChRs (Lippiello and Fernandez, 1986; Pabreza et al., 1990). It has been well established that nAChRs are allosteric proteins that exist in multiple conformational states (resting, open, and desensitized), and these various states can have different affinities for specific ligands (Lena and Changeaux, 1993; Edelstein and Changeux, 1998). Desensitized states of the receptor exhibit significantly higher affinities for agonists than the resting or active states, and are presumably stabilized by the presence of ligands. Therefore, binding affinity is a measure of interactions with one or more of the desensitized states of the receptor and may not accurately reflect potential functional potency and efficacy (Lippiello et al., 1987). The rational design of potent and selective oL4132 nAChR ligands would be facilitated by the identification of a pertinent pharmacophore model (Glennon and Dukat, 1996, 1999). The most useful models would clearly define not only distances between pharmacophoric elements but also regions of receptor tolerance for steric bulk. This is an exceedingly difficult task since (1) the binding site of the oL4132 receptor is not well characterized, (2) numerous nicotinic pharmacophores likely exist given the diversity of nAChR subtypes, and (3) different allosteric states for any specific receptor subtype may possess different pharmacophores (Karlin, 1993). Thus, radioligand binding data might be useful in the development of a pharmacophore model for a desensitized state of the oL4132 receptor, but this model may differ significantly from a pharmacophore model that reflects interactions required for receptor activation. The putative pharmacophoric elements common to all high affinity et4132 nAChR ligands include (1) a basic or quaternized nitrogen atom, and (2) a less basic nitrogen or carbonyl oxygen. However, the interactions between these pharmacophoric elements and complementary sites on the receptor have not been well characterized. It is conceivable that the protonated sp 3 nitrogen may form a hydrogen bond with an anionic site on the protein (Czajkowski and Karlin, 1995) or, alternatively, the positive ammonium charge may be stabilized through cation-Tr interactions with nearby aromatic amino acid side chains (Dougherty, 1996; Schmitt et al., 1998; Zhong et al., 1998), in analogy

to the binding of acetylcholine with acetylcholinesterase (Sussman et al., 1991; Harel et al., 1993). A putative hydrogen bond between the pyridine nitrogen and a complementary hydrogen bond donor site on the receptor is believed to be important for high affinity binding, but the nature of this interaction is not well understood since the binding of many ligands does not appear to be directly dependent on the basicity or p K a of the pyridine nitrogen (Elliott et al., 1996; Dukat et al., 1999). Previous reports from these labs have described several novel series of nAChR ligands that contain a variety of isosteric replacements for the pyridine ring of nicotine and maintain high affinity for c~4132 receptors (Fig. 1). Included among these are pyrrolidinyl isoxazoles (Garvey et al., 1994), such as ABT-418 (3), a compound that exhibits anxiolytic and cognitive enhancing properties (Arneric et al., 1995). A novel series of 3-pyridyl ether compounds, typified by A-84543 (4), has also been reported (Abreo et al., 1996). Several members of this series possess subnanomolar binding affinity (Holladay et al., 1998; Lin et al., 1998), including A-85380 (5) and ABT-594 (6), which have potency for oL4132 receptors rivaling that of epibatidine, the most potent nAChR ligand identified to date. ABT-594 (6) possesses broad-spectrum antinociceptive activity in preclinical models and is the first nAChR-mediated analgesic to enter human clinical trials (Bannon et al., 1998). The furo[3,2-b]pyridine ligand class (exemplified by 7), which was originally designed based on structural elements embedded within the pyridyl ethers, also contains potent nAChR ligands (Elliott et al., 1997). The high nAChR affinity of these three series demonstrates that the isoxazole and furo[3,2-b]pyridine heterocycles and the 3pyridyl ether moiety serve as proficient bioisosteric surrogates for the pyridine ring of nicotine. In an effort to identify additional ligand classes that retain potent oL4132 receptor binding affinity (Fig. 2), we

N H

(S)-nicotine I1) Ki = 1 nM

~NvCI

(-)-epibatidine 121 Ki = 0.04 nM

A-84543 (4)

ABT-418 131 Ki = 4 nM

A-85380 (S)

Ki = 0.15 nM

Ki =.0.05 nM

ABT-594 (6) Ki = 0.04 nM

A-98795 (7) Ki = 2.7 nM

Fig. 1. High-affinity et4132 nAChR ligands.

117


of the various chloropyridyl-substituted 2-azabicyclo [2.2.1]heptanes have been described (Hodgson et al., 1998; Kasyan et al., 1998; Malpass and Cox, 1999). 8

9

H

10

Fig. 2. Recentlyidentifiedhigh-affinityoL4132nAChRligands.

have found that the pyrrolizidine nucleus (exemplified by 8) is a useful bioisosteric replacement for the pyrrolidine ring of nicotine (Wasicak et al., 1998). Another basic amine fragment from which potent analogs may be synthesized is the 2-azabicyclo[2.2.1]heptane skeleton (e.g., 9). Additional SAR has recently been performed in the furopyridine series resulting in the identification of furo[2,3-b]pyridinyl pyrrolidines (e.g., 10) as potent a4132 receptor ligands. A preliminary pharmacophore model using (-)-epibatidine (2) as a template is proposed in which a reasonable superposition of the putative pharmacophoric elements of relatively low energy conformers of the diverse array of high affinity ligands for the oL4132 receptor reported herein may be accommodated.

3. Results and discussion

The structure of 7a-(3-pyrrolidinyl)-hexahydro-1H-pyrrolizine (8) can be conceptualized as a hybrid between the individual enantiomers of nicotine. Pyrrolizidine 8 was synthesized in straightforward fashion by addition of the pyridyl lithium anion to the readily prepared imminium salt (Eq. (1)) (Wasicak et al., 1998). Compound 8 displays subnanomolar binding affinity (K i = 0.32 nM) for oL4132 receptors, which is a 3-fold increase in binding affinity over (S)-nicotine (1) and a 60-fold increase relative to the less potent (R)-nicotine enantiomer. One advantage with heterocyclic substituted pyrrolizidines series over their pyrrolidine counterparts is that no stereochemical issues come into play since they are symmetrical molecules. 0104"

nBuLi, E t 2 0 (58%) Ki = 0.32

2. Methods

2.1. Binding assays The binding affinity of each compound was determined by measuring the displacement of [3H]-(-)-cytisine from a preparation of whole rat brain (Pabreza et al., 1990).

(1) nM

In addition to 3-pyridines, a variety of other aromatic heterocycles, including isoxazoles, quinoline, and furopyridines may be incorporated onto the pyrrolizidine skeleton

2.2. Molecular modeling All compounds included in this study were initially constructed with standard bond lengths and angles, and their geometries were optimized using the DISCOVER force field (CFF98) implemented in Insight II (98.0). The structures were minimized until the derivative of 0.001 kcal mol-1 ~-1 was satisfied with the conjugate gradient method. Minimum energy conformations of all compounds were utilized in the final superposition with the pharmacophoric elements of (-)-epibatidine in the four-point model (see Fig. 3), except for compound 4 (discussed in text). Theoretical receptor binding sites were generated 2.9 from the pyridine nitrogen of (-)-epibatidine in the direction of the lone pair and the same distance from the sp 3 quaternary amine in the direction of the proton proximal to the pyridine substituent.

~ 1 ~

@N N .~

ABT-418 (3) Ki = 4.2 nM

11 Ki = 0.97 nM

12 Ki = 4.5 nM

13 Ki = 2.8 nM

Ki = 2.6 nM

Ki = 0.37 nM

Ki = 158 nM

Ki = 42 nM

Me

2.3. Chemistry Syntheses and characterization of the pyrrolizidine and furopyridine analogs are contained in the following references (Elliott et al., 1998; Wasicak et al., 1998). Syntheses

Fig. 3. [3H]Cytisinebinding affinities of pyrrolidineand pyrrolizidine analogs.

118


epibatidine conformer A

epibatidine conformer B

(PHT)

(_.+)-Pyrido[3,4-b]homotropane

Fig. 4. Approximated structures of families of low energy epibatidine conformers and the structure of PHT.

(Fig. 3). These heterocyclic substituted pyrrolizidine analogs retain high binding affinity, and all exhibited slightly enhanced oL4132 receptor binding potency relative to the analogous pyrrolidine compounds. The importance of the weakly basic nitrogen of the furo[3,2-b]pyridine heterocycle (7 and 14) is exemplified by the benzofuran analogs (15 and 16), which exhibit a > 60-fold decrease in binding potency (Elliott et al., 1997). (-)-Epibatidine (2) was utilized as the reference compound in developing a pharmacophore model for binding to the oL4132 receptor. The positively charged sp 3 nitrogen, its electron-rich complementary protein site, the pyridine nitrogen, and its complementary hydrogen bond donor site point were chosen as pharmacophoric elements for superposition in this 4-point model (Abreo et al., 1996). Epibatidine possesses a rigid 7-azabicyclo[2.2.1]heptane skeleton with a rotatable chloropyridyl substituent. This rotation accommodates a large range of internitrogen distances (Bencherif et al., 1998), a parameter that has been emphasized in various nicotinic pharmacophore models (Beers and Reich, 1970; Sheridan et al., 1986; Dukat et al., 1994; Glennon et al., 1994; Abreo et al., 1996; Barlocco et al., 1998; Koren et al., 1998). Calculations typically generate two families of low energy epibatidine conformers (approximated by conformers A and B, Fig. 4) that differ by roughly 180 ~ in the rotation of the pyridyl ring. Although the internitrogen distances vary greatly (from ~ 4.5 to 5.5 /k), these conformers have quite similar energy and are separated by a very low barrier (Bencherif et al., 1998;

.

Campillo et al., 1998). Therefore, at the outset it is not clear which low energy epibatidine conformer family should be utilized as the reference template when attempting to superimpose the putative pharmacophoric elements of various ligands. A preliminary analysis indicated that a better superposition is obtained between epibatidine conformer A and the conformationally restricted nicotine analog pyrido[3,4-b]homotropane (PHT), which possess oL4132 receptor binding affinity approximately equivalent to nicotine (Kanne and Abood, 1988). Therefore, we chose initially to examine conformer A in greater detail as the template for superposition, orienting the pyridine ring nitrogen proximal to the sp 3 nitrogen ( N - N distance of 4.79 ,~), although a suitable overlap of conformer B with the pharmacophoric elements of various ligands may also be possible. As previously mentioned, the exact nature of the interaction between the charged sp 3 nitrogen and its complementary protein site is not known. Whether the protonated site is viewed as interacting with a "rr cloud of aromatic residues or as a hydrogen bond to a negatively charged amino acid, it is necessary to address the appropriate directionality of that interaction. Therefore, one of two possible protons on the sp 3 nitrogen of epibatidine must be selected, the hydrogen atom either proximal to or distal to the pyridine ring. The heterocyclic substituted pyrrolizidines proved to be a useful series in helping establish that

9 (4)

o o

9 C3)

.

Fig. 5. Pharmacophoric element selection for (-)-epibatidine (2). The nitrogen atoms (dark blue) are pharmacophoric elements (1) and (2), and elements (3) and (4) are points on the receptor (red) with which elements (1) and (2) interact.

.

.

.

.

.

.

.

.

Fig. 6. Superposition of the putative pharmacophoric elements of (-)-epibatidine (pink) and pyrrolizidine 8 (green).

M.J. Dart et al. / Pharmaceutica Acta Helvetiae 74 (2000) 115-123 H

(=)-epibatidine Ki = 0.042 nM

H N

Table 1 oL4132 nAChR binding affinities, distances between pharmocophoric elements, and superposition rms deviations

,

~

9

2-aza epibatidine analogs

H

H N

H

H

N H eXO

H

N (=)-endo.eplbatidine

18

Ki = 7.6 nM

cl

19

cl

Fig. 7. Structures of epibatidine, endo-epibatidine, and 2-aza epibatidine analogs.

the proton proximal to the chloropyridine moiety of epibatidine should be included in the superposition with site points from other ligands. The geometrical constraints contained within the azabicyclic structure of pyrrolizidine 8 enforce a syn relationship between the pyridyl heterocycle and the proton on the basic nitrogen. Fig. 5 illustrates the four points selected as pharmocophoric elements for (-)-epibatidine (2), and Fig. 6 shows the superposition of low energy conformations of 2 with pyrrolizidine 8. This four-point pharmacophore model for binding based on the potent ligand epibatidine also accommodates low energy conformations of the putative pharmacophoric elements of the high affinity pyrrolizidine, pyrrolidine, and azetidine analogs illustrated in Figs. 1 and 3. In the following sections the model is utilized to help explain binding trends observed in some additional structural classes. One of the new series comprises analogs in which the azabicyclic ring nitrogen of epibatidine has been translocated from the 7- to the 2-position of the rigid bicyclo[2.2.1]heptane framework (Fig. 7). The resulting 2-azanorbornane skeleton is an interesting template for

Boc N

21

o

cL

N OH

Compound

K a (nM)

N - N b (,~,) (3)-(4) c (,~)

RMS d (,~)

(-)-epibatidine (S)-nicotine 4 7 8 9 10 17 18 19 26 27 28 29

0.04 1 0.15 2.7 0.32 0.032 3.7 7.6 6.6 30 207 8000 1700 498

4.79 4.48 6.23 5.82 4.40 4.54 5.47 5.93 6.18 6.68 6.63 7.72 6.77 5.43

0.00 0.47 1.07 0.70 0.30 0.72 0.49 1.80 1.68 2.03 1.54 2.65 1.71 1.29

CN2CI2

3.76 4.67 5.74 5.16 3.81 3.94 3.85 8.41 8.00 8.93 7.42 10.36 7.96 3.63

"[3H]Cytisine binding. bDistance between sp3 nitrogen atom (1) and pyridine ring nitrogen (2) (Fig. 3). oDistance between putative receptor binding sites (3) and (4) (Fig. 3). ORMS deviations of the four pharmacophoricelements from those of (-)-epibatidine; lower RMS values imply better fit.

potential epibatidine analogs (Hodgson et al., 1998; Kasyan et al., 1998; Malpass and Cox, 1999). An efficient synthesis of the endo 6-substituted isomer 9 proceeded from the known racemic 6-heptanone intermediate 20 (Eq. (2)) (Hodgson et al., 1998; Malpass and Cox, 1999). Addition of the chloropyridyl lithium reagent generated by treatment of 2-chloro-5-iodopyridine (21) with n-butyl lithium at - 7 8 ~ in C H 2 C 1 2 , to ketone 20 gave a 78% isolated yield of the exo-addition adduct 22. The alcohol was removed using the protocol reported by Trudell in his synthesis of epibatidine (Zhang and Trudell, 1996). Thus, the hindered methyl oxalyl ester was formed in near quantitative yield and subsequently treated with nBu3SnH to afford the desired endo-chloropyridyl substituted azabicycloheptane, which was deprotected to give 9.

1. MeO2CCOCI, DMAP 2. nBu3SnH, AIBN (67%) 3. TsOH,H2 O

nBuki, -78 ~

(•

119

H ..,N... jCI / tl l Nf! / T [ . , ~ . , j ~ .~J H

(2)

endo-isomer 9

(78%) CI

The 6-endo 2-aza epibatidine analog 9 exhibits very high binding affinity for oL4132 receptors (K i = 0.032 nM), comparable to epibatidine. As expected, a unified superposition may be obtained between the pharmacophoric elements of (-)-epibatidine (2) and compound 9 (Fig. 8), and

this may account for their similar oL4132 binding profiles (Table 1). Synthesis of the corresponding exo-substituted 2azabicyclo[2.2.1]heptane analogs 18 and 19 (Eq. (3)) began with reductive Heck-arylation of the known 2-azabicyclo-

120

M.J. Dart et al. / Pharmaceutica Acta Heluetiae 74 (2000) 115-123

/

/

Fig. 8. Superpositionof the putative pharmacophoricelementsof (-)-epibatidine (pink) and 2-azabicycloheptane9 (blue).

Fig. 9. Superpositionof the putative pharmacophoricelements of (-)-epibatidine (thin pink), endo-epibatidine 17 (green), 6-exo analog 18 (blue), and 5-exo analog 19 (orange).

heptene 23 with 2-chloro-5-iodopyridine (21). An approximate 1:1 mixture of the two exo-regioisomers 24 and 25 was obtained, a result that concurs with a recent report from Malpass (Kasyan et al., 1998; Malpass and Cox, 1999). Deprotection, chromatographic separation, and salt

formation afforded the 6-exo isomer 18 and the 5-exo product 19 as crystalline solids. Structural assignments were initially made by 2D NMR spectroscopy and confirmed by X-ray crystallographic analysis of 19.

Boc

H

N

N

TsOH 9

H

Boc

CI Pd(OAc)2, PPh3 HCO2H, piperidine DMF, 8O ~

(+)-23

-1:1 mixture of exo-regioisomers

N 1. TFA 2. S e p a r a t i o n

CI

3. msOHoH20

Boc

N

(3)

H

N

H

. TsOH H

5-exo

Ki = 30 n M

Ci

distances between putative protein site points of 17, 18, and 19 are much longer than the optimum distances for epibatidine (2) or compound 9 (Table 1), and this may account for the reduced oL4132 receptor binding affinity of these analogs. A recent report from these laboratories described the synthesis and preliminary pharmacology of furo[3,2b]pyridinyl pyrrolidine analogs (Elliott et al., 1997). This ligand class was originally designed as conformationally

(+_)-Endo-epibatidine (17) exhibits > 150-fold weaker binding affinity than the natural exo-isomer 2 (Wypij and Shen, 1994), and a similar trend is observed in the 2azabicyclo[2.2.1]heptane epibatidine analogs. Molecular modeling indicates that a very close superposition of the pharmacophoric elements of endo-epibatidine (17) and the two exo-substituted 2-azabicycloheptane compounds 18 and 19 can be obtained; however, the overlap with epibatidine is not good (Fig. 9). The intemitrogen distances and

3-Pyridyl Ether

cl

,,,.._

CI

A-84543 (4)

6-exo ~ . ( " N

Ki = 6.6 nM

H Constraint

Fig. 10. From 3-pyridylethers to furo[3,2-b]pyridineheterocycles.

7 Furo[3,2-b]pyridine Ki : 2.7 nM

M.J. Dart et al. / Pharmaceutica Acta Helvetiae 74 (2000) 115-123 3-Pyridyl Ether

Furo[2,3-c]pyridine

A-84543 (4) Ki = 0.15 nM

Me

4-Pyridyl Ether

26 Ki = 207 nM

Furo[3,2-c]pyridine

Me 27-,,.11"Ki = 8000 nM

Me 28 Ki = 1700 nM

2-Pyridyl Ether

Furo[2,3-b]pyridine

Me 29 ~ Ki = 498 nM

Me

10 Ki = 3.7 nM

Fig. 11. [3H]Cytisinebinding affinities of various pyridyl ethers and their constrained furopyridine analogs.

................

~,%

121

:~ ....

%,

/

\

Fig. 12. Superposition of putative pharmacophoric elements of (-)-epibatidine (pink), furo[3,2-b]pyridine 7, and 84543 (4) (faint blue).

Fig. 13. Superposition of putative pharmacophoric elements of (-)-epibatidine (pink), furo[2,3-c]pyridine 26, and A-84543 (4) (faint blue).

Fig. 14. Superposition of putative pharmacophoric elements of (-)-epibatidine (pink), furo[3,2-c]pyridine 28, and 4-pyridyl ether 27 (blue). constrained 3-pyridyl ethers (Fig. 10). Although generally not as potent as many analogs in the 3-pyridyl ether series, the parent furo[3,2-b]pyridine compound 7 exhibits low nanomolar binding affinity (K i = 2.7 nM) for the oL4132 nAChRs. The 3-pyridyl ether of A-84543 (4) may also be constrained by tethering the oxymethyl group through an sp 2 carbon to the 4-pyridyl position (Fig. 11). The resulting furo[2,3-c]pyridyl pyrrolidine (26) is significantly less potent (K i --207 nM) than the isomeric furo[3,2-b]pyridine analog 7. The constrained furo[3,2-c]pyridine analog 28 of the 4-pyridyl ether 27 displayed only micromolar binding affinity. Whereas the 2-pyridyl ether 29 showed modest binding affinity ( g i --498 nM), the furo[2,3-b]pyridine analog 10 exhibited significantly improved binding affinity ( K i - - 3 . 7 nM) for oL4132 receptors. Thus, both the furo[2,3-b]pyridine and furo[3,2-b]pyridine heterocycles serve as useful bioisosteric replacements for the pyridine ring of nicotine. Figs. 12-15 illustrate the superpositions of ( - ) - e p i batidine (2) with the pyridyl ethers and their corresponding furopyridine analogs. The best overlap of the flexible

Fig. 15. Superposition of putative pharmacophoric elements of (-)-epibatidine (pink), furo[2,3-b]pyridine 10 (green), and 2-pyridyl ether 29 (blue).

122


3-pyridyl ether molecule 4 with (-)-epibatidine (Figs. 12 and 13) was obtained with a comparatively high energy conformation ( A E = 2.2 kcal/mol), which agrees with previous reports (Abreo et al., 1996). In contrast, higher energy conformers of either the 2- or 4-pyridyl ether compounds 29 and 27 did not result in a significantly improved overlap. Low energy conformers of both the furo[3,2-b]pyridine 7 (Fig. 12) and furo[2,3-b]pyridine 10 (Fig. 15) can achieve a reasonable superposition with (-)-epibatidine. In contrast, the less potent furo[2,3c]pyridine 26 (Fig. 13), furo[3,2-c]pyridine 28, and 4pyridyl ether 27 (Fig. 14) compounds contain much longer distances between the putative pharmacophoric elements compared to (-)-epibatidine (Table 1). The pyridine ring of the 2-pyridyl ether 29 does not overlap well with the aromatic ring of either (-)-epibatidine or 10 (Fig. 13) and may occupy space that the receptor does not tolerate, thus accounting for its reduced binding affinity. We have demonstrated that the pyrrolizidine nucleus and 2-azabicyclo[2.2.1]heptane skeleton are two basic amine cores that can be used in the construction of high affinity analogs for oL4132 nAChRs. By conformationally constraining a variety of pyrrolidinyl pyridyl ethers, we have identified the furo[3,2-b]pyridine and furo[2,3b]pyridine heterocycles as effective pyridine bioisosteres. (-)-Epibatidine has been utilized as a reference template in the design of a four-point pharmacophore model for binding to the oL4132 receptor. Trends with respect to binding affinity in the 2-azanorbornane, pyridyl ether, and furopyridine series can be rationalized by the proposed model. In general, the compounds having low energy conformations that achieve the best fit superposition with the putative pharmacophoric elements of (-)-epibatidine also exhibit the highest binding affinity for oL4132 receptors. Therefore, it is conceivable that these high affinity ligands may have similar modes of binding with the oL4132 receptor. Clearly, information regarding the structure of the receptor and residues involved in the binding site would greatly facilitate rational SAR and the design of more precise pharmacophore models for binding and functional activity at oL4132 nAChRs.

References Abreo, M.A., Lin, N.-H., Garvey, D.S., Gunn, D.E., Hettinger, A.-M., Wasicak, J.T., Pavlik, P.A., Martin, Y.C., Donnelly-Roberts, D.L., Anderson, D.J., Sullivan, J.P., Williams, M., Arneric, S.P., Holladay, M.W., 1996. Novel 3-pyridyl ethers with subnanomolar affinity for central neuronal nicotinic acetylcholine receptors. J. Med. Chem. 39, 817-825. Alkondon, M., Pereira, E.F.R., Eisenberg, H.M., Albuquerque, E.X., 1999. Choline and selective antagonists identify two subtypes of nicotinic acetylcholine receptors that modulate GABA release from CA1 interneurons in rat hippocampal slices. J. Neurosci. 19, 26932705. Americ, S.P., Anderson, D.J., Bannon, A.W., Briggs, C.A., Buccafusco, J.J., Brioni, J.D., Cannon, J.B., Decker, M.W., Donnelly-Roberts, D.,

Gopalakrishnan, M., Holladay, M.W., Kyncl, J., March, K.C., Radek, R.J., Rodrigues, A.D., Sullivan, J.P., 1995. Preclinical pharmacology of ABT-418: A prototypical cholinergic channel activator for the potential treatment of Alzheimer's disease. CNS Drug Rev. 1, 1-26. Bannon, A.W., Decker, M.W., Holladay, M.W., Curzon, P., DonnellyRoberts, D., Puttfarcken, P.S., Bitner, R.S., Diaz, A., Dickenson, A.H., Porsolt, R.D., Williams, M., Arneric, S.P., 1998. Broad-spectrum, nonopioid analgesic activity by selective modulation of neuronal nicotinic acetylcholine receptors. Science (Washington, DC) 279, 77-81. Barlocco, D., Cignarella, G., Tondi, D., Vianello, P., Villa, S., Bartolini, A., Ghelardini, C., Galeotti, N., Anderson, D.J., Kuntzweiler, T.A., Colombo, D., Toma, L., 1998. Mono- and disubstituted-3,8-diazabicyclo[3.2.1]octane derivatives as analgesics structurally related to epibatidine: synthesis, activity and modeling. J. Med. Chem. 41, 674-681. Beers, W.H., Reich, E., 1970. Structure and activity of acetylcholine. Nature 228, 917-922. Bencherif, M., Schmitt, J.D., Bhatti, B.S., Crooks, P., Caldwell, W.S., Lovette, M.E., Fowler, K., Reeves, L., Lippiello, P.M., 1998. The heterocyclic substituted pyridine derivative (+_)-2-(-3-pyridinyl)-lazabicyclo[2.2.2]octane (RJR-2429): a selective ligand at nicotinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 284, 886-894. Bitner, R.S., Nikkel, A.L., Curzon, P., Arneric, S.P., Bannon, A.W., Decker, M.W., 1998. Role of the nucleus raphe magnus in antinociception produced by ABT-594: immediate early gene responses possibly linked to neuronal nicotinic acetylcholine receptors on serotonergic neurons. J. Neurosci. 18, 5426-5432. Brioni, J.D., Decker, M.W., Sullivan, J.P., Arneric, S.P., 1997. The pharmacology of (-)-nicotine and novel cholinergic channel modulators. Adv. Pharmacol. (San Diego) 37, 153-214. Campillo, N., Antonio Paez, J., Alkorta, I., Goya, P., 1998. A theoretical study of epibatidine, J. Chem. Soc., Perkin Trans. 2, 2665-2670. Czajkowski, C., Karlin, A., 1995. Structure of the nicotinic receptor acetylcholine-binding site. Identification of acidic residues in the subunit within 0.9 nm of the e~ subunit-binding site disulfide. J. Biol. Chem. 270, 3160-3164. Dougherty, D.A., 1996. Cation-7r interactions in chemistry and biology: a new view of benzene, Phe, Tyr and Trp. Science (Washington, DC) 271, 163-168. Dukat, M., Damaj, M.I., Glassco, W., Dumas, D., May, E.L., Martin, B.R., Glennon, R.A., 1994. Epibatidine: a very high affinity nicotinereceptor ligand. Med. Chem. Res. 4, 131-139. Dukat, M., Dowd, M., Damaj, M.I., Martin, B., E1-Zahabi, M.A., Glennon, R.A., 1999. Synthesis, receptor binding and QSAR studies on 6-substituted nicotine derivatives as cholinergic ligands. Eur. J. Med. Chem. 34, 31-40. Edelstein, S.J., Changeux, J.-P., 1998. Allosteric transitions of the acetylcholine receptor. Adv. Protein Chem. 51, 121-184. Elliott, R.E., Kopecka, H., Gunn, D.E., Lin, N.-H., Garvey, D.S., Ryther, K.B., Holladay, M.W., Anderson, D.J., Campbell, J.E., Sullivan, J.P., Buckley, M.J., Gunther, K.L., O'Neill, A.B., Decker, M.W., Arneric, S.P., 1996. 2-(Aryloxymethyl) azacyclic analogues as novel nicotinic acetylcholine receptor (nAChR) ligands. Bioorg. Med. Chem. Lett. 6, 2283-2288. Elliott, R.L., Ryther, K.B., Anderson, D.J., Piattoni-Kaplan, M., Kuntzweiler, T.A., Donnelly-Roberts, D., Americ, S.P., Holladay, M.W., 1997. Novel 2-(2'-furo[3,2-b]-pyridinyl) pyrrolidines: potent neuronal nicotinic acetylcholine receptor ligands. Bioorg. Med. Chem. Lett. 7, 2703-2708. Elliott, R.L., Ryther, K.B., Holladay, M.W., Wasciak, J.T., Daanen, J.F., Lin, N.-H., Dart, M.J., He, Y., Li, Y., 1998. Furopyridine, thienopyridine, pyrrolopyridine and related pyrimidine, pyridazine and triazine compounds useful in controlling chemical synaptic transmission. WO 9846609. Garvey, D.S., Wasicak, J.T., Elliott, R.L., Lebold, S.A., Hettinger, A.-M., Carrera, G.M., Lin, N.-H., He, Y., Holladay, M.W., Anderson, D.J.,

M.J. Dart et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 115-123

Cadman, E.D., Raszkiewicz, J.L., Sullivan, J.P., Arneric, S.P., 1994. Ligands for brain cholinergic channel receptors: synthesis and in vitro characterization of novel isoxazoles and isothiazoles as bioisosteric replacements for the pyridine ring in nicotine. J. Med. Chem. 37, 4455-4463. Glennon, R.A., Dukat, M., 1996. Nicotine receptor ligands. Med. Chem. Res. 6, 465-486. Glennon, R.A., Dukat, M., 1999. Nicotinic cholinergic receptor pharmacophores. In: Arneric, S.P., Brioni, J.D. (Eds.), Neuronal Nicotinic Receptors. Wiley-Liss, New York, NY, 271-284. Glennon, R.A., Herndon, J.L., Dukat, M., 1994. Epibatidine-aided studies toward definition of a nicotine receptor pharmacophore. Med. Chem. Res. 4, 461-473. Grady, S.R., Grun, E.U., Marks, M.J., Collins, A.C., 1997. Pharmacological comparison of transient and persistent [3H]dopamine release from mouse striatal synaptosomes and response to chronic L-nicotine treatment. J. Pharmacol. Exp. Ther. 282, 32-43. Harel, M., Schalk, I., Ehret-Sabatier, L., Bouet, F., Goeldner, M., Hirth, C., Axelsen, P.H., Silman, I., Sussman, J.L., 1993. Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase. Proc. Natl. Acad. Sci. U.S.A. 90, 9031-9035. Hodgson, D.M., Maxwell, C.R., Matthews, I.R., 1998. An epoxide rearrangement. Radical rearrangement approach to 6-substituted 2azabicyclo[2.2.1 ]-5-heptenes. Synthesis of an epibatidine analog. Synlett., 1349-1350. Holladay, M.W., Wasicak, J.T., Lin, N.-H., He, Y., Ryther, K.B., Bannon, A.W., Buckley, M.J., Kim, D.J.B., Decker, M.W., Anderson, D.J., Campbell, J.E., Kuntzweiler, T.A., Donnelly-Roberts, D.L., Piattoni-Kaplan, M., Briggs, C.A., Williams, M., Arneric, S.P., 1998. Identification and structure-activity relationships of (R)-5-(2azetidinylmethoxy)-2-chloropyridine (ABT-594), a potent, orally active, non-opiate analgesic agent acting via neuronal nicotinic acetylcholine receptors. J. Med. Chem. 41,407-412. Kanne, D.B., Abood, L.G., 1988. Synthesis and biological characterization of pyridohomotropanes. Structure-activity relationships of conformationally restricted nicotinoids. J. Med. Chem. 31,506-509. Karlin, A., 1993. Structure of nicotinic acetylcholine receptors. Curr. Opin. Neurobiol. 3, 299-309. Kasyan, A., Wagner, C., Maier, M.E., 1998. Regiochemistry of the reductive Heck coupling of 2-azabicyclo[2.2.1]hept-5-ene. Synthesis of epibatidine analogs. Tetrahedron 54, 8047-8054. Koren, A.O., Horti, A.G., Mukhin, A.G., Guendisch, D., Kimes, A.S., Dannals, R.F., London, E.D., 1998. 2-, 5-, and 6-Halo-3-(2(S)azetidinylmethoxy)pyridines: synthesis, affinity for nicotinic acetylcholine receptors and molecular modeling. J. Med. Chem. 41, 36903698. Lena, C., Changeaux, J.-P., 1993. Allosteric modulations of the nicotinic acetylcholine receptor. Trends Neurosci. 16, 181-186. Lena, C., Changeax, J.-P., 1999. The role of [32-subunit-containing nicotinic acetylcholine receptors in the brain explored with a mutant mouse. Ann. NY Acad. Sci. 868, 611-616. Lin, N.-H., Gunn, D.E., Li, Y., He, Y., Bai, H., Ryther, K.B., Kuntzweiler, T., Donnelly-Roberts, D.L., Anderson, D.J., Campbell, J.E., Sullivan, J.P., Arneric, S.P., Holladay, M.W., 1998. Synthesis and structure-activity relationships of pyridine-modified analogs of 3-[2-((S)-pyrrolidinyl)methoxy]pyridine, A-84543, a potent nicotinic acetylcholine receptor agonist. Bioorg. Med. Chem. Lett. 8, 249-254. Lippiello, P., Fernandez, K., 1986. The binding of L-[3H]-nicotine to a single class of high affinity sites in rat brain membranes. Mol. Pharmacol. 29, 448-454. Lippiello, P.M., Sears, S.B., Fernandes, K.G., 1987. Kinetics and mechanism of L-[3H]nicotine binding to putative high affinity receptor sites in rat brain. Mol. Pharmacol. 31,392-400. Lu, Y., Grady, S., Marks, M.J., Picciotto, M., Changeux, J.-P., Collins, A.C., 1998. Pharmacological characterization of nicotinic receptorstimulated GABA release from mouse brain synaptosomes. J. Pharmacol. Exp. Ther. 287, 648-657.

123

Malpass, J.R., Cox, C.D., 1999. Synthesis of 5- and 6-chloropyridyl-substituted 2-azabicyclo[2.2.1 ]heptanes; novel epibatidine isomers. Tetrahedron Lett. 40, 1419-1422. Marubio, L.M., del Mar Arroyo-Jimenez, M., Cordero-Erausquin, M., Lena, C., Le Novere, N., de Kerchove d'Exaerde, A., Huchet, M., Damaj, M.I., Changeux, J.-P., 1999. Reduced antinociception in mice lacking neuronal nicotinic receptor subunits. Nature (London) 398, 305-810. Nordberg, A., 1994. Human nicotinic receptors - - their role in aging and dementia. Neurochem. Int. 25, 93-97. Pabreza, L.A., Dhawan, S., Kellar, K.J., 1990. [3H]Cytisine binding to nicotinic cholinergic receptors in brain. Mol. Pharmacol. 39, 9-12. Perry, E.K., Morris, C.M., Court, J.A., Cheng, A., Fairbairn, A.F., McKeith, I.G., Irving, D., Brown, A., Perry, R.H., 1995. Alteration in nicotine binding sites in Parkinson's disease, lewy body dementia and alzheimer's disease: possible index of early neuropathology. Neuroscience 64, 385-395. Picciotto, M.R., Zoli, M., Rimondini, R., Lena, C., Marubio, L.M., Pich, E.M., Fuxe, K., Changeux, J.-P., 1998. Acetylcholine receptors containing the [32 subunit are involved in the reinforcing properties of nicotine. Nature (London) 391, 173-177. Picciotto, M.R., Zoll, M., Lena, C., Bessis, A., Lallemand, Y., LeNovere, N., Vincent, P., Pich, E.M., Brulet, P., Changeux, J.-P., 1995. Abnormal avoidance leaning in mice lacking functional high-affinity nicotine receptor in the brain. Nature (London) 374, 65-67. Schmitt, J.D., Sharpies, C.G.V., Caldwell, W.S., 1998. Aromatic-cation interactions in nicotinic acetylcholine receptor ligand binding. In Book of Abstracts, 216th ACS National Meeting, Boston, American Chemical Society, Washington, DC, MEDI-153. Sheridan, R.P., Nilakantan, R., Dixon, J.S., Venkataraghavan, R., 1986. The ensemble approach to distance geometry: application to the nicotinic pharmacophore. J. Med. Chem. 29, 899-906. Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., Silman, I., 1991. Atomic structure of acetylcholinesterase from torpedo california: a prototypic acetylcholine-binding protein. Science 253, 872-879. Tani, Y., Saito, K., Imoto, M., Ohno, T., 1998. Pharmacological characterization of nicotinic receptor-mediated acetylcholine release in rat brain an in vivo microdialysis study. Eur. J. Pharmacol. 351, 181-188. Wasicak, J.T., Garvey, D.S., Holladay, M.W., Lin, N.-H., Ryther, K.B., 1998. Preparation of 7a-heteroarylhexahydro-lH-pyrrolizines as cholinergic synaptic transmission modulators. US 5733912. Williams, M., Arneric, S., 1996. Beyond the tobacco debate: dissecting out the therapeutic potential of nicotine. Expert Opin. Invest. Drugs 5, 1035-1045. Wonnacott, S., Soliakov, L., Wilkie, G., Redfern, P., Marshall, D., 1996. Presynaptic nicotinic acetycholine receptors in the brain. Drug Dev. Res. 38, 149-159. Wypij, D.M. and Shen, T.Y. (1994) Epibatidine and related analogs 3 compete with [ H]-cytisine with high affinity for binding to rat brain cortical membrane preparations. In: Clarke, P.B.S., Quik, M., Thurau, K., Adlkofer, F., (Eds.), International Symposium on Nicotine: The Effects of Nicotine on Biological Systems II Montreal, P32. Zhang, C., Trudell, M.L., 1996. A short and efficient total synthesis of ( _+)-epibatidine. J. Org. Chem. 61, 7189-7191. Zhong, W., Gallivan, J.P., Zhang, Y., Li, L., Lester, H.A., Dougherty, D.A., 1998. From ab initio quantum mechanics to molecular neurobiology: a cation-~r binding site in the nicotinic receptor. Proc. Natl. Acad. Sci. U.S.A. 95, 12088-12093. Zoli, M., Lena, C., Picciotto, M.R., Changeux, J.-P., 1998. Identification of four classes of brain nicotinic receptors using [32 mutant mice. J. Neurosci. 18, 4461-4472. Zoli, M., Picciotto, M.R., Ferrari, R., Cocchi, D., Changeux, J.-P., 1999. Increased neurodegeneration during ageing in mice lacking high-affinity nicotine receptors. EMBO J. 18, 1235-1244.




Recombinant human receptors and functional assays in the discovery of altinicline (SIB-1508Y), a novel acetylcholine-gated ion channel (nAChR) agonist Nicholas D.P. Cosford *, Leo B leicher, Jean-Michel Vernier, Laura Chavez-Noriega, Tadimeti S. Rao, Robert S. Siegel, Carla Suto, Mark Washburn, G. Kenneth Lloyd, Ian A. McDonald Merck Research Laboratories San Diego, 505 Coast Blvd. So., La Jolla, CA 92037, USA

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are a class of ion channels with significant potential as molecular targets for the design of drugs to treat a variety of CNS disorders. The discovery that neuronal nAChRs are further subdivided into multiple subtypes suggests that drugs which act selectively at specific nAChR subtypes might effectively treat Parkinson's disease (PD), Alzheimer's disease (AD), schizophrenia, ADHD, depression, anxiety or pain without the accompanying adverse side effects associated with non-selective agents such as nicotine (1) and epibatidine. Altinicline (SIB-1508Y) is a novel, small molecule designed to selectively activate neuronal nAChRs and is undergoing clinical evaluation for the treatment of PD. It was selected from a series of compounds primarily on the basis of results from functional assays, including (a) measurement of Ca 2+ flux in stable cell lines expressing specific recombinant human neuronal nAChR subtypes; (b) determination of in vitro and in vivo neurotransmitter release; (c) in vivo models of PD. Biological data on both altinicline and the series of compounds from which it was selected are reported. 9 2000 Elsevier Science B.V. All rights reserved. Keyworcls: Recombinant human receptors; Functional assays; Altinicline; Nicotine; Parkinson's disease; nAChR subtypes; Nicotinic acetylcholine receptors; Nicotine; Ca2+ flux; SIB-1765F; Cognitive deficits

1. Introduction

The last few years have witnessed an explosion of interest in the modulation of neuronal nicotinic acetylcholine receptors (nAChRs) as a therapeutic point of intervention for the treatment of several disease states (McDonald et al., 1995, 1996; Lloyd et al., 1998; Holladay et al., 1999). This interest has been stimulated by the demonstration that nicotine and other nAChR agonists exhibit efficacy in animal, and in some cases human, models of Parkinson's disease (PD; Newhouse et al., 1996; Maggio et al., 1998; Schneider et al., 1999), Alzheimer's disease (AD; Buccafusco et al., 1996; Newhouse et al., 1996) and pain (Rao et al., 1996; Lawand et al., 1999). Evidence that a specific nAChR subtype could be linked to a specific disease state, such as o~7 and schizophrenia (Freedman et al., 1997), has further strengthened the case for drug discovery programs focused on selective agonists and antagonists of nAChR. Furthermore, the identification of * Corresponding author.

potent and, in some cases, selective nAChR agonists from natural sources, e.g., anatoxin-a (Thomas et al., 1993), epibatidine (Spande et al., 1992) and from SAR-based medicinal chemistry research, e.g., ABT-418 (Potter et al., 1999), ABT-089 (Arneric et al., 1997), ABT-594 (Holladay et al., 1998), RJR-2403 (Bencherif et al., 1996), RJR-2429 (Bencherif et al., 1998) and GTS-21 (De Fiebre et al., 1995) suggests that the design of drugs which exert their actions through nAChR is a realistic goal. Biochemical and molecular studies have shown that neuronal nAChR are composed of two types of subunit which combine to form the pentameric ion channel (Changeux, 1993; Sargent, 1993). The ligand-binding ot subunit contains vicinal cysteine residues in the N-terminal region. The non-ligand binding 13 subunit, when present in the assembled receptor, may be considered to be a modulating subunit. The stoichiometry is believed to be two oL and three [3 subunits that are arranged, barrel-like, to form a pore in the cell membrane. Activation of the receptor by an agonist leads to opening of the pore which allows the passage of positively charged ions such as Na + and Ca 2+.


126

N.D.P. Cosford et al. / Pharmaceutica Acta Helvetiae 74 (2000) 125-130

To date, nAChR genes encoding eight a subunits (or 2-or 9) and three [3 subunits (132-[34) have been isolated. These subunits may coassemble in a theoretically large array of combinations to form a multitude of potential nAChRs. For the most part, the exact composition of native neuronal nAChRs is unknown. The exception to this are the putative homomeric nAChR subtypes, such as oL7, that are formed by the coassembly of five oL7 subunits and which are labelled in rat brain by the selective antagonists oLbungarotoxin (Alkondon et al., 1998; Dajas-Bailador et al., 1998) and methyllycaconitine (Alkondon et al., 1998; Davies et al., 1999). When research aimed at the discovery of subtype-selective nAChR agonists was initiated in this laboratory there was very limited information in the literature on the biological activity of nicotine derivatives. Of the analogues that had been reported there was little to indicate that changes to the basic structure of nicotine would lead to compounds with improved therapeutic properties (Lin et al., 1994). The use of nicotine as a drug is severely limited for a number of reasons, including toxicity (due to cardiovascular and respiratory side effects and, at higher doses, convulsions followed by death), rapid metabolism (short half-life) and limited gastrointestinal absorption. The side effects of nicotine appear to be mediated through its action on peripheral (neuromuscular junction) nAChRs as well as ganglionic and central nAChRs (Lee and Hou, 1996). The aim of our research, therefore, was to discover compounds that retained the therapeutically beneficial effects of nicotine but that also possess attractive drug-like properties such as tolerability, good oral bioavailability and extended duration of action. In order to accomplish this goal, we focused on the design of novel analogues of nicotine which were evaluated in a number of biological assays, including functional assays that measure nAChR subtype selectivity.

2. D i s c u s s i o n

At the outset, two broad strategies were pursued to achieve the goal of identifying safe, selective nAChR modulating compounds. The first approach was to prepare conformationally restricted analogues of nicotine, such as SIB-1663 (2), with the idea that locking the molecule into a specific conformation would favour the activation of a subset of nAChR subtypes, and thus eradicate, or at least reduce, unwanted side effects (Vernier et al., 1998). The second approach was to prepare nicotine analogues possessing free rotation about the pyridine-pyrrolidine ring bond, allowing the compounds to access numerous possible conformations, and then examine the effect on biological activity (including subtype selectivity) of various substituents in the molecule (Cosford et al., 1996). The starting point for this research was (R/S)-5-bromonicotine (3), a compound that had been reported in the literature some years previously (Rondahl, 1977). In this study, a

series of 5-halogenated nicotine derivatives was prepared and tested on mammalian tissue preparations (contraction of guinea pig vas deferens). Of the compounds evaluated, it was found that 3 had a significantly reduced contractile response compared with nicotine, indicating the likelihood of reduced peripheral side effects. We postulated that the differential activity observed in the earlier study was an indication of an altered nAChR subtype selectivity of 3 compared with nicotine. In order to test this hypothesis, a series of analogues of 3 was prepared and evaluated in various biological assays. N ~N~

CH3

1 (nicotine)

H

~

~O 2 (SIB-1663)

H

HN 3

(S)-6 (SlB-1508Y)

R

H,

N

R=Br, 3 R = Et, 4 R = CHCH2, 5 R = CCH, 6 (SIB-1765F) R=CN, 7

: Ni H (R)~

Two primary assays were utilized to evaluate analogues. The first was a binding assay that tested the ability of compounds to displace [3H]nicotine from a rat cortical membrane preparation (Flynn and Mash, 1986). The second was a functional assay that measured changes in intracellular Ca 2§ ([Ca 2§ ]i), in cells recombinantly expressing specific human nAChR subtypes, using fluorescence detection. This cell-based functional assay has been incorporated into an automated high-throughput screening system that employs the Ca2+-sensitive dye, fura 2 (Velicelebi et al., 1998). This type of assay has distinct advantages over traditional binding assays, in which the test compound displaces a radiolabelled ligand from the high affinity binding site, present when the receptor is in a deactivated, or desensitized state. In the Ca 2+ flux assay the receptors are in a fully functional resting state prior to being challenged with a test compound. Thus, the assay is highly sensitive and versatile, and will identify compounds with differing mechanisms of action; i.e., competitive, non-competitive or uncompetitive agonists or antagonists, potentiators or allosteric modulators. Compounds identified with activity in the primary assays were tested in a superfused rat striatal slice preparation, a functional assay that measures the ability of test compounds to stimulate dopamine (DA) release in vitro (Sacaan et al., 1995). Activity in this assay led to evaluation of compounds in a battery of in vivo models. 3. R e s u l t s m in vitro

The biological data from the primary assays are presented in Table 1. In the series of racemic 5-substituted

N.D.P. Cosford et al./ Pharmaceutica Acta Heluetiae 74 (2000) 125-130

127

Table 1 All data represent the mean values from at least two independent experiments performed in duplicate (SEM values omitted for clarity) Compound

IC50 (nM)[ 3H]nica

1 3 4 5 6 7 (S)-6 ( R)-6

4 19 11 nd 4.6 nd 3 75

Release [ 3H]DAc

EC50 (IxM) ot 2[34 b

o~3[34b

o~4134b

o~4[3 2 b

o~3[32 b

1.58 4 4 6.3 4 12.6 3.16 4

7.94 inactive inactive inactive 15.8 31.6 10 inacti ve

0.8 12.6 3.16 5 3.16 31.6 2 inactive

2 31.6 inactive 31.6 0.2 19.9 0.316 12.5

6.3 3.98 7.94 12.6 5 31.6 3.16 31.6

100 69 27.5 nd 143 82 163 55

aBinding affinities (nM) at nAChRs were determined by measuring the displacement of [3H]nicotine ([3H]nic) from a preparation of rat cortical membranes (Flynn and Mash, 1986). bpotency data (IxM) derived from measured changes in [Ca 2+ ]i in HEK 293 cells expressing the designated human recombinant nAChR subtypes in response to the test compounds. CThe efficacy of the compounds (300 txM) to stimulate [3H]DA release was determined using a preparation of superfused rat striatal slices as described by Sacaan et al. (1995). The data are expressed as a percentage of the [3H]DA release elicited by a maximally efficacious dose of nicotine (10 txM).

nicotine derivatives the substituent at the 5-position of the pyridyl ring is changed from H to Br, Et, vinyl, ethynyl and CN, substituents that project into the same region of space but with differing steric attributes and electronic characteristics. It is clear from these data that, while the analogues exhibit low nanomolar potency in the binding assay, there is no obvious correlation between binding potency and the potency of compounds to activate nAChR subtypes in the Ca 2+ flux assay, or between binding potency and striatal DA release efficacy. However, examination of the Ca 2+ flux assay data reveals that the compound that is most potent at the oL4132 subtype, 6 (SIB1765F), is the most efficacious at stimulating in vitro DA release. Furthermore, synthesis and biological testing of the individual enantiomers of racemic 6, (S)-6 (altinicline, SIB-1508Y) and (R)-6, demonstrated that the (S)-enantiomer, which has the same configuration as (-)-nicotine, possesses essentially all of the biological activity present in 6. This is graphically illustrated by the concentrationresponse curves for 6, (S)-6 and (R)-6 in the striatal slice assay (Fig. 1). In support of the above observations, recent data from the literature indicates a possible connection between [32-

containing nAChR subtypes and DA release. Thus, it was shown that nicotine stimulates DA release in the ventral striatum of wild-type mice, but not in [32-knockout mice, as measured in live animals by in vivo microdialysis (Picciotto et al., 1998). In another study, it was found that the ot 3132-selective antagonist ot-conotoxin MII blocked 34%-49% of nicotine-stimulated DA release in rat striatal synaptosomes (Kulak et al., 1997). The latter result indicates that one third to one half of the DA release elicited by nicotine in rodent striatum is mediated by ot3132-containing nAChRs. We were intrigued by the possible relevance of these findings to our own compounds, and focusing on the data for the [32-containing subtypes, the Ca 2+ flux assay data (Table 1) indicate that both nicotine and (S)-6 are more potent at e~4132 than at ot3132 (3- and 10-fold, respectively). The concentration-response curves (Figs. 2 and 3) illustrate the preferential activation of oL4132 compared to ot3132 graphically. Thus, at lower

Nicotine 100-

,4: z~

"

O

~2 o ~2

U! 6

a (s)-e v (R)-e

X

o~

m ~,t

9"

r

0,

IL

-9

0. -6

-~ Log [Agonist] (M)

Fig. 1. Concentration-response curves for 6, (S)-6 and (R)-6 in the superfused rat striatal slice assay measuring the ability of test compounds to stimulate [3H]DA release.

Fig. 2. Concentration-response curve for 1 and (S)-6 in the Ca 2+ flux assay. Data are derived from measured changes in [Ca 2+ ]i in HEK 293 cells expressing the designated human recombinant nAChR subtypes in response to the test compounds. Each data point represents the mean value from at least two independent experiments performed in duplicate.

128


(s)~

(s).s .. 500t._

.t. t

0

o

(z3~2 o (z4~2

.~ 25-

--o

75-

(x3p2 v (x3J32(x5

I= O

(3 ,~

50-

o (z4132

m o

0

D.

O,. 25o

0

0.

-8

o7'

-9

Concentration (log M)

concentrations, e.g., 300 nM (log -6.5), the oL4132 subtype is selectively activated by nicotine and by (S)-6, while at this concentration neither compound acts as an agonist at c~3132. At higher concentrations nicotine and (S)-6 become increasingly efficacious at the c~3132 subtype compared with the c~4132 subtype. Interestingly, when nicotine and (S)-6 were profiled against a three way subunit combination, the oL3132et 5 subtype, in the Ca 2§ flux assay it was found that (S)-6 was more potent and efficacious at this nAChR subtype than at either c~4132 or oL3[32 (Figs. 4 and 5). In order to further characterize nAChR subtype selectivity, (S)-6 was tested in an electrophysiological assay. Voltage clamp recordings in Xenopus oocytes expressing human oL3132 and a4132 nAChR subtypes were obtained. The inward current elicited by (S)-6 was normalized to the ECs0 response for acetylcholine (ACh) in oocytes. At a concentration of 10 IxM, (S)-6 produced a response that was 33% of the EC80 for ACh in oocytes expressing the oL4132 nAChR subtype, while at the same concentration

Nicotine

zx ~t3~2

/~/~"~

0 ~_~_.~'~--.~ --~'''= -9 -8 -7 -6

~auon

-5

.~

.'6

.'5

.'4

Concentration (log

Fig. 3. Concentration-response curve for 1 and (S)-6 in the Ca 2+ flux assay. Data are derived from measured changes in [Ca 2§ ]i in HEK 293 cells expressing the designated human recombinant nAChR subtypes in response to the test compounds. Each data point represents the mean value from at least two independent experiments performed in duplicate.

lO0.

.'e

-4

(k~ M

Fig. 4. Concentration-response curve for 1 and (S)-6 in the Ca 2§ flux assay. Data are derived from measured changes in [Ca 2§ ]i in HEK 293 cells expressing the designated human recombinant nAChR subtypes in response to the test compounds. Each data point represents the mean value from at least two independent experiments performed in duplicate.

Fig, 5. Concentration-response curve for 1 and (S)-6 in the Ca 2+ flux assay. Data are derived from measured changes in [Ca 2§ ]i in HEK 293 cells expressing the designated human recombinant nAChR subtypes in response to the test compounds. Each data point represents the mean value from at least two independent experiments performed in duplicate.

(S)-6 elicited a response that was 7% of the EC80 for ACh in oocytes expressing the oL3132 nAChR subtype. No detectable inward currents were observed for 6 at the human ct7 receptor subtype (data not shown), which contrasts with nicotine which is a weak agonist at the oL7 receptor (ECs0 = 113 txM; Chavez-Noriega et al., 1997). These data support the conclusion that 6 and (S)-6 are most active at the et4132 nAChR subtype. Thus, the Ca 2+ flux assay, striatal slice assay and electrophysiology data considered together are consistent with the hypothesis that nAChR agonist-stimulated DA release is mediated through [32-containing nAChR subtypes, and that it is through this mechanism that (S)-6 stimulates DA release in vivo.

4. R e s u l t s m in vivo

Since 6 and (S)-6 had proven to be active in a number of in vitro assays, the compounds were further characterized in vivo. A summary of the results from in vivo experiments is presented in Table 2. A number of different nAChR agonists have been shown to either suppress or enhance the locomotor response in rats following subcutaneous administration (Menzaghi et al., 1999, 1997). The locomotor activity of nicotine (1, 6 and (S)-6 was measured in rats habituated to the test cages at a range of doses. It was found that 6 and (S)-6 were much more efficacious than 1 at producing a motor stimulant response (Table 2) and demonstrated a longer duration of action (Menzaghi et al., 1997). Furthermore, 6 and (S)-6 were more efficacious than 1 over a broader range of doses, i.e., 10-75 m g / k g (6), 3-23 m g / k g [(S)-6] compared with 0.2-0.6 m g / k g for 1, and were generally better tolerated. These data suggest that 6 and (S)-6 are nAChR agonists with longer lasting CNS stimulant effects and fewer adverse side-effects compared with nicotine.

129


Table 2 In vivo assay (species)

Compound

Dose (mg/kg), route of administration

Results

1 6 (S)-6 1

0.4, s.c. 40, s.c. 12, s.c.

140 crossovers/120 raina 620 crossovers/120 min 700 crossovers/120 rain 1O0c

6 L-dopa

25, s.c. 10, i.m.

263 c 10% at 20 min; 4% at 60 mind

hippocampus

L-dopa (S)-6 and L-dopa 1

25, i.m. 1 and 10, i.m. 0.4, s.c.

58% at 20 min; 68% at 60 min 46% at 20 min; 41% at 60 min 2-folde

hippocampus frontal cortex frontal cortex

(S)-6 1 (S)-6

12.5, s.c. 0.4, s.c. 12.5, s.c.

3-4-fold 2-3-fold 6-7-fold

Brain region

Locomotor activity (rat) m

Turning model (unilateral 6-OHDA lesioned rats)

substantia nigra, striatum

Parkinsonian behavioral rating (MPTP monkeys)

substantia nigra, striatum, VTA etc.

ACh release in rat (in vivo microdialysis)

0.4, s.c. b

aData represent total crossovers (photocell interruptions) over 120 min immediately following drug administration at given dose (Menzaghi et al., 1997; Menzaghi et al., 1999). bMaximally effective dose (intolerable side effects were observed for 1 at 1 mg/kg). CIpsilateral rotations measured over a period of 120 min, expressed as a percentage of the value for 1 (Cosford et al., 1996). dData are presented as percent of motor improvement over pre-drug baseline at the indicated time points (Menzaghi et al., 1999). eData are presented as increase in levels of ACh over baseline (Menzaghi et al., 1999).

The efficacy of 6 and (S)-6 at stimulating DA release from rat striatal slices suggested potential as a therapy for patients in which dopaminergic transmission is compromised. C o m p o u n d 6 was therefore evaluated in a specific animal model of PD. The effect of 6 on ipsilateral rotations of rats with unilateral nigrostriatal 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) lesions (rat turning model; Ungerstedt and Arbuthnott, 1970) is shown in Table 2. At a dose of 25 m g / k g s.c., 6 produced a significant increase in ipsilateral rotations compared with saline treated animals, an effect which is blocked by the non-selective n A C h R antagonist mecamylamine. As with the locomotor activity, 6 was more efficacious than nicotine at the m a x i m u m effective dose with minimal adverse side effects. The data from rodent models prompted the evaluation of (S)-6 in a primate model of PD. The symptoms of MPTP-treated monkeys, which produces Parkinson-like motor dysfunction, were assessed using a m o t o r / b e h a v ioral rating scale (Menzaghi et al., 1999). The animals were then given different doses of L-dopa either alone or in combination with (S)-6 (1 m g / k g ) and the behavioral ratings reassessed at 20 and 60 min post-injection (Table 2). This led to the critical observation that (S)-6 coadministered with subtherapeutic doses of L-dopa (e.g., 10 m g / k g ) produced an i m p r o v e m e n t in behavioral rating similar to that of much higher doses of L-dopa. These results indicate a therapeutically relevant synergistic effect between (S)-6 and L-dopa. The ability of (S)-6 to stimulate ACh release was measured using in vivo microdialysis and the results, presented in Table 2, were compared with nicotine (1). It was found that (S)-6 was more efficacious than 1 at stimulating ACh release in both brain regions examined

(hippocampus, frontal cortex). These results suggest that (S)-6 has the potential to ameliorate the cognitive deficits of patients with PD, as well as improving motor function.

5. Conclusions C o m p o u n d (S)-6 (altinicline, SIB-1508Y) is a selective n A C h R agonist that was discovered by employing a medicinal chemistry approach based on modifying the structure of nicotine. Using a variety of biological assays, including functional assays using human recombinant n A C h R subtypes, novel analogues were evaluated and 6 was identified as a c o m p o u n d with an attractive pharmacological profile. Optical resolution of the enantiomers of 6 and further biological studies led to the pre-clinical, and ultimately clinical development of (S)-6 for the treatment of PD.

References Alkondon, M., Pereira, E.F.R., Albuquerque, E.X., 1998. Brain Res. 810, 257-263. Arneric, S.P., Campbell, J.E., Carroll, S., Daanen, J.F., Holladay, M.W., Johnson, P., Lin, N.-H., Marsh, K.C., Peterson, B., Qui, W., Roberts, E.M., Rodrigues, A.D., Sullivan, J.P., Trivedi, J., Williams, M., 1997. Drug Dev. Res. 41, 31-43. Bencherif, M., Lovette, M.E., Fowler, K.W., Arrington, S., Reeves, L., Caldwell, W.S., Lippiello, P.M., 1996. J. Pharmacol. Exp. Ther. 279, 1413-1421. Bencherif, M., Schmitt, J.D., Bhatti, B.S., Crooks, P., Caldwell, W.E., Lovette, M.E., Fowler, K., Reeves, L., Lippiello, P.M., 1998. J. Pharmacol. Exp. Ther. 284, 886-894.

130

N.D.P. Cosford et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 125-130

Buccafusco, J.J., Prendergast, M.A., Terry, A.V. Jr., Jackson, W.J., 1996. Drug Dev. Res. 38, 196-203. Changeux, J.-P., 1993. Sci. Am. 269, 58-62. Chavez-Noriega, L.E., Crona, J.H., Washburn, M.W., Urrutia, A., Elliott, K.E., Johnson, E.C., 1997. J. Pharmacol. Exp. Ther. 280, 346-356. Cosford, N.D.P., Bleicher, L., Herbaut, A., McCallum, J.S., Vernier, J.-M., Dawson, H., Whitten, J.P., Adams, P., Chavez-Noriega, L., Correa, L.D., Crona, J.H., Mahaffy, L.S., Menzaghi, F., Rao, T.S., Reid, R., Sacaan, A.I., Santori, E., Stauderman, K.A., Whelan, K., Lloyd, G.K., McDonald, I.A., 1996. J. Med. Chem. 39, 3235-3237. Dajas-Bailador, F., Costa, G., Dajas, F., Emmett, S., 1998. Neurochem. Int. 33, 307-312. Davies, A.R.L., Hardick, D.J., Blagbrough, I.S., Potter, B.V.L., Wolstenholme, A.J., Wonnacott, S., 1999. Neuropharmacology 38, 679-690. De Fiebre, C.M., Meyer, E.M., Henry, J.C., Muraskin, S.I., Kem, W.R., Papke, R.L., 1995. Mol. Pharmacol. 47, 164-171. Flynn, D.D., Mash, D.C., 1986. J. Neurochem. 47, 1948-1954. Freedman, R., Coon, H., Myles-Worsley, M., Orr-Urtreger, A., Olincy, A., Davis, A., Polymeropoulos, M., Holik, J., Hopkins, J., Hoff, M., Rosenthal, J., Waldo, M.C., Reimherr, F., Wender, P., Yaw, J., Young, D.A., Breese, C.R., Adams, C., Patterson, D., Adler, L.E., Kruglyak, L., Leonard, S., Byerley, W., 1997. Proc. Natl. Acad. Sci. U. S. A. 94, 587-592. Holladay, M.W., Wasicak, J.T., Lin, N.-H., He, Y., Ryther, K.B., Bannon, A.W., Buckley, M.J., Kim, D.J.B., Decker, M.W., Anderson, D.J., Cambell, J.E., Kuntzweiler, T.A., Donnely-Roberts, D.L., Piottoni-Kaplan, M., Briggs, C.A., Williams, M., Arneric, S.P., 1998. J. Med. Chem. 41,407-412. Holladay, M.W., Cosford, N.D.P., McDonald, I.A., 1999. In: Arneric., S.P., Brioni, J.D. (Eds.), Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities. Wiley-Liss, pp. 253-273. Kulak, J.M., Ngugen, T.A., Olivera, B.M., McIntosh, J.M., 1997. J. Neurosci. 17, 5263-5270. Lawand, N.B., Lu, Y., Westlund, K.N., 1999. Pain 80, 291-299. Lee, T.S., Hou, X.H., 1996. Chest 109, 584-585. Lin, N., Carrera, G.M. Jr., Anderson, D.J., 1994. J. Med. Chem. 37, 3542-3553. Lloyd, G.K., Menzaghi, F., Bontempi, B., Suto, C., Siegel, R., Akong, M., Stauderman, K., Velicelebi, G., Johnson, E., Harpold, M.M., Rao, T.S., Sacaan, A.I., Chavez-Noriega, L.E., Washburn, M.S., Vernier,

J.M., Cosford, N.D.P., McDonald, I.A., 1998. Life Sci. 62, 16011606. Maggio, R., Riva, M., Vaglini, F., Fornai, F., Molteni, R., Armogida, M., Racagni, G., Corsini, G.U., 1998. J. Neurochem. 71, 2439-2446. McDonald, I.A., Cosford, N.D.P., Vernier, J.-M., 1995. Annu. Rep. Med. Chem. 30, 41-50. McDonald, I.A., Vernier, J.-M., Cosford, N.D.P., Corey-Naeve, J., 1996. Curr. Pharm. Des. 2, 357-366. Menzaghi, F., Whelan, K.T., Risborough, V.T., Rao, T.S., Lloyd, G.K., 1997. J. Pharmacol. Exp. Ther. 280, 384-392. Menzaghi, F., McClure, D.E., Lloyd, G.K., 1999. In: Arneric, S.P., Brioni, J.D. (Eds.), Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities. Wiley-Liss, pp. 379-394. Newhouse, P., Potter, A., Corwin, J., 1996. Drug Dev. Res. 38, 278-289. Picciotto, M.R., Zoli, M., Rimondini, R., Lena, C., Marubio, M.L., Pich, E.M., Fuxe, K., Changeux, J.-P., 1998. Nature 391, 173-177. Potter, A., Corwin, J., Lang, J., Piasecki, M., Lenox, R., Newhouse, P.A., 1999. Psychopharmacology 142, 334-342. Rao, T.S., Correa, L.D., Reid, R.T., Lloyd, G.K., 1996. Neuropharmacology 35, 393-405. Rondahl, L., 1977. Acta Pharm. Suec. 14, 113-118. Sacaan, A.I., Dunlop, J.L., Lloyd, G.K., 1995. J. Pharmacol. Exp. Ther. 274, 224-230. Sargent, P.B., 1993. Annu. Rev. Neurosci. 16, 403-443. Schneider, J.S., Tinker, J.P., Van Velson, M., Menzaghi, F., Lloyd, G.K., 1999. J. Pharmacol. Exp. Ther. 290, 731-739. Spande, T.F., Garraffo, H.G., Edwards, M.W., Yeh, H.J.C., Pannell, L., Daly, J.W., 1992. J. Am. Chem. Soc. 114, 3475-3478. Thomas, P., Stephens, M., Wilkie, G., Amar, M., Lunt, G.G., Whiting, P., Gallagher, T., Pereira, E., Alkondon, M., Albuquerque, E.X., Wonnacott, S., 1993. J. Neurochem. 60, 2308-2311. Ungerstedt, V., Arbuthnott, G., 1970. Brain Res. 24, 485-493. Velicelebi, G., Stauderman, K.A., Varney, M.A., Akong, M., Hess, S.D., Johnson, E.C., 1998. In: Conn, P.M. (Ed.), Methods in Enzymology, Vol. 294C. Academic Press, San Diego, pp. 20-47. Vernier, J.M., Holsenback, H., Cosford, N.D.P., Whitten, J.P., Menzaghi, F., Reid, R., Rao, T.S., Sacaan, A.I., Lloyd, G.K., Suto, C.M., Chavez-Noriega, L.E., Washburn, M.S., Urrutia, A., McDonald, I.A., 1998. Bioorg. Med. Chem. Lett. 8, 2173-2178.



Receptors in neurodegenerative diseases, muscarinic cholinergic receptors Piero Angeli

*

Dipartimento di Scienze Chimiche, Uniuersit~t di Camerino, Via S. Agostino, 1, 62032 Camerino, Italy

Keywords: Muscarinic therapeutic agents; Muscarinic receptor subtype characterization; Selective muscarinic receptor antagonist

Muscarinic acetylcholine receptors are present in the mammalian central nervous system (CNS) and in the periphery. Their stimulation produces inhibitory responses such as bradycardia and vascular smooth muscle dilatation, and excitatory responses such as ganglionic depolarization and smooth muscle contraction. Muscarinic receptors of the CNS are involved in motor control, temperature and sleep regulation, and in processes including higher cognitive functions such as memory and learning. The latest picture of muscarinic receptors, based on modern pharmacology, molecular biology, and molecular modeling studies, shows five subtypes (M1-M 5) which are typical members of the superfamily of G protein coupled receptors (GPCRs), composed of seven oL-helically arranged transmembrane domains (TM I-VII), connected by three extracellular (o) and three intracellular (i) loops (Fig. 1). Mutagenesis studies improved our knowledge of the molecular mechanisms underlying the function of muscarinic receptors. The main indications suggest that: Different receptor subsites are involved in the binding of muscarinic receptor agonists and antagonists

Ligand binding appears to be initiated by ion-ion interaction between the positively charged amino head group present in virtually all muscarinic receptor ligands and a conserved Asp residue located in TM III

(Wess, 1993);

The agonist binding site is located in a narrow cleft defined bYoseveral TM domains (primarily TM I I I - V I I ) , about 15 A away from the extracellular surface

(Wess, 1993);

The receptor domains involved in the binding of muscarinic receptor antagonists are only poorly defined. Particularly, it remains unclear which muscarinic receptor subsites are recognized by the bulky hydrophobic

(Wess, 1993); o, The acetylcholine-receptor complex is characterized by an intricate network of hydrogen bond interactions, rather than by a few well-defined points of contact

"

0,

Extracellular

er (111 I~I '

o~

(Wess, 1993); 9

* Tel.: -t-0039-0737-40-2236;fax: -t-0039-0737-637345;e-mail: angeli@camserv,unicam.it 0031-6865/00/$ - see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S0031-6865(99)00025-4

. ~-

~

lntracellular

Fig. 1. Model of the seven transmembranedomains.

P. Angeli / Pharmaceutica Acta Helvetiae 74 (2000) 131-134

132

ring systems or sidechains present in virtually all potent muscarinic receptor antagonists (Wess, 1993); Different receptor domains are involved in conferring subtype selectivity on structurally different muscarinic receptor antagonists (Wess, 1993). The possibility of increasingly efficacious muscarinic therapeutic agents greatly depends on a sound knowledge of the functional role of the multiple muscarinic receptors expressed in the brain and in the periphery. In fact, many diseases including Alzheimer and Parkinson's, urinary incontinence, irritable bowel syndrome, and chronic obstruc-

CH2CH21~(C2Hs)3

F

~

OCH2CH2~(C2H5) 3

+ oc.2cH2CH2-

-NHCOOCH2C=CCH2N~CH3) 3 McN-A-343

C1/

OCH2CH2]~r(C2H5)3

tive pulmonary disease, await new potent and selective therapeutics which will take shape from the muscarinic agonists and antagonists synthesized by medicinal chemists. Medicinal chemists, in fact, have given an important contribution to muscarinic receptor subtype characterization. The historical evolution of muscarinic receptor classification starts in 1951 with gallamine (Riker and Wescoe, 1951), a cardioselective compound acting as a selective allosteric antagonist at M 2 receptors (Fig. 2). The first agonist which stimulated selectively sympathetic ganglionic muscarinic receptors was McN-A-343, a butynyl compound studied by Roszkowski (1961) (Fig. 2). Barlow et al. (1976) demonstrated that the pharmacological properties of ileal and atrial muscarinic receptors are different. In fact, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) (Fig. 2) proved to be almost 20-fold more selec-

Gallamine

p-fluorohexahydrosila-difenidol HC_COO__(F---~N-'CH3 4-DAMP

O ~ C.H2N(CH2CH2CH3)2 HN>_.~NCONHCH2CH2- - N ~

~._._ffN Pirenzepine

N

~'~

HO--Si~CH2CH2CH2 - N ~

r

FIN

.NCOCH 2-Nk___/)

CN

Hexahydrosila-difenidol

C.H2N(C2Hs)2

AF-DX116

CH3 CH3

o

~ N C O C. H HN

AF-DX384

' ~ ~ ' 2NH (CH 2)6N(CH2)8N(CH2)6N-~H2COCN. NH

~~/N

Tripitramine

NX~/

2

H3CO.

,CH

~N..,]

ttC

\

/

OCH3

COOC2H5

O~~N

CH3

H PD102807

O

Himbacine CH2NH(CH2)rNH(CH2)sNH(CHE)6NHCH2 -~ OCH3 H3CO Methoctramine

~

~ ~ / N NCOCH2-N .N-.C.--NH2 x___/ ~H Darifenacin

Fig. 2. Structural formulae of the muscarinic ligands discussed.

Guanylpirenzepine


tive for ileal than atrial muscarinic receptors. The existence of muscarinic receptor subtypes was definitely ascertained in 1980 with the advent of pirenzepine (Hammer et al., 1980), an antiulcerogenic drug which inhibits gastric secretion (Fig. 2). The affinities of this tricyclic compound, obtained using binding studies and isolated tissues, indicated differences between neural and peripheral muscarinic receptors. Further evidence coming from other authors in the period 1982-1984 (Hammer and Giachetti, 1982; Gilbert et al., 1984) pointed to a muscarinic receptor classification into two types: M 1 receptors, located on neural tissue and secretory glands, characterized by a high affinity for pirenzepine and stimulated by McN-A-343; M 2 receptors, located on neural and effector tissue, characterized by a low affinity for pirenzepine. The first International Symposium on Subtypes of Muscarinic Receptors (Boston, 1983) collected many papers dealing with the concept of the existence of more than one type of muscarinic receptor (TIPS, 1984). Among them, a paper from Mutschler and Lambrecht (1984) presented antagonists of the difenidol type, showing much higher affinity for muscarinic receptors in the ileum than for those in the atria. Hexahydrosila-difenidol proved to be the most selective compound (Fig. 2), with a 27-fold difference. Later, Hammer et al. (1986) presented a study with evidence for the heterogeneity of the M 2 muscarinic receptor using a new antagonist, AF-DX 116, with a pyridobenzodiazepinone tricyclic structure (Fig. 2). In fact, the binding profile of this cardioselective muscarinic antagonist suggests a further subclassification of M 2 receptors such as M 2 cardiac type and M 2 glandular type muscarinic receptors. This new subclassification is also confirmed by the study of Anwar-ul et al. (1986) on the cardioselective alkaloid himbacine (Fig. 2). Again, an article by Kubo et al. (1986) in Nature "Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor" opened new prospects on receptor study and let us know in depth the molecular basis of their function. Tetraamines were shown to be competitive antagonists of muscarinic receptors. Melchiorre et al. (1987) presented in J. Med. Chem. the most potent and cardioselective compound of this class, methoctramine (Fig. 2), displaying a selectivity ratio (atria vs. ileum or bladder) of ca. 270. TIPS (1989) showed a picture of muscarinic receptor nomenclature recommended by the Fourth International Symposium on Subtypes of Muscarinic Receptors (Wiesbaden, 1989) where three pharmacologically defined muscarinic subtypes (M1-M 3) and five cloned muscarinic subtypes are placed side by side. Among the selective antagonists showed in this classification, p-fluorohexahydrosila-difenidol appears (Lambrecht et al., 1989) as a new M3-selective compound (Fig. 2). In the aforementioned Symposium, Eberlein et al. (1989) presented AF-DX 384 (Fig. 2), a successor of AF-DX 116. -

-

133

In late 1994, polymethylene tetraamines once again played the main role in muscarinic receptor classification (Minarini et al., 1994). One of them, tripitramine (Fig. 2), represents, in fact, the most potent and selective muscarinic M 2 receptor antagonist which significantly discriminates between M 2 and M 4 sites, so confirming the pharmacological identification of this fourth muscarinic subtype. PD102807, the most potent and M 4 selective compound of a series of benzoxazine isoquinolines (Fig. 2), was identified and characterized by Augelli-Szafran et al. (1997) and, in the same year, Alabaster (1997) presented darifenacin (Fig. 2), a potent and selective M 3 antagonist with preferential affinity for gut and bladder relative to salivary gland. During the past years, other antagonists have been included in the TIPS classification; among them guanylpirenzepine (Fig. 2) (Micheletti et al., 1990) and toxins MT 3 and MT 7 (Jerusalinsky and Harvey, 1994; Adem and Karlsson, 1997) have to be mentioned. The introduction of a guanyl moiety on the pirenzepine molecule, in fact, modifies the selectivity profile of pirenzepine and allows this new antagonist to discriminate between duodenum and vas deferens receptors. The findings that small proteins contained in mamba snake venom are highly specific for muscarinic receptors led to the identification of MT 7 and MT 3 as very selective M 1 and M 4 antagonists, respectively, and opened the way to a new approach to drug design.

R e f e r e n c e s

Adem, A., Karlsson, E., 1997. Muscarinic receptor subtype selective toxins. Life Sci. 60, 1069-1076. Alabaster, V.A., 1997. Discovery and development of selective M 4 antagonists for clinical use. Life Sci. 60, 1053-1060. Anwar-ul, S., Gilani, H., Cobbin, L.B., 1986. The cardio-selectivity of himbacine: a muscarine receptor antagonist. Naunyn-Schmiedegerg's Arch. Pharmacol. 332, 16-20. Augelli-Szafran, C.E., Moreland, D.W., Nelson, C.B., Penvose-Yi, J.R., Schwarz, R.D., Jaen, J.C., 1997. Identification and characterization of m 4 selective muscarinic antagonists. Life Sci. 60, 1168. Barlow, R.B., Berry, K.J., Glenton, P.A.M., Nikolaou, N.M., Soh, K.S., 1976. A comparison of affinity constants for muscarine-sensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29~ and 37~ Br. J. Pharmacol. 58, 613-620. Eberlein, W.G., Engel, W., Mihm, G., Rudolf, K., Wetzel, B., Entzeroth, M., Mayer, N., Doods, H.N., 1989. Structure-activity relationships and pharmacological profile of selective tricyclic antimuscarinic. TIPS, Suppl. 50-54. Gilbert, R., Rattan, S., Goyal, R.K., 1984. Pharmacologic identification, activation and antagonism of two muscarinic sensitive receptor subtypes in the lower oesophageal sphincter. J. Pharmacol. Exp. Ther. 230, 284-291. Hammer, R., Giachetti, A., 1982. Muscarinic receptor subtypes: M 1 and M 2. Biochemical and functional characterization. Life Sci. 31, 29912994. Hammer, R., Berrie, C.P., Birdsall, N.J.M., Burgen, A.S.V., Hulme, E.C., 1980. Pirenzepine distinguishes between different subclasses of muscarinic receptors. Nature 283, 90-92. Hammer, R., Giraldo, E., Schiavi, G.B., Monferini, E., Ladinsky, H., 1986. Binding profile of a novel cardioselective muscarine receptor

134


antagonist, AF-DX 116, to membranes of peripheral tissues and brain in the rat. Life Sci. 38, 1653-1662. Jerusalinsky, D., Harvey, A.L., 1994. Toxins from mamba venoms: small proteins with selectivities for different subtypes of muscarinic acetylcholine receptors. TIPS 15, 424-430. Kubo, T., Fukuda, K., Mikami, A., Maeda, A., Takahashi, H., Mishina, M., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., Numa, S., 1986. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature 323, 411-416. Lambrecht, G., Feifel, R., Moser, U., Wagner-RSder, M., Choo, L.K., Camus, J., Tastenoy, M., Waelbroeck, M., Strohmann, C., Tacke, R., Rodrigues de Miranda, J.F., Christophe, J., Mutschler, E., 1989. Pharmacology of hexahydro-difenidol, hexahydro-sila-difenidol and related selective muscarinic antagonists. TIPS, Suppl. 60-64. Melchiorre, C., Cassinelli, A., Quaglia, W., 1987. Differential blockade of muscarinic receptor subtypes by polymethylene tetraamines. Novel class of selective antagonists of cardiac M-2 muscarinic receptors. J. Med. Chem. 30, 201-204. Micheletti, R., Schiavone, A., Angelici, O., Duranti, P., Giudici, L.,

Cereda, E., Donetti, A., 1990. Affinity profile of the novel muscarinic antagonist, guanylpirenzepine. Life Sci. 47, PL55-PL58. Minarini, A., Bolognesi, M.L., Budriesi, R., Canossa, M., Chiarini, A., Spampinato, S., Melchiorre, C., 1994. Design, synthesis and biological activity of methoctramine-related tetraamines bearing an 11acetyl-5,11-dihydro-6 H-pyrido[2,3-b][ 1,4]benzodiazepin-6-one moiety: structural requirements for optimum occupancy of muscarinic receptor subtypes as revealed by symmetrical and unsymmetrical polyamines. J. Med. Chem. 37, 3363-3372. Mutschler, E., Lambrecht, G., 1984. Selective muscarinic agonists and antagonists in functional tests. TIPS, Suppl. 39-44. Riker, W.F., Wescoe, W.C., 1951. The pharmacology of flaxedil with observations on certain analogs. Ann. N.Y. Acad. Sci. 54, 373-394. Roszkowski, A.P., 1961. An unusual type of sympathetic ganglionic stimulant. J. Pharmacol. Exp. Ther. 132, 156-170. TIPS, 1984. Supplement (January). TIPS, 1989. Supplement (December). Wess, J., 1993. Molecular basis of muscarinic acetylcholine receptor function. TIPS 14, 308-313.



Design and development of selective muscarinic agonists for the treatment of Alzheimer's disease" characterization of tetrahydropyrimidine derivatives and development of new approaches for improved affinity and selectivity for M 1 receptors William S. Messer Jr. *, W.G. Rajeswaran, Yang Cao, Hai-Jun Zhang, Afif A. E1-Assadi, Colleen Dockery, Jill Liske, John O'Brien, Frederick E. Williams, Xi-Ping Huang, Mary E. Wroblewski, Peter I. Nagy, Steven M. Peseckis Department of Medicinal and Biological Chemistry, College of Pharmacy, The Uniuersity of Toledo, 2801 W. Bancroft St., Toledo, OH 43606 USA

Abstract

Cholinergic neurons degenerate in Alzheimer's disease, resulting in cognitive impairments and memory deficits, and drug development efforts have focused on selective M~ muscarinic agonists. 5-(3-Ethyl-l,2,4-oxadiazol-5-yl)-l,4,5,6-tetrahydropyrimidine trifluoroacetic acid (CDD-0102) stimulates M~ muscarinic receptors in rat brain [Messer, W.S., Jr., Abuh, Y.F., Liu, Y., Periyasamy, S., Ngur, D.O., Edgar, M.A., E1-Assadi, A.A., Sbeih, S., Dunbar, P.G., Roknich, S., Rho, T., Fang, Z., Ojo, B., Zhang, H., Huzl, J.J., III, Nagy, P.I., 1997a. J. Med. Chem. 40, 1230-1246.] and improves memory function in rats with lesions of the basal forebrain cholinergic system. Moreover, CDD-0102 exhibits oral bioavailability, few side effects and low toxicity, and thus represents a viable candidate for clinical studies. Despite the development of functionally selective agonists such as xanomeline and CDD-0102, there is room for improvements in ligand affinity and selectivity. The high degree of amino acid homology within transmembrane domains has hindered the development of truly selective agonists. Site-directed mutagenesis, biochemical and molecular modeling studies have identified key amino acid residues such as Thr192 and Asn382 in the binding of agonists to M~ receptors [Huang, X.P., Nagy, Pi., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1999. Br. J. Pharmacol. 126, 735-745.]. Recent work has implicated residues at the top of transmembrane domain VI in the binding of muscarinic agonists and activation of M~ receptors [Huang, X.P., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1998. J. Pharmacol. Exp. Ther. 286, 1129-1139.]. Thus, residues such as Ser388 represent molecular targets for the further development of agonists with improved M~ receptor affinity, selectivity and activity. 9 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Muscarinic receptor; Alzheimer's disease; Xanomeline; CDD-0102; Site-directed mutagenesis; Molecular modeling; Memory function

1. Introduction

Alzheimer's disease is a neurological disorder characterized by progressive decline in cognitive function and memory deficits. Pathological hallmarks of Alzheimer's disease include neurofibrillary tangles and amyloid plaques. A marked loss of cholinergic neurons is a consistent neurochemical finding associated with the disorder, and the loss of cholinergic activity could contribute to the behavioral manifestations of Alzheimer's disease (Perry et al., 1977, 1999; Whitehouse et al., 1982; Coyle et al., 1983).

* Corresponding author. Tel.: +1-419-530-1958; fax: +I-419-530-7946

The development of selective muscarinic agonists, thus, has been a major focus of the pharmaceutical industry for the past decade. Many compounds have been synthesized and tested for muscarinic activity, and a few compounds have been introduced into clinical trials. Although compounds like xanomeline (see Fig. 1) have exhibited some beneficial activity in Alzheimer's patients, unwanted side effects have limited their utility. In an attempt to develop ligands with improved activity and selectivity, we synthesized a series of amidine derivatives, including 5-(3ethyl - 1,2,4- oxadiazo1-5-yl ) - 1,4,5,6- tetrahydropyrimidine trifluboroacetic acid (CDD-0102), and compared its pharmacological, behavioral and toxicological properties with xanomeline.

0031-6865/00/$ - see front matter 9 2000 Published by Elsevier Science B.V. All rights reserved. PII: S003 1-6865(99)00026-6

136

W.S. Messer Jr. et al. / Pharmaceutica Acta Helvetiae 74 (2000) 135-140

N.-.--S

~

CH3

C6H13~O

~.,,," N~CH3 HN,,,,,~N Xanomeline

CDD-0102

Fig. 1. Chemical structure of xanomeline and CDD-0102.

To date, no muscarinic agonist has been approved for treatment of Alzheimer's disease. Perhaps, the main difficulty in the development of selective muscarinic agonists has been the issue of subtype selectivity. There are five subtypes of muscarinic receptors, with M 1, M 3 and M 5 receptors coupling through the Gq family of G proteins to the activation of phospholipase C. Both M 2 and M 4 receptors couple to the inhibition of adenylyl cyclase through the Gi/o family of G proteins. Drug development efforts have focused on identifying M~ agonists, based on the preferential localization of M~ receptors in forebrain regions such as the cerebral cortex and hippocampus, and behavioral studies implicating M1 receptors in memory function. M 3 receptors mediate smooth muscle contraction and exocrine gland secretion. M 3 receptors are highly homologous to M 1 receptors, particularly within transmembrane domain regions believed to comprise the binding site for acetylcholine and other muscarinic agonists. Thus, most muscarinic agonists activate M 3 receptors to varying degrees, thereby producing unwanted side effects. Site-directed mutagenesis and molecular modeling studies of muscarinic receptors have helped identify the amino acid residues important for agonist binding and receptor activation. A conserved aspartate residue in transmembrane domain (TM) III (Asp l05 in M 1 receptors) appears to bind to the positively charged onium group in acetylcholine and other quaternary muscarinic agonists (Fraser et al., 1989). In M 3 receptors, conserved threonine, tyrosine and asparagine residues in TM V and TM VI appear to contribute hydrogen bonds to the ester moiety of acetylcholine (Wess et al., 1991, 1992; Bluml et al., 1994). Moreover, mutations of residues at the top of TM VI result in constitutively active M 5 receptors (Spalding et al., 1995, 1997). In order to understand the relative importance of these conserved residues in M 1 receptors, we constructed a series of mutant M 1 receptors, and examined ligand binding properties and functional activity following expression in A9 L cells. In addition, we initiated a series of molecular modeling studies to help understand the relative contribution of individual residues to the receptor binding process. The combination of approaches provides insight into the mode of agonist binding to receptors, and offers some understanding of the mechanisms of receptor activation. Portoghese et al. (1987) have utilized a bivalent ligand approach in the design of selective ligands for opioid

receptors. We adopted a similar strategy for developing more selective muscarinic agonists, starting with the M 1 agonist, xanomeline. As compared with the parent compound, xanomeline, the bivalent ligands display markedly increased binding affinity for M~ receptors. These compounds provide a framework for discussing the underlying mechanisms of ligand binding and receptor activation, and suggest new strategies for developing truly subtype-selective muscarinic agonists.

2. Experimental procedures 2.1. Synthetic chemistry

CDD-0102 (Messer et al., 1997a) and xanomeline (Sauerberg et al., 1992) were synthesized according to published methods. Bivalent ligands were synthesized according to the generalized procedure shown in Scheme 1. Detailed procedures will be reported elsewhere. 2.2. Biochemical and molecular modeling studies

Receptor binding and agonist activity were measured in A9 L cells expressing human M 1 muscarinic receptors. All receptor binding assays and studies of phosphoinositide metabolism were conducted as outlined previously (Messer et al., 1997a). The site-directed mutagenesis studies also have been described previously (Huang et al., 1998, 1999). Molecular modeling studies employed the seven-transmembrane-domain model of the human M 1 receptor developed by Nordvall and Hacksell (1993), as described previously (Messer et al., 1997a). 2.3. In vivo studies

Pharmacological and toxicological screens of muscarinic agonists were conducted in mice, according to published methods (Messer et al., 1997b). At least three animals were administered i.p. either 0.1, 1.0 or 10 m g / k g of either CDD-0102 or xanomeline in saline. Separate animals were injected with saline i.p. as controls. In toxicity studies, mice were administered CDD-0102 or xanomeline either i.p. or p.o. (two animals per dose per route) with doses from 10 to 1000 mg/kg. From these studies, a narrow dose range was utilized to determine the LDs0 for each drug by both routes of administration. Representational memory function (a type of working memory) was tested in young, male Sprague-Dawley rats as described previously (Messer et al., 1997b). Briefly, animals were trained to perform a paired-run alternation task in a T-maze for a food reward. Animals were trained until they have achieved greater than 90% correct for 3 consecutive days, then administered 192 IgG-saporin by stereotaxic surgical techniques. The 192 IgG-saporin is a

137

W.S. Messer Jr. et al. / Pharmaceutica Acta Helvetiae 74 (2000) 135-140 Sx

N" N C1

N HO.

Nail / THF, A

(kkl-12)n

IQN/'--~ O N N~ \S

(CHz)n

HO' n = 2-10, 12

~ ~"(

S N" "N

.S, N N

N-S-N

(qH2)n 178

> 178

160

aAll values shown represent oral doses. bMouse water maze performance in hippocampally deficient mice. cMouse water maze performance in nbM-lesioned rats. dEDs0 values reflect the dose required to induce 50% of the pellets to leave the stomach or to move the lead pellet 50 cm within the intestines, respectively. eIndicates oral dose required to induce salivation in 6/6 mice. f Dose at which maximal improvementof mouse water maze performance was observed (mean latency to find the hidden platform decreased from 90 s to about 60 s).

H. Tecle et al. / Pharmaceutica Acta Helvetiae 74 (2000) 141-148 12

E

147

4. Conclusion

10

8

.... ~

~ 2 [~ o

1

2

,,c,-,o171 .Control

5

W

6

Days of culture

The pharmacologic profile of the CI-1017, unlike the profile of classical muscarinic agonist, indicates that it should be useful in the treatment of cognitive disorders such as AD. CI-1017 was found to be well-tolerated in Phase I clinical trials and is proceeding into Phase II studies.

Fig. 2. CI-1017 (10 p~M) inhibits A-beta secretion.

References

M 1 receptors. In the case of (R)-6m, activation of both M~ and M 4 receptors was observed, although the compound was slightly more potent at the former than the latter. Compound (S)-6m activated M~ receptors selectively over M 3 and M 5 receptors, but was about 10 times less potent at M~ receptors than (R)-6m. Additional evidence for muscarinic potency and M~ selectivity of (R)-6m was obtained from a cell amplification assay utilizing NIH 3T3 cells transfected with Hm~Hm 4 receptors (Table 11). In this assay, as in the previously described two functional assays, (R)-6m was significantly more selective for the M~ receptor than for the non-M~ receptors. (R)-6m enhanced amplification of Hm~- and Hm4-transfected cells with approximately equal potency (EDs0 values of 42 and 51 riM, respectively). Concentrations required to stimulate amplification of Hm 2and Hm3-transfected cells were significantly higher. Interestingly, (S)-6m did not display the same level of selectivity in the cell amplification assay as was observed in the second-messenger assays. The in vivo pharmacology of (R)-6m is in accordance with its ability to discriminate among the receptor subtypes (Table 12). Thus, very minor peripheral side effects (salivation, GI motility mediated by non-M~ receptors) were detected only at very high (178 m g / k g ) doses. By contrast, the central effect mediated by M j receptors (improved spatial memory) was evident at oral doses of 0.03-3.2 mg/kg. Typically, several of the classical muscarinic agonists (e.g., RS-86, arecoline, or oxotremorine) display pronounced parasympathetic side effects (salivation, lacrimation, urination, gastrointestinal effects, etc) at doses lower than those required for central effects. Recent studies have shown that activation of muscarinic receptors can alter processing of the APP and production of beta-amyloid protein (Robertson and Harrell, 1997; Haring et al., 1998). Using transfected Hm~ CHO cells, CI-1017 (10 p~M) was found to markedly increase the soluble form of APP (APPs) in contrast to a small increase observed with carbachol (10 p~M). Further, CI-1017 was able to significantly decrease A-beta production (Fig. 2). Thus, CI- 1017 may alter the underlying pathology of AD in addition to affecting the symptoms (memory deficits) of AD.

Baker, R., Saunders, J., 1989. Central muscarinic ligands and receptors. In: Allen, R.C. (Eds.), Annu. Rep. Med. Chem., Vol. 24, pp. 31-40 and references therein. Bolden, C. et al., 1992. Antagonism by antimuscarinic and neuroleptic compounds at the five cloned human muscarinic cholinergic receptors expressed in Chinese hamster ovary cells. J. Pharmacol. Exp. Ther. 517, 576-580. Bonner, T.I. et al., 1987. Identification of a family of muscarinic acetylcholine receptor genes. Science 237, 527-532. Braeuner-Osborne, H., 1995. Annulated heterocyclic bioisosteres of norarecoline. Synthesis and molecular pharmacology at five recombinant human muscarinic acetylcholine receptors. J. Med. Chem. 38, 21882195. Braeuner-Osborne, H., Brann, M.R., 1996. Pharmacology of muscarinic acetylcholine receptor subtypes (m 1-m5): high throughput assays in mammalian cells. Eur. J. Pharmacol. 295, 93-102. Buckley, N.J., 1990. Molecular pharmacology of cloned muscarinic receptors. In: Nahorski, S.R., (Ed.), Transmembrane Signaling, Intercellular Messengers and Implications for Drug Development. Wiley, pp. 11-31. Buckley, N.J. et al., 1989. Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Mol. Pharmacol. 35, 469-476. Christie, J.E. et al., 1981. Physostigmine and arecoline: effects of intravenous infusions in Alzheimer presenile dementia. Br. J. Psychiatry 138, 46-50. Davies, P., Maloney, A., 1976. Selective loss of central cholinergic neurons in Alzheimer's disease. Lancet 2, 1403. Davis, R., et al., 1993. Subtype-selective muscarinic agonists: potential therapeutic agents for Alzheimer's disease. In: Cuello, C.A., (Ed.), Cholinergic Function and Dysfunction, Progress in Brain Research. Elsevier, Amsterdam, pp. 439-445. Doerje, F. et al., 1991. Antagonist binding profiles of five cloned human muscarinic receptor subtypes. Pharmacol. Exp. Ther. 256, 727-733. Haring, R. et al., 1998. Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the m~ muscarinic receptor to [3-amyloid precursor protein secretion. J. Neurochem. 7, 20942103. Hulme, E.C. et al., 1990. Muscarinic receptor subtypes. Annu. Rev. Pharmacol. Toxicol. 30, 633-673. Jones, S.V.P., et al., 1992. Muscarinic acetylcholine receptors. In: Brann, M.R. (Ed.), Molecular Biology of G-Protein Receptors. Birkhauser, Boston, pp. 170-197. McConnell, H.M., 1992. The cytosensor microphysiometer: biological applications of silicon technology. Science 257, 1906-1912. Mutschler, E., Hultzsch, K., 1973. Uber struktur-wirkungs-beziehungen von ungesattigten estern des arecaidins und dihydroarecaidins. Arsneim-Forsch. (Drug Res.) 23, 732-737. Nabeshima, T. et al., 1991. Staurosporine facilitates recovery from the basal forebrain-lesion-induced impairment of learning and deficit of cholinergic neuron in rats. J. Pharmacol. Exp. Ther. 257, 562-566.

148

H. Tecle et al. / Pharmaceutica Acta Helvetiae 74 (2000) 141-148

Perry, E.K. et al., 1978. Correlation of cholinergic abnormalities with senile plaques and mental test scores in dementia. Br. Med. J. 2, 1457-1459. Robertson, M.C., Harrell, L.E., 1997. Cholinergic activity and amyloid precursor protein metabolism. Brain Res. Rev. 25, 50-69. Schwarz, R.D., 1993. Characterization of muscarinic agonists in recombinant cell lines. Life Sci. 52, 465-472. Street, L.J. et al., 1990. Synthesis and biological activity of 1,2,4, oxadiazole derivatives: highly potent and efficacious agonists for cortical muscarinic receptors. J. Med. Chem. 33, 2690-2697. Symons, J.P. et al., 1988. Water maze learning and effects of cholinergic drugs in mouse strains with high and low hippocampal pyramidal cell counts. Life Sci. 42, 375-383. Tecle, H., et al., 1989. Abstract of papers. 197th National Meeting of the American Chemical Society, Dallas, TX. American Chemical Society, Washington, DC, MEDI-47. Tecle, H. et al., 1993. Synthesis and SAR of bulky 1-azabicyclo[2.2.1]-3one oximes as muscarinic receptor-subtype-selective agonists. Life Sci. 52, 505.

Venter, J.C., Fraser, C.M., 1990. Molecular biology of receptors and drug design. In: Clark, C.R., Moos, W.H. (Eds.), Drug Discovery Technologies. Ellis Horwood, Chichester, pp. 231-255. Vickroy, T.W. et al., 1984. Pharmacological differences between the high-affinity muscarinic agonist binding states of the rat heart and cerebral cortex labeled with (+)-[3H]-cis-methyldioxolane. J. Pharmacol. Exp. Ther. 229, 747-755. Watson, M. et al., 1986. [3H]Pirenzepine and (-)-[3H]quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites: I. Characterization and regulation of agonist binding to putative muscarinic subtypes. J. Pharmacol. Exp. Ther. 237, 411418. Whishaw, I.Q. et al., 1985. Disruption of central cholinergic systems in the rat basal forebrain lesions or atropine: effects on feeding, sensorimotor behavior, locomotor activity and spatial navigation. Behav. Brain Res. 17, 103-115. Whitehouse, P. et al., 1982. Alzheimer' s disease and senile dementia: loss of neurons in the basal forebrain. Science 215, 1237-1239.



Ligands for the common allosteric site of acetylcholine M2-receptors" development and application U. Holzgrabe a,*, W. Bender a, H.M. Botero Cid a, M. Staudt a, R. Pick a, C. Pfletschinger a E. Balatkovfi b, C. Triinkle b, K. Mohr b a Department of Pharmaceutical Chemistry, Institute of Pharmacy and Food Chemistry, University of Wi~rzburg, Am Hubland, 97074 Wi~rzburg, Germany b Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany

Abstract

Ligands for the allosteric site of acetylcholine M 2 receptors are able to retard the dissociation of simultaneously bound ligands for the orthosteric site. This effect promotes receptor occupation by the orthosteric ligand. The allosteric effect opens various therapeutic perspectives, e.g., in organophosphorus poisoning. The aim of our studies was to optimize the affinity of the modulators for the common allosteric binding site of muscarinic M 2 receptors, the orthosteric site of which was liganded with the N-methylscolopamine. The phthalimido substituted hexane-bisammonium compound W84 served as a starting point. Previous molecular modelling studies revealed two positive charges and two aromatic imides in a sandwich-like arrangement to be essential for a high allosteric potency. A three-dimensional quantitative structure activity relationship (3D QSAR) analysis predicted compounds with substituents of increasing size on the lateral imide moieties to enhance the affinity for the allosteric binding site. Thus, we synthesized and pharmacologically evaluated compounds bearing "saturated" phthalimide moieties as well as phthalimidines with substituents of systematically increasing size in position 3 or on the aromatic ring at one or both ends of the molecule. Within each series, QSAR could be derived: 1. "Saturation" of the aromatic ring of the phthalimide moiety results in less potent compounds. 2. Increasing the size of the substituents in position 3 of the phthalimide enhances the potency. 3. Putting substituents on the aromatic part of the phthalimide increases the potency more effectively: the introduction of a methyl group in position 5 gave a compound with a potency in the nanomolar concentration range which was subsequently developed as the first radioligand for the allosteric binding site. 9 2000 Elsevier Science B.V. All rights reserved.

Keywords: Allosteric site; Acetylcholine M 2 receptor; 3D QSAR analysis; N-methylscopolamine; W84; Organophosphorus poisoning; Allosteric modulation; Pain; Dementia

1. Introduction Whereas the concept of allosteric modulation for enzymes (Koolman and RiShm, 1996) and for ion channel receptors is well-established, this p h e n o m e n o n is hardly recognized in the case of G-protein coupled receptors. As a

* Corresponding author. Tel.: +49-931-888-5460; fax: +49-931-8885494; e-maih [email protected]

paradigm we consider the interaction at acetylcholine M 2 receptors between structurally heterogeneous allosteric modulators and orthosteric antagonists, such as N-methylscopolamine (NMS), (Christopoulos et al., 1998; Holzgrabe and Mohr, 1998) or agonists (Gharagozloo et al., 1999). The allosteric modulators can influence both the ligand association and dissociation resulting either in a reduction or in an elevation of ligand equilibrium binding. In combination with antagonists such as atropine, the therapy of organophosphorus poisoning can take advantage

0031-6865/00/$ - see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S0031-6865(99)00028-X

150

U. Holzgrabe et al. / Pharmaceutica Acta Helvetiae 74 (2000) 149-155

of the retardation of the dissociation. The allosteric elevation of endogenous acetylcholine binding (Birdsall et al., 1999) might be beneficial in the treatment of pain and dementia. The goal of the investigations, presented here, was to optimize the affinity of the modulators for the common allosteric binding site of muscarinic M 2 receptors, the orthosteric site of which was liganded with the antagonist N-methylscolopamine. The phthalimido substituted alkane-bisammonium compound W84 (Fig. 1) was taken as a starting point. Previous molecular modelling studies (Holzgrabe et al., 1996) revealed two positively charged nitrogens and two aromatic imides in a sandwich-like arrangement to be essential for a high allosteric potency (see Fig. 2). An additionally performed three-dimensional quantitative structure activity relationship (3D QSAR) analysis (Holzgrabe and Hopfinger, 1996) predicted compounds with substituents of increasing size on the lateral imide moieties to enhance the affinity for the allosteric binding site. This pharmacophore hypothesis was supported by the result of a study in which the length of the spacers between the pharmacophoric elements was varied (NassifMakki et al., 1999). The distance between othe positive charges was determined to be at least 10 A, which is equivalent to about seven methylene groups; the spacer between the positive charge and the lateral imide moiety has to be long enough to allow the phthalimides to properly reach the aromatic positions. In the case of the W84 compounds a length of three methylene groups is necessary. Taking the findings of the theoretical studies into account, three projects were initiated: (1) In order to check whether aromatic imides in lateral positions are necessary for a high allosteric potency the phthalimide moieties were replaced with corresponding "saturated" imides and, additionally, the hexahydrophthalimide was stepwise reduced to a succimide ring. (2) Considering the QSAR analysis the allosteric potency should increase with increasing steric size of the substituent on the phthalimide. Thus, various substituents were introduced in position 3 and 5 of the phthalimide. (3) Since it is unlikely that the allosteric binding site is symmetrical the structural variations mentioned above were performed at one end and at both ends of the symmetrical parent compound W84.

0 R~~,.~ L ].~/N~~

E)?H 3 ~

CH3 IN-N/ '

R = H W84 R = CH3 dimethyI-W84

Ok~.~,,~....,,,~. R l" II (~

2 Br"

Fig. 1. Structural formula of the parent compound W84 and dimethyl-W84.

1oA

~-.

~9

flexible

spacer

Fig. 2. Pharmacophore model.

In each project it was tried to built series of systematically varied compounds which can be used to establish QSAR analyses.

2. Common allosteric site QSAR analyses of series of compounds are only valid when all compounds bind in the same mode to the same binding site. Generally, the allosteric action is evaluated as the quantification of an effect; the ECs0,diss value is the concentration of the modulator at which the rate of radioligand dissociation is reduced by 50% (Jepsen et al., 1988). However, experiments to antagonize allosteric effects revealed that structurally closely related allosteric agents may nevertheless reveal a divergent mode of allosteric action (Tr~inkle and Mohr, 1997). In order to gain a more direct insight into the binding events, a high affinity radioligand for the allosteric binding site was developed. Introduction of a methyl group in position 5 of the phthalimide ring (Tr'finkle et al., 1998) yielded a compound, designated as dimethyl-W84 (see Fig. 1), with particularly high allosteric potency (for comparison with other compounds see Tr~inkle et al., 1996). Tritiation of dimethylW84 gave the first radioalloster suitable for direct binding measurements at the allosteric site (Tr~inkle et al., 1998). Competition binding experiments with the prototype modulators alcuronium, W84 and gallamine went parallel with the inhibition of the [3H]NMS dissociation which indicated that the high affinity binding of [3H]dimethyl-W84 reflects the occupation of the common allosteric site of the M 2 receptor.

3. Replacement of the phthalimide with corresponding ' 6saturated 79 moieties In previous studies, the unilateral replacement of the phthalimide with a morpholino or hydroxy substituents (Holzgrabe et al., 1997) or shortening of the lead compound W84 down to monoquaternary compounds (Kostenis

151

U. Holzgrabe et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 149-155

et al., 1994) resulted in a considerable reduction of the allosteric potency indicating the importance of the lateral aromatic rings. However, the question remains whether an aromatic annelation of the imide is necessary or whether a hydrophobic moiety is sufficient for the interaction with the receptor protein. Thus, a cis and trans-annelated cyclohexane and a trans-cyclohexene imide compound 1-3 in addition to a dimethyl- (4), a monomethyl- (5) and an unsubstituted (6) succimide derivative were synthesized by conversion of the corresponding 1,2-dicarbonic acids to the anhydrides, condensation of the N',N'-dimethylaminopropanamine with the anhydrides giving the imides and connecting two imide units with dibromoheptane (see Fig. 3). The allosteric potencies of these new compounds were compared to the C7/3'-phth compound (Choo and Mitchelson, 1989) which was previously reported to have a higher potency than the corresponding hexane compound (Triinkle et al., 1996) (ECs0 value of 390 nM vs. 1300 nM). In comparison to C7/3'-phth all concentration-effect-curves except with the cyclohexene derivative compound 3 were shifted in a parallel fashion to the right indicating the same mode of action but a lower potency (see Fig. 3). Interestingly, the decrease in potency resembles the diminution of the size of the lateral moiety in

going from the saturated cyclohexane-l,2-dicarbonic acid imide through the dimethyl- and monomethyl- to the nonsubstituted succimide. A positive correlation between the allosteric potency and the lipophilicity of the lateral moiety was found suggesting that with increasing size of the hydrophobic imides the contact area with the receptor protein is enlarged. log(1/ECs0 ) = 1.412( _+0.61)logP + 4.195(_+0.57) n=6; r 2=0.91;s=0.33;F=40.1;Q2=0.83; S p R E S S -'-

0.452

(1)

where n is the number of compounds, r 2 is the square of the correlation coefficient, s the standard deviation, F the ratio of explained to unexplained variance, Q2 is the cross validated r 2 by using the leave-one-out procedure (Q2 c a n adopt values between 1 and zero) and SpRESs is the standard deviation from the predictive residual sum of squares. Since the cyclohexene derivative shows a steeper concentration-effect-curve, the data of this compound were omitted.

o

o

I

2 Bf

o

I

o

l

1

X

C3/7"-phth

X

H3C~.....~O

H3C~.~ O

H3C,,,.L~ N ~ ; H

0

H

1

0

H

2

0

L ~ N'M~' "

o

3

o

4

5

75,-.

50-

C7/3"-phth~ \\ 25O-

o

6

EC5o [p M]

100-

v

~~

\

\

A 1

0.8

9 2.

4.5

9 3

2.2

[] 4

12.1

0 5

32.0

9 6

151

(mean + SEM; n=3-4) ,'

,

,

-8

-7

-6

; -

' -4

1

-

-2

test compound (log M) Fig. 3. Structural formula of the "saturated" imides and concentration-effect-curves for reducing the apparent rate constant of [3H]NMS dissociation from M 2 receptors in porcine heart membranes.

152


within this series of compounds, this stereochemical finding is a hint that the phthalimide binds into a pocket at the entrance of the receptor which has steric restrictions. Thus, in the next step, it was aimed to probe the size of the hypothesized binding pocket.

. ..........

r

1 5"1 4

oTM

3~....-"~unsubS i:ituted

-1.0

cis-cycl0hex~~ ..... .-. ~~.e " crs-cycl~ ..... ~ h y l ......diaethyl

-0.5

0.0

0.5 1.0 IogP-values

1.5

4. Symmetrical substituent variations at the phthalimide skeleton

2.0

Fig. 4. Correlationbetween the allosteric potency of C3/7'-phth and 1, 2, 4-6 and the lipophilicity.

Even though the correlation is found to be highly significant, the difference in potency between the transand c i s - d i a s t e r e o m e r s of the cyclohexane compound cannot be explained by the calculated lipophilicity (see Fig. 4). Since the trans isomer of the cyclohexane-imide sterically resembles the phthalimide more than the cis isomer, the higher potency of the t r a n s - i s o m e r can be easily understood. However, even though no significant correlation of the potency with steric parameters was found

Since the preceding study had revealed a cycloalkane moiety to be disadvantageous as a lateral system, in the following the aromatic phthalimide skeleton was kept the same whilst one carbonyl functional was replaced with hydrogens (7), as well as hydroxy (8) and alkoxy groups (9) and with phenyl (10) and benzyl (11) and benzylidene rings (12) (see Fig. 5). Whereas the hydroxy and alkoxy derivatives can be easily obtained by reduction of the carbonyl group with NaBH 4 and conversion with the corresponding alcohol in acidic media, other synthesis pathways had to be developed for the other compounds. The synthesis of the phenyl substituted compound 10 started off with the reduction of phenylphthalazone with Zn/HC1 to give the phthalimidine which was alkylated

0 3

\~

1.

W84

O

#

2 Br O

--I

"

CH 3

,,

ff

v

O

.CH3

q

2

R = H 7 = OH 8

= OCH 3 = OC2H 5 = O-i-C3H 7

9a 9b 9r

= S-i-C3H7

9(t

= C6H 5

10

E E , ZZ,

EZ

12

= CH2-C6H 5 11

Fig. 5. Structural formulaof the symmetricalphthalimido substitutedallosteric modulators7-12.

2 Br O

U. Holzgrabe et al./ Pharmaceutica Acta Helvetiae 74 (2000) 149-155

with dibromoalkane in presence of Nail. Two of these molecules were connected with 1,6-bis(dimethylamino)hexane to obtain the bisammonium salt. The benzyl and benzylidene compounds 11 and 12, resp., were achieved via the benzalphthalide which was converted to the imidine; this imidine can be either reduced to give the benzyl product 11 or the obtained E / Z isomers separated for the preparation of the benzylidene bisammonium salts 12 (Botero Cid et al., 1999). The parallel concentration-effect-curves for these compounds exhibit an allosteric potency of the same order of magnitude as the parent compound W84. Whereas most of the alkoxy substituted compounds 9 are less potent than W84, the benzyl and phenyl substituted ones 10 and 11 show a slightly higher potency; interestingly, all benzylidene isomers 12 are more than one order of magnitude more potent than W84. Again, the correlation between the potency and the lipophilicity (Tr) was checked, but the regression analysis results in a poor correlation. The benzylidene derivatives turned out to be outliers. Omitting these compounds gave a significant, but still poor positive correlation: log( 1(EC 50) = 0.304( _+0.15) 7r + 5.464( _+0.19) n = 8;

r 2 --

0.80"

s

--

0.167 ; F = 23.4; Q2 = 0.67 ;

s PRESS= 0.21

(2)

In addition, steric parameters, such as volume, surface and refractivity calculated by means of Hyperchem/ Chemplus, were checked. Using a parabolic model a poor correlation with the volume was found wherein the benzyl compound was an outlier. Omitting this compound resulted in a striking correlation (Fig. 6): log( 1/ECs0) = + 0.0000867( + 0.000026)vol 2 - 0 . 0 9 7 7 ( + 0.031)vol + 32.76(+9.26) n=ll;

r2=0.95;s=0.18;

SpRESs = 0.24

8,00

153

Volume (parabolic

correlation)

7,50 7,00 tj -

6,5o

B

6,00 5,50 5,00 4,50 4,00 - 400,00

450,00

500,00

550,00

600,00

650,00

700,00

750,00

Volume

Fig. 6. Correlation between the allosteric potency and the volume of the lateral substituents of the compounds7-10 and 12.

benzyl substituted compound is one order of magnitude less potent than the benzylidene compounds although the volume of both substituents is almost the same. The striking difference between these compounds is the difference in rigidity caused by the double bond. In the case of the benzylidene compounds the smaller loss of entropy on binding to the receptor protein (Andrews, 1993) might be the reason for the high potency. Remarkably, the stereochemistry of the benzylidene groups attached to the phthalimide skeleton, EE, ZZ and EZ, does not influence the potency. Since the alkane bisammonium compounds were found to be highly flexible (Holzgrabe and Hopfinger, 1996), it is tempting to speculate that either the aromatic ring of the phthalimide or of the benzylidene ring can serve as the partner for interaction with the binding site. Molecular modelling investigations supported this hypothesis (Botero Cid et al., 1999). Summarizing this part of the study: since no steric restriction and no stereoselectivity concerning the substituent in position 3 could be found the binding pocket is supposed to be rather large.

F=80.4;Q2=0.91; (3)

From this analysis the minimum potency was calculated to be related with a volumes in a range between 540 and 575 ,~3, which corresponds to the methoxy group. Groups of smaller and bigger size induced a higher potency. The increase in potency going along with the diminution of the substituent size might be caused by a different sort of interaction between this part of the phthalimide (carbonyl and hydroxy groups versus alkoxy groups) and the receptor protein. The increase in potency connected with the expansion of the alkane ether chain goes along with the elevation of the intermolecular hydrophobic contact surface with the receptor protein (B/Shin and Klebe, 1996) and, therefore, with the enhancement of the affinity of the compounds to the receptor binding site. Interestingly, the

5. Asymmetrical substituent variations at the phthalimide skeleton Due to the simplicity of the synthesis most of the newly synthesized allosteric modulators were symmetrical. However, the binding site of the modulators is unlikely to be symmetrical. In order to check whether both ends of the modulators have a different contribution to the potency we aimed to synthesize asymmetrical compounds. Due to the results of the 3D QSAR analysis is was predicted that enlargement of one phthalimide moiety should increase the potency even with the contralateral phthalimide kept unchanged. On the one hand the synthesis started off with the alkylation of dimethylaminopropylphthalimide with a huge excess of dibromohexane without any solvent in order to

154

U. Holzgrabe et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 149-155

avoid the bisamination of dibromohexane. On the other hand, a corresponding 1,2-dicarbonic acid was converted via the anhydride to the dimethylaminopropylimide which can be connected to the above obtained bromo-compound to give the asymmetrical compounds 13 (Fig. 7). With exception of the "pyridophthalimide" all aromatic compounds were found to have a higher potency than the parent compound W84. Especially the naphthalimide compound shows a very high potency. In order to quantify the potency, it was checked whether a correlation between the potency and lipophilic or steric parameters could be found. Again, the correlation with the lipophilicity was poor in comparison with the volume of the lateral substitutent (Bender et al., 1999) (Fig. 8).

Iog(1/ECso) 7.5 7.3

913g

7.1

1 3 e ~

6.9 6.7

.....

6.5

"

1~,~O/

//

6.3 6.1 5.9

--

5.7

/

Z v 13b

\.,

W84

4'13a

5.5 470

520

570

620

670

720

V o l u m e [A3]

log( 1/ECs0) = -0.000089( _ 0.000036)vol 2

Fig. 8. Correlation between the allosteric potency and the volume of the lateral substituents of the compounds 13.

+ 0.108( + 0 . 0 4 1 ) v o l - 25.85( ___12.2) n = 8; r 2 = 0 . 9 3 ; s = 0.151; F = 41.6; Q2 =0.898; SpRESs = 0.199

(4)

In contrast to Eq. (3), the allosteric potency is running through an maximum volume which amounts to 600 to 650 /~3. These findings clearly indicate that the hypothesised binding area has a defined shape: On the one hand, there is obviously much space for hydrophobic substituents at position 3 whereas on the other hand there is pocket which can perfectly take in and interact with the naphthalimide (or phenothiazine moiety (Holzgrabe et al., 1997)). The interactions with smaller and bigger substituents are less strong causing a lower potency. At this point, the questions still remains, whether the allosteric modulators have to be symmetrical or asymmetrical. Therefore, pairs

0

CH 3

,-,H3

I

/

W84

O O N~~

I

R

-

O -

o

13a '/CH3 13b

13c

O

O O _N~'~ /7

O -

O

v

F

13d

13g O!~

CI "CI

6.1. Comparison of the differences in potency upon symmetrical and asymmetrical variations of lateral moieties The diminution of one phthalimide to a succimide resulted in the series of W84 compounds in a loss of potency of Alog(1/ECs0)=0.88 (Bender et al., 1999). Replacement of both phthalimides with the succimide in the corresponding heptane series induced about twice the loss of potency: Alog(1/ECs0)= 2.54. This clearly indicates that the phthalimide moiety is important in both places.

6.2. Stepwise replacement of the phthalimide with analogue imides

R o

6. Symmetrical versus asymmetrical substituent variations

0

|

Lead structure

of corresponding symmetrical and asymmetrical phthalimides will be compared in Section 6.

13e

-

13f

-

13h

O

13i

Fig. 7. Structural formulae of the asymmetrically substituted phthalimide compounds 13.

Preliminary investigations showed that the replacement of one phthalimide in W84 with the benzylidene phthalimidine moiety results in an increase in potency in comparison to W84 which could be further enhanced by the replacement of the second phthalimide with benzylidene phthalimidine moiety (Holzgrabe et al., 1997). Similar observations were made for the stepwise replacement of the phthalimide with the above discussed optimal naphthalimide substituent. The symmetrical compound appears to be about 100 times more potent than W84 and seems to be the most active compound reported till now. It is interesting to find out in the next step whether an asymmetrical compound composed of an hexane-bisammonium-chain connecting a benzylidene phthalimidine and a naphthalim-


ide will show a h i g h e r or l o w e r potency. Investigations in this direction are in progress.

7. Conclusions T a k e n together, it can be c o n c l u d e d that the 3D Q S A R analysis, w h i c h predicted h i g h e r affinity to the allosteric binding site by e n l a r g e m e n t of the n o n - o v e r l a p v o l u m e of the modulators, has led to highly potent c o m p o u n d s characterized by a rigid h y d r o p h o b i c m o i e t y in position 3 of the p h t h a l i m i d e and a large aromatic area annellated to the imide. T h e s e findings initiate the synthesis of n e w m o d u l a tors of h i g h e r allosteric p o t e n c y and m a y open the perspective to find c o m p o u n d s suitable for therapeutic purposes.

Acknowledgements T h a n k s are due to the D e u t s c h e F o r s c h u n g s g e m e i n schaft D F G and to the F o n d s der C h e m i s c h e n Industrie, D e u t s c h l a n d , for financial support, to the K A A D for the grant given to H M B C , to the E U for E r a s m u s grants given to EB, P M and CP, as well as to I r m e l a S c h w e s i g , Ilona K n o b l a u c h , F r a u k e MiSrschel and Iris W i t t e n for their skillful technical assistance and to H u g o K u b i n y i ( B A S F , L u d w i g s h a f e n , F R G ) for providing the B I L I N program.

References Andrews, P.R., 1993. Drug-receptor interactions. In: Kubinyi, H. (Ed.), 3D QSAR in Drug Design Theory Methods and Applications. ESCOM, Leiden, pp. 13-40. Bender, W., Staudt, M., Triinkle, C., Mohr, K., Holzgrabe, U., 1999. Probing the size of a hydrophobic binding pocket within the allosteric site of muscarinic acetlylcholine M2-receptors. Life Science, in press. Birdsall, N.J.M., Farries, T., Gharagozloo, P., Kobayashi, S., Lazareno, S., Sugimoto, M., 1999. Subtype-selective positive cooperative interactions between brucine analogs and acetylcholine at muscarinic receptors: functional studies. Mol. Pharmacol. 4, 778-787. B~ihm, H.-J., Klebe, G., 1996. Was liiBt sich aus der molekularen Erkennung in Protein-Ligand-Komplexen fiir das Drug-Design neuer Wirkstoffe lernen?. Angew. Chem. Int. Ed. Engl. 35, 2588-2614.

155

Botero Cid, H.M., Pick, R., Holzgrabe, U., Mies-Klomfal3, E., Kostenis, E., Mohr, K., 1999. Structure-activity relationships in a series of bisquaternary phthalimidine derivatives modulating the muscarinic M2-receptor allosterically. J. Med. Chem., submitted. Choo, L.K., Mitchelson, F., 1989. Characterization of the antimuscarinic effect of heptane-l,7-bis(dimethyl-3'-phthalimidopropyl ammonium bromide. Eur. J. Pharmacol. 162, 429-435. Christopoulos, A., Lanzafame, A., Mitchelson, F., 1998. Allosteric interactions of muscarinic cholinoceptos. Clin. Exp. Pharmacol. Physiol. 25, 185-194. Gharagozloo, P., Lazareno, S., Popham, A., Birdsall, N.J., 1999. A1losteric interactions of quaternary strychnine and brucine derivatives with muscarinic acetylcholine receptors. J. Med. Chem. 42, 438-445. Holzgrabe, U., Hopfinger, A.J., 1996. Conformational analysis, molecular shape comparison and pharmacophore identification of different allosteric modulators of muscarinic receptors. J. Chem. Inf. Comp. Sci. 36, 1018-1024. Holzgrabe, U., Mohr, K., 1998. Allosteric modulation of antagonist binding to muscarinic receptors. Drug Discovery Today 3, 214-222. Holzgrabe, U., Wagener, M., Gasteiger, J., 1996. Variation of the oxime function in bispyridinium-type allosteric modulators of M2-cholinoceptors. J. Mol. Graph. 14, 185-193. Holzgrabe, U., Staudt, M., Bender, W., Mesagna, P., Pfletschinger, C., Triinkle, C., Mohr, K., 1997. Allosteric modulation of antagonist binding to the acetylcholine M2-receptor: unilateral structural variations of alkane bisammonium-type modulators. Abstract DPhG and SGPhW Symposium, Ziirich. Jepsen, K., Liillmann, H., Mohr, K., Pfeffer, J., 1988. Allosteric stabiliza3 tion of H-N-methylscopolamlne binding in guinea-pig myocardium by an antidote against organophosphate intoxication. Pharmacol. Toxicol. 63, 163-168. Koolman, J., RiShm, K.-H., 1996. Color Atlas of Biochemistry. Thieme Verlag, Stuttgart, pp. 107-108. Kostenis, E., Holzgrabe, U., Mohr, K., 1994. Allosteric effect on M 2cholinoceptors of derivatives of the alkane-bis-ammonium compound W84 comparison with bispyridinium-type allosteric modulators. Eur. J. Med. Chem. 29, 947-953. Nassif-Makki, T., Triinkle, C., Bejeuhr, G., Cambareri, A., Pfletschinger, C., Kostenis, E., Mohr, K., Holzgrabe, U., 1999. Bisquaternary ligands of the common allosteric site of M 2 acetylcholine receptors: optimization of the distances between the pharmacophoric elements. J. Med. Chem. 42, 849-858. Triinkle, C., Mohr, K., 1997. Divergent modes of action among cationic allosteric modulators of muscarinic M 2 receptors. Mol. Pharmacol. 51,674-682. Triinkle, C., Kostenis, E., Burgmer, U., Mohr, K., 1996. Search for lead structures to develop new allosteric modulators of muscarinic receptors. J. Pharmacol. Exp. Ther. 279, 926-933. Triinkle, C., Mies-KlomfaB, E., Botero Cid, M.H., Holzgrabe, U., Mohr, K., 1998. Identification of a [3H]ligand for the common allosteric site of muscarinic acetylcholine M 2 receptors. Mol. Pharmacol. 54, 139145.




Receptors in cardiovascular disease" review and introduction Amedeo Leonardi *, Giorgio Sironi, Gianni Motta Pharmaceutical R&D Department, Recordati, Via M. Civitali 1, Milan 20148, Italy

Abstract Despite recent encouraging declines, cardiovascular disease (CVD) is still responsible for about 50% of premature death in the Western industrialized countries, greater than cancer, AIDS and accidents, combined. Different aspects of the disease have been considered and the main currently available and possible future drugs whose effect is based on interaction with a receptor have been reviewed. Catecholamines receptors ligands, mainly [3-blockers, and the new angiotensin II antagonists represent the most important classes among the established therapies. Investigational approaches such as the oral glycoprotein GPIIb/IIIa antagonists and endothelin, adenosine and neuropeptide Y receptors ligands are discussed. Receptorology represents just a part of the therapeutical approach to CVD, where other classes of drugs with enzyme or ionic channel based mechanisms are largely used and innovative therapies based on the most advanced research techniques could early become reality. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: Cardiovascular disease; [3-blockers; Mixed mechanism; Angiotensin II; Glycoprotein GpIIb/IIIa; Endothelin (ET)-I; Adenosine; Neuropeptide

Y

1. Introduction Despite the significant progress made in preventing and treating cardiovascular disease (CVD), it still is the leading cause of death in the developed world, and death due to CVD is increasing in these nations. Approximately 130 million people in the seven major pharmaceutical markets (the United States, Japan, France, Germany, Italy, Spain and the United Kingdom) suffer from CVD. In 1995, approximately 2.6 million people in these countries died from CVD. Diseases of the heart and vascular system encompass a variety of conditions, the most relevant of them being strictly interrelated, as show in Fig. 1. Hypertension and atherosclerosis are central to the pathogenesis of coronary artery disease (ischemia, angina, myocardial infarction) heart failure, cerebral (stroke) and peripheral vascular disease. Since these two risk factors have been widely investigated in the last years, many different therapeutic approaches are now available, in particular for hypertension, and this is reflected in the conspicuous market share


foreseen for them for the end of 1999, as shown in Table 1. The search for new approaches in the field of hypertension and atherosclerosis is still of interest; also if a major research investment is being devoted to the other conditions of CVD and, in particular, to their prevention. It is interesting to remark that the total cardiovascular market has been US$65 billion in 1998 and that 48 out of the top 50 pharmaceutical companies have research projects in the CVD field.

2. Receptors as therapeutic targets in CVD The main established, marketed therapeutics, having a receptor as their target, are shown in Table 2, subdivided by classes and paired to the investigational approaches (Fig. 2) in the same classes, encompassing some representative new drug candidates at different development levels. Among the oldest established therapies, catecholamine receptor ligands are the most important, with the [3-blockers as the major class. Although ~ - b l o c k e r s have been in use for a long time, their mechanism of action in hypertension is uncertain yet, although a number of possible mechanisms are likely to be involved. There are many [3-blockers now available and


158

A. Leonardi et al. / Pharmaeeutica Acta Helvetiae 74 (2000) 157-161

RENALDATMAGE~

ANGINA~

MI* ~ "

CEREBRALi AMAGE(STROKE)

HEART ~ FAILURE

LV HYPERTROPHY

ARRHYTHMIA 9MYOCARDIAL INFARCTION

Fig. 1. Cardiovasculardisease map.

their clinical use is based on three main characteristics: cardioselectivity, intrinsic sympathomimetic activity (ISA) and lipid solubility. Despite these differences, they are approximately equipotent as antihypertensive agents and the choice should be oriented toward cardioselective agents with ISA and low lipophilicity, offering the likelihood of minimizing side effects and poor interaction with lipid and carbohydrate metabolism upon once-a-day administration (Kaplan, 1997). Furthermore, the antiarrhythmic (class II) and antianginal effects of these drugs make them especially valuable in hypertensive patients with coexisting coronary disease. Mixed mechanism adrenergic agents are also in use as antihypertensives, like oL+ [3 adrenoceptor blockers, the most known being labetalol and carvedilol, which probably by virtue of its additional antioxidant activity is being used also for prevention and therapy of heart failure (Lowes et al., 1999). Other mixed mechanism drugs used in hypertension are the OLz-adrenergic + imidazoline 11 agonists (clonidine, guanfacil, rilmenidine, moxonidine) and the c~1-adrenergic + 5-HT 2 serotonergic antagonist, ketanserin. A new compound of this type has been recently reported (SUN 9221) (Mizuno et al., 1999). Also the oL1-adrenergic + Ca 2+channel blocker agents, monatepil (Honda et al., 1995) and S-2150 (Kimoto et al., 1997), have shown interesting results in animal models. The only receptor-targeted antihypertensive drugs in the renin-angiotensin system (RAS) are the antagonists of the angiotensin II (AT1) receptor. Angiotensin II is the final active component in the RAS and plays a major role in the regulation of blood pressure and renal function by interacting with the AT~ receptor, the AT 2 type being possibly involved in foetal/early live and wound healing (Reid, 1996). After the good results obtained in hypertensive patients with saralasin, a peptide analog of angiotensin II with partial agonist activity (Nicholls et al., 1994), many laboratories were involved in the search for non-peptidic antagonists of the AT~ recep-

tors and the first marketed drug of this class, losartan, has been followed by some others, with further compounds in final development stages. Angiotensin II antagonists confirmed the better side-effects profile expected by lack of interaction with the kallikrein-kinin-prostanoids system but in some cases, limited efficacy was shown and several associations with diuretics and Ca 2+ antagonists are under evaluation. Glycoprotein G P l l b / I l l a antagonists currently available for intravenous use as antithrombotics following coronary angioplasty (PTCA) represent a recently introduced class of drugs and contain one of the few monoclonal antibodies used as therapeutic, abciximab. Coronary thrombosis is largely platelet-dependent, through platelet adhesion, activation and aggregation. Aspirin and ticlopidine predominantly target platelet activation pathways but because of the variety of means through which platelet can be activated, the blockade of any single platelet activation pathway can have limited efficacy. The final, obligatory step in the formation of platelet aggregates is the cross-linking of platelets by plasma fibrinogen, or other matrix proteins. This process is mediated by the GPIIb/IIIa receptor located in the platelets, which becomes competent to bind fibrinogen upon platelet activation (Abrams and Shattil, 1997). Several antagonists of the platelet GPIIb/IIIa receptor endowed with oral activity are currently undergoing clinical studies (Olson et al., 1999) and their availability will surely help to lower the risk of thrombotic events in unstable angina and following PTCA.

3. Investigational approaches based on receptors Endothelin (ET)-I is the most potent vasoconstricting and hypertensive agent so far reported. It is produced by endothelial cells and targets with the same affinity two receptors: ETA and ET B, which are located in smooth muscle and endothelial cells of the vascular system and myocardium (Quadri et al., 1998). Several nonpeptidic antagonists selective for the ET-A are under evaluation mainly for their potential use in primary pulmonary hypertension, a lethal disease without

Table 1 Distribution by value of the current CVD therapies Cardiovascular agents Market share (%) Antihypertensives 50 Vasodilators 6 Hypolipidemics-antiatherosclerosis 20 Anticoagulants 12 Cardiac stimulants 3 Antiarrhythmics 4 Othersa 5 aCardiovascular shock, cardiac protectants.

159

A. Leonardi et al. / Pharmaceutica Acta Helvetiae 74 (2000) 157-161

Table 2 Main receptors in cardiovascular disease Drug class

Established targeted receptor

Drug

Investigational approach

Compound

Antihypertensives

[3-adrenergic antagonists [3-adrenergic antagonists (ISA)

propranolol, nadolol, timolol pindolol, carteolol, penbutolol

adenosine A~ antagonists endothelin A antagonists

[31-adrenergic cardioselective antagonist (no ISA) e~~-adrenergic antagonists

bevantolol, metoprolol, atenolol

neuropeptide Y antagonists

KW-3902 LU135252, TBC 11251, PD 163070 BIBP-3226

prazosin, terazosin, doxazosin

c~1-adrenergic + Ca 2+-channel antagonists

Monatepil, S-2150

oL+ [3-adrenergic antagonists 2-adrenergic + imidazoline I I agonists oL~-adrenergic + 5-HT 2serotoninergic antagonists dopamine D~ agonists angiotensin II-AT~ antagonists

labetalol, carvedilol clonidine, guanfacil, rilmenidine, moxonidine ketanserin

o~1-adrenergic + 5-HT2 antagonists

SUN-9221

adenosine A2 agonists (?)

CGS 22989

adenosine A 1 agonists glycoprotein IIb/IIIa antagonists

CVT-510 lefradafiban, sibrafiban, xemilofiban ramatroban, bosentan, SB 209670 GP-531

Coronary vasodilators Antianginal Hypertensives Antiarrhythmics Inhibitors of platelets function

aldosterone antagonists oL1-adrenergic antagonists

fenoldopam losartan, valsartan, irbesartan, eprosartan, candesartan spironolactone prazosin

[3-adrenergic antagonists oL-adrenergic agonists dopamine agonists [3-adrenergic antagonists glycoprotein IIb/IIIa antagonists

atenolol, acebutolol ephedrine, metaraminol dopamine acebutolol, propranolol abciximab, eptifibatide, tirofiban

Heart failure

established therapy (Prie et al., 1997), and their potential is being evaluated also as antihypertensives. In addition to its hypertensive action, ET-1 also exerts long-term negative effects such as hypertrophy and cellular injury in myocytes in the myocardium where both ET-A and ET-B receptors are present. In the failing heart, the production of ET-1 is markedly increased and long-term treatment with ET-1 antagonists greatly improved survival rate in different animal models of chronic heart failure. Whether ET-A-selective or dual ET-A/B antagonists could be better for the treatment of heart failure still has to be clearly defined (Miyauchi and Goto, 1999). Adenosine has many cardiovascular effects that lead to vasodilatation and hypotension accompanied by cardiac depression. The research in this field led to the discovery of different subtypes for these agonists and evolved toward the study of subtypes-selective agonists and antagonists. Due to the presence of the A 2 subtype in the coronary arteries, where adenosine exerts a vasodilator effect, the search for selective A 2 agonists was very intensive and produced many candidates, but most of them were discontinued (Quadri et al., 1998). Selective A~ antagonists, such as KW-3902, have shown potent diuretic activity and other compounds of this class

TxA 2 + PGH 2 antagonist endothelin antagonists (A or A + B?) adenosine agonist

are under clinical development for different cardiovascular indications. An adenosine A 1 agonist, CVT-510, is in clinical trials for use as an antiarrhythmic and another imidazole derivative with agonistic properties, GP-668, has shown interesting cardioprotective properties (Kurz et al., 1997). Neuropeptide Y (NPY) is one of the most abundant neuromodulatory peptides with potent effects on blood pressure homeostasis through both central and vascular sites of action. At least six subtypes of the NPY receptor have been so far identified, with Y1 as the subtype mainly involved in blood pressure control (Quadri et al., 1998). Peptide and non-peptide antagonists of the NPY~ receptor are available and all of them counteract the hypertensive effects of NPY but not of other mediators such as noradrenaline and AGII. Whether NPY 1 antagonists could become general-use antihypertensive drugs still has to be clarified. Receptorology played and is playing a relevant role in CVD moving from [3-blockers to newer approaches. It must be, however, emphasized that the classes of drugs currently most frequently used in CVD have different mechanisms, namely, they are inhibitors of several enzymes such as ACE, HMG-CoA reductase, COX, carbonic

160

A. Leonardi et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 157-161

H2 H2N" ~ /

~

~'""~,,,., H 0

Lefradafb ian

, , O , v , COOE,

--

~

Xemilofiban

H2

~.

I~H

MeO~~UOH

Ramatroban H ~

3H3C

Bosentan.~,~F

H2

H

NH2

H~ ~

Monatepil

GP-531 HaC~ Me~~N~~I

MeO

H

CH3

SUN-9221

S-2150

H3

C

~

H

LU135252

~H3

MeNo ~" ~v' 1 3 H 3

PD163070

TBC11251 H OH H

BIBP-3226 Fig. 2. Compounds under investigation approach.

anhydrase, PDE or ion channels modulators like the blockers of the Ca 2+, Na + or C1- channels or activators of the K § channels.

Furthermore, enzymes are utilized as life-saving drugs such as streptokinase or tissue plasminogen activator (TPA) in myocardial infarction, and mucopolysaccharides such as

A. Leonardi et al. / Pharmaceutica Acta Helvetiae 74 (2000) 157-161

heparins and also monoclonal antibodies are in use or under evaluation in cardiovascular shock.

4. Conclusion The classical receptorial approach is just a part of the research in CVD, where the search for new biological targets by use of the most advanced techniques is a reality and will yield innovative therapies in the future.

References Abrams, C., Shattil, S.J., 1997. The platelet integrin, GP IIb/IIIa (e~iib /[33). Adv. Mol. Cell. Biol., Vol. 18. JAI Press, Greenwich, CT, pp. 67-107. Honda, Y., Masuda, Y., Yoshida, T., Sato, F., Kurokawa, M., Hosoki, K., 1995. Studies on calcium antagonistic and oL~-adrenergic receptor blocking activities of monatepil maleate, its metabolites and their enantiomers. Arzneim.-Forsch./Drug Res. 45 (II), 1057-1060. Kaplan, N.M., 1997. Systemic hypertension: therapy. In: Braunwald, E. (Ed.), Heart Disease. W.B. Saunders, pp. 853-854. Kimoto, S., Haruna, M., Matsuura, E., Uno, O., Ishii, M., Hirono, S., Yoshimura, K., Ueda, M., Iwaki, K., 1997. Pharmacological studies on a new antihypertensive agent, S-2150, a benzothiazepine derivative: 3. Hypotensive and antimyocardial-stunning effects in dogs. J. Cardiovasc. Pharmacol. 29 (2), 180-187. Kurz, M.A., Bullough, D.A., Bugge, C.J., Mullane, K.M., Young, M.A., 1997. Cardioprotection with a novel adenosine-regulating agent medi-

161

ated by intravascular adenosine. Eur. J. Pharmacol. 322 (2-3), 211220. Lowes, B.D., Gill, E.A., Abraham, W.T., Larrain, J.R., Robertson, A.D., Bristow, M.R., Gilbert, E.M., 1999. Effects of carvedilol on left ventricular mass, chamber geometry, and mitral regurgitation in chronic heart failure. Am. J. Cardiol. 83 (8), 1201-1205. Miyauchi, T., Goto, K., 1999. Heart failure and endothelin receptor antagonists. TIPS 20, 210-217. Mizuno, A., Inomata, N., Miya, M., Kamei, T., Shibata, M., Tatsuoka, T., Yoshida, M., Takiguchi, C., Miyasaki, T., 1999. Synthesis and pharmacological evaluation of pyrroloazepine derivatives as potent antihypertensive agents with antiplatelet aggregation activity. Chem. Pharm. Bull. 47 (2), 246-256. Nicholls, M.G., Charles, C.J., Crozier, I.G., Espiner, E.A., Ikram, H., Rademaker, M.J., Richards, A.M., Yandle, T.G., 1994. Blockade of the renin-angiotensin system. J. Hypertens. 12 (10), $95-S 103, Suppl. Olson, R.E., Sielecki, T.M., Wityak, J., Pinto, D.J., Batt, D.G., Frietze, W.E., Liu, J., Tobin, A.E., Orwat, M.J., Di Meo, S.V., Houghton, G.C., Lalka, G.K., Mousa, S.A., Racanelli, A.L., Hausner, E.A., Kapil, R.P., Rabel, S.R., Thoolen, M.J., Reilly, T.M., Anderson, P.S., Wexler, R.R., 1999. Orally active isoxazoline glycoprotein IIb/IIIa antagonists with extended duration of action. J. Med. Chem. 42, 1178-1192. Prie, S., Leung, T.K., Cernacek, P., Ryan, J.W., Dupuis, J., 1997. The orally active ET(A) receptor antagonist (+)-(S)-2-(4,6-dimethoxypyrimidin-2-yloxy)-3-methoxy-3,3-diphenyl-propionic acid (LU 135252) prevents the development of pulmonary hypertension and endothelial metabolic dysfunction in monocrotaline-treated rats. J. Pharmacol. Exp. Ther. 282 (3), 1312-1318. Quadri, L., Gobbini, M., Monti, L., 1998. Recent advances in antihypertensive therapy. Curr. Pharm. Des. 4, 489-512. Reid, J.L., 1996. New therapeutics for hypertension. Br. J. Clin. Pharmacol. 42 (I), 37-41.



Pharmaceutica Acta Helvetiae 74 (2000) 163-171 www.elsevier.corn/locate/pharmactahelv

Adrenoceptor subclassification" an approach to improved cardiovascular therapeutics J.

Paul Hieble

*

Division of Pharmacological Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA

Abstract The subdivision of 0/ adrenoceptors into the 0/1 and 0/2 classes was the impetus for the design of the selective 0/1-adrenoceptor antagonists, which remain useful antihypertensives. 0/2-Adrenoceptor agonists also have application as antihypertensive drugs, based on their ability to reduce sympathetic outflow. Likewise, subdivision of the /3 adrenoceptors has lead to the development of selective /3~-adrenoceptor antagonists as antihypertensives and selective /32 agonists as bronchodilators. In the past decade, both the 0/1 and 0/2 adrenoceptors have been further subdivided, each into three subclasses. In addition, there is strong functional evidence to suggest the presence of additional adrenoceptor subtypes, such as the "0/~L" adrenoceptor and "/34" adrenoceptor. 0/IA (or 0/~L)-Adrenoceptor antagonists have been evaluated for benign prostatic hyperplasia (BPH), and selective 0/~A agonists for stress incontinence. Gene knockout experiments in mice suggest an important role for the 0/1B adrenoceptor in the control of vascular tone. Hence, selective 0/~B antagonists may offer a new approach toward hypertension. Although targeting of specific adrenoceptors can be used to optimize the therapeutic profile of a drug, there are also cases where blockade of multiple adrenoceptors is desirable, as with the 0///3-adrenoceptor antagonist carvedilol in congestive heart failure. It is possible that combination of affinities for selected adrenoceptor subtypes within a single molecule may be desirable for certain applications. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: Hypertension; Benign prostatic hyperplasia; Congestive heart failure; Cardiac arrhythmia; O~ILAdrenoceptor; ]~4 Adrenoceptor

1. Subclassification of adrenoceptors The receptors for the catecholamines adrenaline and noradrenaline, designated as the adrenoceptors, have been studied for almost a century and have provided many targets for drug action. Before the advent of molecular biology, functional assays were used to initially divide adrenoceptors into the major classes o~ and /3 (Ahlquist, 1948) and then into al and o~2 (Langer, 1974; Starke et al., 1974) and /31 and /32 (Lands et al., 1967). This subclassification was supported by the identification of selective agonists and antagonists for most of these subtypes, and by the use of radioligand binding techniques to provide a more direct measure of receptor affinity. These radioligand binding assays provided evidence that further division of the al and og2 classes was possible (Bylund, 1985; Morrow and Creese, 1986). The adrenoceptors were one of the first targets for the cloning techniques devel-

* Tel.: +1-610-270-6053; fax: +1-610-270-5080

oped in the 1980's. At present, nine human adrenoceptors have been cloned. These fall into three groups ( a 1, o/2 and /3). After some initial confusion (see Hieble et al., 1995), there now appears to be a correlation between the recombinant adrenoceptors and those previously characterized in native tissues. The cloning of a third /3-adrenoceptor subtype (Emorine et al., 1987) supported functional studies (Arch et al., 1984; Wilson et al., 1984) characterizing an "atypical" /3 adrenoceptor and showing that its selective activation by novel agonists was possible. In addition to the human receptors, adrenoceptors from several nonhuman species have also been cloned. Table 1 compares amino-acid identity between the nine rat and human adrenoceptors. Several observations can be made from this Table. (1) There is at least 30% amino acid identity when comparing any adrenoceptor of either rat or man. This is expected, since all of these receptors are activated by the same catecholamine ligands. (2) Based on amino acid identity, the adrenoceptors fall into three groups, a 1, o/2 and /3, with about 50% identity between the three mem-


164

J.P. Hieble / Pharmaceutica Acta Helvetiae 74 (2000) 163-171

Table 1 Comparison of amino acid identities between human and rat recombinant adrenoceptor subtypes

Immmm//llm -.-

~ t

50 ~

'

-.-_-

-_-_-_..-.-

-_-_-_- -_.=-_-_-.-_-.-

- -.-_-.-

-_-.-

-.- .-.-.-.-.-

- - -.-..

- - -_-_-.-.-.-.-

-_-_-.-.-

-.-_-.-_-.-_-

i:i:i:i:i:!:i:i:!:l~ i1

36

40

37

36

34

35 ~ ~ ~ ~

38 52

38 55

36 37

35 36 34 I_ !

h~2b ~i!!~i~g,..~i ~.i;". 55 hO~2c

35

32

~.,..~5

h~ld ~ hc~2a

hi31

!i ! !!i ! !i))!i!iiiililili))ii)))iii!iii))iii)iii)ii!iii!i iii!i)iiiiiiiiiiiiiiiil)iiiiiiii iiiiiiiiiii)iiiiiiiiiiiiiiilililiiiiimlilili

l

/~l

~ ~i~ii::::::::::i::i::::::i~ ii::i::ii::::i!::!::!i!i!!::!::ii::::i:::::::::: ::::::::iii::::::::::i::::::::::::::::ii::::::::::::i:::: i::ili::i:::::::::::::::::::::: ::i::::::i:::::::::[l::::::i:::::::i

/lllRlBll/ll

2Z

ra2b r~2c

r~l r~2 r~3

bers of each group and 30%-40% identity between receptors in different groups. This is consistent with functional adrenoceptor pharmacology, which suggests much greater divergence between a l, a 2 and 13 adrenoceptors than between subtypes of either class. (3) There is a high degree of amino acid identity between rat and human homologs of each adrenoceptor subtype. The possibility remains that additional adrenoceptors may exist. It is well known that there is pronounced variability in the sensitivity of a 1-mediated responses to prazosin, a potent and selective a 1 antagonist (Drew, 1985). This variability led to the proposal for division of the class into a li~ and a l l adrenoceptors, based on sensitivity to prazosin (Flavahan and Vanhoutte, 1986; Muramatsu et al., 1990). When the recombinant a 1 adrenoceptors were identified, it was found that both a la, O~lb and ald subtypes had " a l H " characteristics, based on radioligand binding assays to transfected cells. Antagonists were identified, such as RS 17053 (Ford et al., 1996), which had high potency and selectivity for the a la subtype, but about 100-fold lower potency in functional assays reflecting " a l L " affinity. Other antagonists appear to show the opposite selectivity pattern (Hieble and Ruffolo, 1997). Based on functional assays using cells transfected with the recombinant a la adrenoceptor, and on the effect of receptor environment on antagonist affinity, it has been proposed that the a l l adrenoceptor represents an affinity state of the ala adrenoceptor (Williams et al., 1996; Ford et al., 1997). This may explain why efforts to clone a discrete protein having a l l characteristics have been unsuccessful.

Several /3-adrenoceptor antagonists such as pindolol, are known to produce cardiac stimulation, both in man and experimental animals. This intrinsic sympathomimetic activity does not appear to be mediated by either the 131- or 132- adrenoceptor (Kaumann, 1989). While it was initially thought that the /33 adrenoceptor might be involved, it now appears that a novel 13 adrenoceptor may be present in cardiac tissue, mediating the inotropic response to these atypical agonists (Kaumann, 1998). This receptor has been studied primarily with CGP-12177, a phenoxypropanolamine having potent antagonist activity at /31 and 132 adrenoceptors, and partial agonist activity at the /33 subtype. In vitro studies using rat or human atrium show an inotropic response to CGP-12177 which is insensitive to propranolol, but can be blocked by bupranolol (Kaumann and Molenaar, 1997). This response is not due to 133adrenoceptor activation, since it is present in mice where the 133 adrenoceptor has been genetically eliminated (Kaumann et al., 1998). This putative receptor is currently designated as the "/34" adrenoceptor.

2. Selective adrenoceptor agonists/antagonists as therapeutic agents

2.1. a2-Adrenoceptor agonists Clonidine was one of the first effective antihypertensive drugs. While actions at imidazoline receptors have been postulated (Ernsberger et al., 1992), it is likely that clonidine and related agents lower blood pressure via an action


on brainstem O~2 adrenoceptor to reduce sympathetic outflow to heart and blood vessels. Activation of o~2 adrenoceptors at other sites within the central nervous system results in sedation and analgesia. These actions have also been exploited clinically, although sedation is an unwanted side-effect commonly associated with clonidine therapy. Studies using mice having specific ce2-adrenoceptor subtypes knocked out or mutated have established that the sympathoinhibitory action of an az-adrenoceptor agonist results from cezA-receptor activation (MacMillan et al., 1996; Rohrer and Kobilka, 1998). Interestingly, the initial pressor response commonly observed upon intravenous administration of an c~2-adrenoceptor agonist is absent in O~2b-knockout mice (Link et al., 1996). This is the first evidence for a cardiovascular response mediated by the O~2B subtype. The sedation and analgesia associated with central a2-adrenoceptor activation had been proposed to result from cezA-adrenoceptor activation (Mizobe et al., 1996). Experiments in mice with mutated C~2a adrenoceptors are consistent with this proposal (MacMillan et al., 1996). A Ce2A involvement in both the hypotensive and sedative effects of a centrally acting c~2-adrenoceptor agonist would explain the failure of extensive structure-activity studies in this pharmacological class to identify agonists capable of dissociating these two actions. Even if the cardiovascular and sedative/analgesic actions cannot be dissociated, a subtype selective Ceza-adrenoceptor agonist may offer therapeutic advantages for both indications, since the unwanted vasoconstrictor action would be eliminated. Other therapeutic actions have been identified for centrally active Cez-adrenoceptor agonists, for example, behavioral improvement in attention deficit hyperactivity

165

disorder (Hunt et al., 1990; Hunt et al., 1995). The subtype involved in this action has not been identified; hence the possibility remains that a subtype selective agonist might offer improved therapeutic efficacy.

2.2. ce2-Adrenoceptor antagonists Although some drugs have ce2-antagonist activity as a significant component of their pharmacological profile, e.g., antidepressants such as mirtazepine, no selective a zantagonist has been successfully developed for any cardiovascular (or noncardiovascular) indication. Potential targets for an az-antagonist include hypertension (Sawyer et al., 1985; Roesler et al., 1986), obesity (Hieble and Ruffolo, 1991; Hieble and Kolpak, 1992), non-insulin-dependent diabetes (Angel et al., 1993) and erectile dysfunction (Munoz et al., 1994). The antagonists evaluated for the above indications did not differentiate between ce2-adrenoceptor subtypes. Indeed, with the exception of the O~2a-antagonist BRL 48962 (Beeley et al., 1995; Table 2), o/2 antagonists showing pharmacologically significant selectivity between subtypes have not been identified. It is possible that postsynaptic a 2 adrenoceptors may have c~2B (Link et al., 1996) or a zc (Craig et al., 1995) characteristics. Since the presynaptic O/2 adrenoceptors regulating neurotransmitter release from sympathetic neurons appear to have CgZA pharmacology in almost all species and tissues examined (Trendelenburg et al., 1997), it is likely that a selective aZB or O~2C antagonist would not elevate plasma catecholamines, an action which has limited the development and clinical application of non-subtype selective antagonists.

Table 2 Affinities of adrenoceptor antagonists (Ki, nM) for recombinant human adrenoceptors, as determined by radioligand binding assay. Except where noted, data determined in our laboratories, using [3H] prazosin, [3H] rauwolscine and [125I] iodocyanopindolol to label cel, o~2 and /3 adrenoceptors, respectively Compound

ce l a

~

O~ld

19/2a

O~2b

O~2c

/31

/32

/33

SNAP 5089 SNAP 5540 a Ro 70-004 b L-765,314 c ( + ) Cyclazosin d BMY 7378 BRL 48962 CGP 20712 ICI 118551 Rec 15/2739 SL 89 0591 Tamsulosin Prazosin Terazosin Doxazosin Carvedilol

1.4 4 1.3 420 33 443 1881 3000 1960 0.7 3.2 0.05 0.6 6.9 2.4 2.8

79 4920 79 2.0 0.7 109 2721 > 5000 > 5000 19 11 0.63 0.3 2.2 0.4 0.9

205 7740 63 34 27 0.6 1785 > 5000 > 5000 6.4 2.7 0.09 0.3 2.4 0.8 2.2

> 5000 ND ND > 70 ND 1135 30 > 5000 3000 57 353 46 2134 3700 729 26

682 ND ND > 70 ND 729 1317 300 > 5000 491 79 174 365 418 > 5000 27

1374 ND ND > 70 ND 270 2867 1180 778 39 174 73 95 213 279 11

> 5000 ND ND ND ND > 5000 > 5000 12 114 ND ND 571 > 5000 > 5000 > 5000 0.4

> 5000 ND ND ND ND > 5000 > 5000 2507 1.2 ND ND 1800 > 5000 > 5000 > 5000 0.2

> 5000 ND ND ND ND > 5000 ND > 5000 2263 ND ND > 5000 > 500O > 5000 > 5000 35

aData bData CData d Data

from from from from

Nagarathnam et al. (2998). Tang et aI. (1997). Patane et al. (2998). Giardina et al. (1996).

166


2.3. Cel-Adrenoceptor agonists In addition to their use for maintenance of systemic blood pressure during surgery, a 1 agonists may be useful to increase the tone of the urethral sphincter as therapy for stress incontinence. A drug for this indication, midodrine, presumably acts via its active metabolite, an analog of methoxamine. Several reports suggest that is possible to increase urethral tone, both in vitro and in vivo, without an associated increase in systemic blood pressure (Taniguchi et al., 1996). An a l agonist having this selectivity profile, NS-49, is currently in clinical trials for stress incontinence. It has been proposed that the urethral selectivity of NS-49 is a consequence of its ability to selectively stimulate the OllA adrenoceptor (Taniguchi et al., 1997). However, many noncatechol agonists at the O~1 adrenoceptor have this pattern of subtype selectivity (Minneman et al., 1994) and evaluation of additional agonists will be required to determine the relationship between agonist selectivity between a~ subtypes and urethral selectivity.

2.4. al-Adrenoceptor antagonists 2.4.1. Hypertension The nonselective a adrenoceptor antagonists phenoxybenzamine and phentolamine were evaluated clinically as antihypertensive drugs many years ago. Despite their ability to block the vasoconstrictor actions of norepinephrine in vitro and in animal models, they failed to produce a sustained reduction in supine blood pressure. The introduction of prazosin, the first highly selective C~l-adrenoceptor antagonist, is the classic example of improved therapeutic efficacy as a result of subtype selectivity. Prazosin proved to be a highly effective antihypertensive drug, and its congeners, doxazosin and terazosin, which offer similar pharmacology with a longer duration of action, remain important options in the treatment regimen available for hypertension. In addition to blood pressure reduction, a 1antagonists offer the advantages of a favorable effect on plasma lipoproteins and a low incidence of sexual dysfunction (lower than placebo in some studies) (Kirby and Pool, 1997). Based on determination of mRNA levels, blood vessels can possess all three a l-adrenoceptor subtypes, with the predominant receptor varying between vessel location and species. The only a 1-knockout experiments which have been reported show elimination of the a~B adrenoceptor to reduce the response to a l-adrenoceptor activation both in vitro and in vivo (Cavalli et al., 1997). Selective a~Badrenoceptor antagonists, such as (+)-cyclazosin and L765,314 (Table 2) have only recently become available, and their cardiovascular profile has not been extensively characterized. Selective alg antagonists have been studied in more detail. These compounds appear to have much less effect on blood pressure than non-subtype selective antagonists such as prazosin.

2.4.2. Cardiac arrhythmia There is a substantial literature showing that C~l-adrenoceptor antagonists such as prazosin are effective against ischemia-induced arrhythmias in a variety of animal models (Corr et al., 1989). There is no corresponding clinical data; a potent, non-subtype selective, a l-adrenoceptor antagonist, abanoquil (UK 52046) was evaluated for this indication but development was terminated before any efficacy data was reported. Recent data would suggest that activation of the a lA subtype may be responsible for ischemia-induced cardiac arrhythmia, based on blockade by WB-4101, an antagonist with moderate selectivity for a l A VS. a l B adrenoceptors, of phenyelphrine (Yasutake and Avkiran, 1995) or cocaine-induced (Billman, 1993) potentiation of ventricular fibrillation in ischemic models. Hearts from transgenic rats expressing constitutively active c~IB adrenoceptors, and having 50% reduced a lg-mRNA levels, were less sensitive to ischemia-induced ventricular tachycardia than normal rats (Harrison et al., 1998). This suggests that activation of the t~la subtype may exert a protective effect. A similar conclusion was postulated from studies on phenylephrine-induced electrophysiological changes in isolated purkinje fibers (Lee and Rosen, 1993). It would appear that evaluation of the anti-arrhythmic activity of one of the highly selective a lg-adrenoceptor antagonists in the models where prazosin has been shown to be effective (Corr et al., 1989) is warranted, especially since these compounds apparently lack the potent hypotensive activity associated with the non-subtype selective antagonists. 2.4.3. Other cardiac indications Many studies have shown that chronic a~-adrenoceptor activation can induce cardiac hypertrophy. Studies in cells transfected with the recombinant a 1 adrenoceptors show that all three subtypes can increase levels of early gene markers of cellular proliferation (Garcia-Sainz et al., 1998). Use of subtype selective antagonists indicated that phenyelphrine-induced hypertrophy in neonatal rat ventricular myocytes was mediated by the alA subtype (Knowlton et al., 1993). Another study using these myocytes showed that chronic exposure to norepinephrine selectively increased a~a-mRNA levels (Rokosh et al., 1996). Interestingly, several nonadrenergic hypertrophic stimuli, including increasing cardiac work by aortic banding, also selectively increased C~a mRNA. In both genetic (Kagiya et al., 1991) and pressure overload-induced (Tamai et al., 1989) cardiac hypertrophy, myocardial a l-adrenoceptor density was increased, and hypertrophy could be reduced by chronic a l-adrenoceptor blockade. The contribution of individual a l-adrenoceptor subtypes was not examined in these studies. Transgenic mice with specific cardiac expression of constitutively active Ollb adrenoceptors showed cardiac hypertrophy, as reflected by an increased heart/body weight ratio and increases in diacylglycerol and atrial


natriuretic factor (Milano et al., 1994). In contrast, overexpression of wild-type C~b adrenoceptors in mice does not result in cardiac hypertrophy. Studies in isolated working hearts from these mice show depressed resting function, which can be normalized by chronic treatment with prazosin (Rohrer and Kobilka, 1998). The above data suggests an important role for the a~ adrenoceptor in cardiac hypertrophy induced by a variety of stimuli, including genetic models. It is not yet clear which subtype or subtypes are involved; however, subtype selective o~ antagonists are now available, and their application in these models may suggest a viable therapeutic approach.

2.4.4. Benign prostatic hyperplasia Phenoxybenzamine and phentolamine have been shown to be effective in the relief of urethral obstruction induced by benign prostatic hyperplasia (BPH). Norepinephrine-induced contraction of isolated human prostate was shown to result from c~1-adrenoceptor activation (Hieble et al., 1985), and selective ce~-adrenoceptor antagonists have been proven to be effective drugs for B PH (Eri and Tveter, 1995). Over the past decade, there has been an intensive search for "uroselective" ce~-adrenoceptor antagonists for BPH which would avoid the cardiovascular side-effects, such as dizziness and orthostatic hypotension, sometimes associated with the currently used cel-adrenoceptor antagonists. Compounds have been evaluated for potency against agonist or stimulation-induced increases in urethral pressure relative to blood pressure reduction or blockade of agonist-induced blood pressure increases. Many selective antagonists have been identified (Hieble and Ruffolo, 1997; Kenny et al., 1997). These include highly selective CelA antagonists, representing several structural classes (Table 2). This selectivity profile was targeted as a result of molecular biological and radioligand binding studies which showed the CglA adrenoceptor to predominate in human prostate, and correlations of antagonist potency with affinity for recombinant ce~ adrenoceptors which showed the best correlation with the Ce~a subtype (Forray et al., 1994; Gluchowski et al., 1995). Nevertheless, data with compounds such as RS 17053 (Ford et al., 1996; Kava et al., 1998) as well as other antagonists showing low affinity for the "Ce~L" adrenoceptor (Leonardi et al., 1997) would indicate that the response of lower urinary tract tissues of human and other species has O~L characteristics. A number of Cel-adrenoceptor antagonists which show only moderate (e.g., Rec 15/2739) or little (e.g., SL 89.0591) selectivity for OflA adrenoceptors vs. the other two subtypes (Table 2) also show a high degree of uroselectivity in animal models (George et al., 1995; Testa et al., 1997). Several of the antagonists found to be uroselective in the dog have been evaluated in B PH patients, but no positive data has yet been reported. Hence, the question remains whether the uroselectivity predicted in animals models will lead to improved therapy.

167

The most recently introduced ce1 antagonist for B PH, tamsulosin, has selectivity for C~IA and CelD adrenoceptors, vs. CelB (Table 2). Tamsulosin shows either no selectivity (Kenny et al., 1994; Blue et al., 1997) or slight urethral selectivity (Brune et al., 1996; Testa et al., 1997) in conscious or anesthetized dogs. A small study comparing tamsulosin and terazosin showed a lower incidence of side effects with tamsulosin (Lee and Lee, 1997), although a similar comparison between tamsulosin and alfuzosin showed a similar incidence (Wilde and McTavish, 1996). Unlike most of the a 1-antagonists, tamsulosin does not require dose-titration, since blood pressure is not significantly reduced at doses effective for BPH. However, this may be a consequence of the delayed-release preparation used, since early studies with standard formulations in normal volunteers showed a hypotensive effect (Tsunoo et al., 1990). c~-Adrenoceptor antagonists have been shown to decrease firing rate in peripheral sympathetic neurons (Ramage and Wyllie, 1995). It is possible that the efficacy of c~,-antagonists in B PH may be a consequence of an action within the central nervous system (brain or spinal cord) to decrease sympathetic outflow to the prostate. The receptor subtypes involved in such an action are as yet unknown.

2.5. ~-Adrenoceptor agonists Agonists such as BRL 37144 showing moderate selectivity for /33 vs. /~2 and /31 adrenoceptors have been shown to produce both thermogenesis and lipolysis in rats (Arch and Wilson, 1996). These actions contribute to the ability of these agonists to produce weight loss in obese rats and reduce hyperglycaemia in diabetic rats (Sennitt et al., 1985; Cawthorne, 1990). Several /33 agonists have been evaluated in man as potential anti-obesity and anti-diabetic drugs. The results of these clinical trials have been disappointing, in part due to/3z-mediated side effects, such as tremor. Studies in isolated adipose tissue from human and rodent sources showed BRL 37144 to promote lipolysis only in the rodent tissue (Langin et al., 1991). When the recombinant /3 adrenoceptors became available, it was confirmed that most of the compounds showing agonist activity on rodent /33 adrenoceptors showed affinity, but little efficacy, at the human receptor. Compounds showing potent and selective agonist activity at the human /33 adrenoceptor have been identified (Sennitt et al., 1998); clinical data on the efficacy of such compounds is not yet available. /33-adrenoceptor m-RNA has been identified in human bladder detrusor, and isoproterenol-induced relaxation in this tissue was insensitive to blockade by either/31 or ~2 antagonists (Takeda et al., 1996). Hence it is possible that /33 agonists may also have application in disorders associated with detrusor hyperactivity, such as urge incontinence.

168


3. Targeted combination of affinities for adrenoceptor subtypes As noted above, there are several examples of improvement of clinical efficacy via introduction of selectivity for a particular adrenoceptor subtype. However, there are also examples of drugs where an action at multiple adrenoceptors is likely to contribute to the therapeutic profile. Carvedilol has affinity < 100 nM for all nine adrenoceptor subtypes, as determined by binding assays to cells expressing recombinant human receptors (Table 2). Although other /3-adrenoceptor antagonists have been found to be effective in reducing mortality and morbidity in congestive heart failure, none thus far have matched the 65% reduction in mortality and 30% reduction in hospital admissions seen with carvedilol (Packer et al., 1996). Although a~-adrenoceptor antagonists produce a favorable acute effect on hemodynamics in heart failure patients, chronic a 1-blockade shows no beneficial effect on mortality (Cohn et al., 1986). It is possible that the a~- and /3adrenoceptor antagonist actions of carvedilol are synergistic in the therapy of heart failure patients. In several animal models of cardiac ischemia, carvedilol produced a significantly greater cardioprotective effect than /3-adrenoceptor antagonists such as metoprolol (Feuerstein et al., 1998) or propranolol (Hamburger et al., 1991; Bril et al., 1992). There is some rationale for the design of an antagonist with a specific spectrum of antagonist activity. There are therapeutic targets for a~-adrenoceptor antagonists where the addition of a2-antagonist activity may improve therapeutic efficacy, e.g., hypertension and B PH. Blockade of prejunctional a 2 adrenoceptors is probably unacceptable in either of these conditions, as a consequence of sympathoactivation. However, if the postjunctional a 2 adrenoceptor to be targeted is the a2B adrenoceptor (which may be the case in hypertension), it may be advantageous to selectively introduce this activity. Compounds could be targeted toward hypertension ( a l B / a 2 B ) or BPH (alL / a2B). Antagonists such as prazosin and terazosin do indeed have some a2B-antagonist activity (Table 2). Although this activity can be demonstrated in vivo (McCafferty et al., 1999), it is several orders of magnitude weaker than that at the a 1 adrenoceptor, and is unlikely to contribute to the antihypertensive activity of these compounds.

4. Conclusions In view of the diverse actions of the catecholamine neurotransmitters, the design of subtype-selective agonists and antagonists is a logical approach in drug design. The original subdivision of adrenoceptors into the a and /3 classes has yielded important drugs acting on both classes. Likewise, targeting specific a- or/3-adrenoceptor subtypes has proven useful.

The introduction of additional selectivity via limiting activity to a single subtype of either a 1 or a 2 adrenoceptors should lead to more effective drugs and perhaps even to new therapeutic targets. However, hypothesis remains to be proven in man. Medicinal chemists have been able design highly potent and selective a lA antagonists, but their efficacy in BPH has not yet been convincingly demonstrated. It is possible that the currently utilized non-subtype selective antagonists may be required to block some adrenoceptor mediated actions which involve multiple subtypes. The ability to identify the specific adrenoceptor subtypes in a tissue and characterize their functions may allow the design of drugs having the optimum spectrum of pharmacological activity. It would also appear that additional receptor subtypes remain to be targeted. Even if the alL adrenoceptor is not a discrete protein, but rather an affinity state of the a~A adrenoceptor, it appears that its selective blockade is possible (Hieble and Ruffolo, 1997). There is evidence that the intrinsic sympathomimetic activity of some /3-adrenoceptor antagonists may be deleterious under some conditions (Willette et al., 1998). Since the "/34" adrenoceptor appears to be involved in this action it is possible that 134 blockade may be beneficial, especially if the 134 receptor is activated in heart failure patients.

References Ahlquist, R.P., 1948. A study of the adrenotropic receptors. Am. J. Physiol. 153, 586-600. Angel, I., Grosset, A., Perrault, G., Schoemaker, H., Langer, S.Z., 1993. In vivo pharmacological profile of SL 84.0418, a new selective, peripherally active a2-adrenoceptor antagonist. Eur. J. Pharmacol. 234, 137. Arch, J.R., Wilson, S., 1996. Prospects for /33-adrenoceptor agonists in the treatment of obesity and diabetes. Int. J. Obes. Relat. Metab. Disord. 20, 191-199. Arch, J.R.S., Ainsworth, A.T., Cawthorne, M.A., Piercy, V., Sennitt, M.V., Thod, V.E., Wilson, C., Wilson, S., 1984. Atypical /3-adrenoceptors on brown adipocytes as target for anti-obesity drugs. Nature 309, 163-165. Beeley, L.J., Berge, J.M., Chapman, H., Hieble, P., Kelly, J., Naselsky, D.P., Rockell, C.M., Young, P.W., 1995. Synthesis of a selective alpha-2A adrenoceptor antagonist, BRL 48962, and its characterization at cloned human alpha-adrenoceptors. Bioorg. Med. Chem. 3, 1693-1698. Billman, G.E., 1993. The effect of adrenergic receptor antagonists on cocaine-induced ventricular fibrillation: alpha but not beta adrenergic receptor antagonists prevent malignant arrhythmias independent of heart rate. J. Pharmacol. Exp. Ther. 269, 409-416. Blue, D.R., Daniels, D.V., Ford, A.P.D.W., Williams, T.J., Eglen, R.M., Clarke, D.E., 1997. Pharmacological assessment of the a l-adrenoceptor antagonist tamsulosin. Am. J. Urol. 157, 190, Suppl. Bril, A., Slivjak, M., DiMartino, M., Feuerstein, G.Z., Linee, P., Poyser, R.H., Ruffolo, R.R. Jr., Smith, E.F., 1992. Cardioprotective effects of carvedilol, a novel fl-adrenoceptor antagonist with vasodilating properties, in anesthetized minipigs: comparison with propranolol. Cardiovasc. Res. 26, 518-525. Brune, M.E., Katwala, S.P., Milicic, I., Buckner, S.A., Ireland, L.M., Kerwin, J.F. Jr., Hancock, A.A., 1996. Effects of selective and


nonselective alpha-1 adrenoceptor antagonists on intraurethral and arterial pressures in intact conscious dogs. Pharmacology 53,356-368. Bylund, D.B., 1985. Heterogeneity of ~x2-adrenergic receptors. Pharmacol. Biochem. Behav. 22, 835-843. Cavalli, A., Lattion, A.L., Hummler, E., Nenniger, M., Pedrazzini, T., Aubert, J.F., Michel, M.C., Yang, M., Lembo, G., Vecchione, C., Mostardini, M., Schmidt, A., Beermann, F., Cotecchia, S., 1997. Decreased blood pressure response in mice deficient of the alphalbadrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 94, 11589-11594. Cawthorne, M.A., 1990. Thermogenic drugs. The potential of selective /3-adrenoceptor agonists as anti-obesity drugs. In: Oomura, Y. et al. (Eds.), Progress in Obesity Research. John Libbey, pp. 567-574. Cohn, J.N., Archibald, D.G., Ziesche, S., Franciosa, J.A., Harston, W.E., Tristani, F.E., Dunkman, W.B., Jacobs, W., Francis, G.S., Flohr, K.H., Goldman, S., Cobb, F.R., Shah, P.M., Saunders, R., Fletcher, R.D., Loeb, H.S., Hughes, V.C., Baker, B., 1986. Effect of vasodilator therapy on mortality in chronic congestive heart failure. N. Engl. J. Med. 314, 1547-1552. Corr, P.B., Heathers, G.P., Yamada, K.A., 1989. Mechanisms contributing to the arrhythmogenic influences of alpha l-adrenergic stimulation in the ischemic heart. Am. J. Med. 87 (Suppl. 2A), 19S-25S. Craig, D., Iacolina, M., Forray, C., 1995. ce2c-Adrenoceptors mediate norepinephrine-induced contraction of rat caudal artery. FASEB J. 9, A106. Drew, G.M., 1985. What do antagonists tell us about ce-adrenoceptors? Clin. Sci. 68 (Suppl. 10), 15s-19s. Emorine, L.J., Marullo, S., Delavier-Klutchko, C., Kaveri, S.V., DurieuTrautmann, O., Strosberg, A.D., 1987. Structure of the gene for human /32-adrenergic receptor: expression and promoter characterization. Proc. Natl. Acad. Sci. U.S.A. 84, 6995-6999. Eri, L.M., Tveter, K.J., 1995. c~-Blockade in the treatment of symptomatic benign prostatic hyperplasia. J. Urol. 154, 923-934. Ernsberger, P., Westbrooks, K.L., Christen, M.O., Schafer, S.G., 1992. A second generation of centrally acting antihypertensive agents act on putative I l imidazoline receptor. J. Cardiovasc. Pharmacol. 20 (Suppl. 4), S1-S10. Feuerstein, G., Liu, G.-L., Yue, T.-L., Hheny, H.-Y., Hieble, J.P., Arch, J.R.S., Ruffolo, R.R. Jr., Ma, X.-L., 1998. Comparison of metoprolol and carvedilol pharmacology and cardioprotection in rabbit ischemia and reperfusion model. Eur. J. Pharmacol. 351, 341-350. Flavahan, N.A., Vanhoutte, P.M., 1986. o~-Adrenoceptor subclassification in vascular smooth muscle. Trends Pharmacol. Sci. 7, 347-349. Ford, A.P., Arredondo, N.F., Blue, D.R. Jr., Bonhaus, D.W., Jasper, J., Kava, M.S., Lesnick, J., Pfister, J.R., Shieh, I.A., Vimont, R.L., Williams, T.J., McNeal, J.E., Stamey, T.A., Clarke, D.E., 1996. RS- 17053, (N-[2-(2-cyclopropylmethoxi-phenoxy)ethyl]-5-chloro-alpha, alpha-dimethyl-lH-indole-3ethanamine hydrochloride), a selective C~A-adrenoceptor antagonist, displays low affinity for functional ce~-adrenoceptors in human prostate: implications for adrenoceptor classification. Mol. Pharmacol. 49, 209-215. Ford, A.P.D.W., Daniels, D.V., Chang, D.J., Gever, J.R., Jasper, J.R., Lesnick, J.D., Clarke, D.E., 1997. Pharmacological pleiotropism of the human recombinant CelA-adrenoceptor: implications for ce~-adrenoceptor subclassification. Br. J. Pharmacol. 121, 1127-1135. Forray, C., Bard, J.A., Wetzel, J.M., Chiu, G., Shapiro, E., Tang, R., Lepor, H., Hartig, P.R., Weinshank, R.L., Branchek, T.A., Gluchowski, C., 1994. The c~l-adrenergic receptor that mediates smooth muscle contraction in human prostate has the pharmacological properties of the cloned human CelC subtype. Mol. Pharmacol. 45,703-708. Garcia-Sainz, J.A., Alcantara-Hernandez, R., Vazquez-Prado, J., 1998. c~1-Adrenoceptor subtype activation increases proto-oncogene mRNA levels. Role of protein kinase C. Eur. J. Pharmacol. 342, 31-317. George, P., Borg, F., O'Connor, S., Lechaire, J., Arbilla, S., Pimoule, C., Graham, D., Wedzirowski, T., Langer, S.Z., 1995. Uroselective a jadrenoceptor antagonists for the treatment of benign prostatic hypertrophy. Eur. J. Med. Chem. 30, 299s-310s. Giardina, D., Crucianelli, M., Romanelli, R., Leonardi, A., Poggesi, E.,

169

Melchiorre, C., 1996. Synthesis and biological profile of the enantiomers of [4-(4-amino-6,7-dimethoxyquinazolin-2-yl)-cis-octahydroqunioxalin-l-yl]furan-2-ylmethanone (Cyclazosin), a potent competitive ~ 1B-adrenoceptor antagonist. J. Med. Chem. 39, 4602-4607. Gluchowski, C., Forray, C.C., Chiu, G., Branchek, T.A., Wetzel, J.M., Hartig, P.R., 1995. Use of C~lc specific compounds to treat benign prostatic hyperplasia. US Patent No 5,403847, April 4, 1995. Hamburger, S.A., Barone, F.C., Feuerstein, G.Z., Ruffolo, R.R. Jr., 1991. Carvedilol (Kredex) reduces infarct size in a canine model of acute myocardial infarction. Pharmacology 43, 113-120. Harrison, S.N., Autelitano, D.J., Wang, B.H., Milano, C., Du, X.J., Woodcock, E.A., 1998. Reduced reperfusion-induced Ins(1,4,5)P3 generation and arrhythmias in hearts expressing constitutively active alpha 1B adrenergic receptors. Circ. Res. 83, 1232-1240. Hieble, J.P., Kolpak, D.C., 1992. In vivo effects of SK&F 86466 on lipolysis in the rat and hamster. Pharmacol. Commun. 1, 109-118. Hieble, J.P., Ruffolo, R.R., Jr., 1991. Effects of c~- and /3-adrenoceptors on lipids and lipoproteins. In: Witiak, D.T., Newman, H.A.I., Feller, D.R. (Eds.), Antilipidemic Drugs: Medicinal Chemical and Biochemical Aspects. Elsevier, Amsterdam, pp. 301-344. Hieble, J.P., Ruffolo, R.R. Jr., 1997. Recent advances in the identification of c~1- and c~2-adrenoceptor subtypes: therapeutic implications. Expert Opin. Invest. Drugs 6, 367-387. Hieble, J.P., Caine, M., Zalaznik, E., 1985. In vitro characterization of the ~-adrenoceptors in human prostate. Eur. J. Pharmacol. 107, 111-117. Hieble, J.P., Bylund, D.B., Clarke, D.E., Eikenberg, D.C., Langer, S.Z., Lefkowitz, R.J., Minneman, K.P., Ruffolo, R.R. Jr., 1995. International Union of Pharmacology Nomenclature of Adrenoceptors. Recommendation for nomenclature of ce~-adrenoceptors: consensus update. Pharmacol. Rev. 47, 267-270. Hunt, R.D., Capper, L., O'Connell, P., 1990. Clonidine in child and adolescent psychiatry. J. Child and Adolescent Psychopharmacol. 1, 87-102. Hunt, R.D., Arnsten, A.F.T., Asbell, M.D., 1995. An open trial of guanfacine in the treatment of attention-deficit hyperactivity disorder. J. Am. Acad. Child. Adolesc. Psy. 34, 50-54. Kagiya, T., Hori, M., Iwakura, K., Iwai, K., Watanabe, Y., Uchida, S., Yoshida, H., Kitabatake, A., Inoue, M., Kamada, T., 1991. Role of increased ceI adrenergic activity in cardiomyopathic Syrian hamster. Am. J. Physiol. 29, H80-H88. Kaumann, A.J., 1989. Is there a third heart /3-adrenoceptor?. Trends Pharmacol. Sci. 10, 316-320. Kaumann, A.J., Molenaar, P., 1997. Modulation of human cardiac function through 4 /3-adrenoceptor populations. Naunyn-Schmiedeberg's Arch. Pharmacol. 355, 667-681. Kaumann, A.J., Preitner, F., Sarsero, D., Molenaar, P., Revelli, J.P., Giacobino, J.P., 1998. ( - ) - C G P 12177 causes cardiostimulation and binds to cardiac putative /34 adrenoceptors in both wild-type and /3 3 adrenoceptor knockout mice. Mol. Pharmacol. 53, 670-675. Kava, M.S., Blue, D.R. Jr., Vimont, R.L., Clarke, D.E., Ford, A.P.D.W., 1998. CelL-Adrenoceptor mediation of smooth muscle contraction in rabbit bladder neck: a model for lower urinary tract tissues of man. Br. J. Pharmacol. 123, 1359-1366. Kenny, B.A., Naylor, A.M., Wyllie, M.G., 1994. The effect of al-adrenoceptor antagonists on blood pressure and prostatic pressure in the anesthetized dog. J. Urol. 44, 52-57. Kenny, B., Ballard, S., Blagg, J., Fox, D., 1997. Pharmacological options in the treatment of benign prostatic hyperplasia. J. Med. Chem. 40, 1293-1315. Kirby, R.S., Pool, J.L., 1997. Alpha adrenoceptor blockade in the treatment of benign prostatic hyperplasia: past present and future. Br. J. Urol. 80, 521-532. Knowlton, K.U., Michel, M.C., Itani, M., Shubeita, H.E., Ishihara, K., Brown, J.H., Chien, K.R., 1993. The c~jA-adrenergic receptor subtype mediates biochemical, molecular and morphologic features of cultured myocardial cell hypertrophy. J. Biol. Chem. 268, 15374-15380.

170


Lands, A.M., Arnold, A., McAuliff, J.P., Luduena, F.P., Brown, T.G., 1967. Differentiation of receptor systems activated by sympathomimetic amines. Nature 214, 597-598. Langer, S.Z., 1974. Presynaptic regulation of catecholamine release. Br. J. Pharmacol. 60, 481-497. Langin, D., Portillo, M.P., Saulnier-Blanche, J., Lafontan, M., 1991. Coexistence of three /3-adrenoceptor subtypes in white fat cells of various mammalian species. Eur. J. Pharmacol. 199 (1974), 291-301. Lee, E., Lee, C., 1997. Clinical comparison of selective and non-selective a lA-adrenoceptor antagonists in benign prostatic hyperplasia: studies on tamsulosin in a fixed dose and terazosin in increasing doses. Br. J. Urol. 80, 606-611. Lee, J.H., Rosen, M.R., 1993. Modulation of delayed after depolarizations by og1 adrenergic receptor subtypes. Cardiovasc. Res. 27, 839844. Leonardi, A., Hieble, J.P., Guarneri, L., Naselsky, D.P., Poggesi, E., Sironi, G., Sulpizio, A.C., Testa, R., 1997. Pharmacological characterization of the uroselective al-antagonist Rec 15/2739 (SBf216469): role of the alL-adrenoceptor in tissue selectivity: Part I. J. Pharmacol. Exp. Ther. 271, 1272-1283. Link, R.E., Desai, K., Hein, L., Stevens, M.E., Chruscinski, A., Bernstein, D., Barsh, G.D., Kobilka, B.K., 1996. Cardiovascular regulation in mice lacking a2-adrenergic receptor subtypes b and c. Science 273, 803-805. MacMillan, L.B., Hein, L., Smith, M.S., Piascik, M.T., Limbird, L.E., 1996. Central hypotensive effects on the a2a-adrenergic receptor subtype. Science 273, 801-803. McCafferty, J.P., Naselsky, D.P., Hieble, J.P., 1999. Characterization of postjunctional c~-adrenoceptors in the pithed mouse. Gen. Pharmacol. 33, 99-105. Mizobe, T., Maghsoudi, K., Sitwala, K., Tianzhi, G., Ou, J., Maze, M., 1996. Antisense technology reveals the alpha 2A adrenoceptor to be the subtype mediating the hypnotic response to the highly selective agonist, dexmedetomidine, in the locus coeruleus of the rat. J. Clin. Invest. 98, 1076-1080. Morrow, A.L., Creese, I., 1986. Characterization of a l-adrenergic receptor sybtypes in rat brain: a re-evaluation of 3H-WB 4101 and 3Hprazosin binding. Mol. Pharmacol. 29, 321-330. Munoz, M., Bancroft, J., Beard, M., 1994. Evaluating the effects of an alpha-2-adrenoceptor antagonist on erectile function in the human male. Psychopharmacology 115, 471-477. Muramatsu, I., Ohmura, T., Kigoshi, S., Hashimoto, S., Oshita, M., 1990. Pharmacological subclassification of a l-adrenoceptors in vascular smooth muscle. Br. J. Pharmacol. 99, 197-201. Nagarathnam, D., Wetzel, J.M., Miao, S.W., Marzabadi, M.R., Chiu, G., Wong, W.C., Hong, X., Fang, J., Forray, C., Branchek, T.A., Heydorn, W.E., Chang, R.S.L., Broten, T., Schorn, T.W., Gluchowski, C., 1998. Design and synthesis of novel ala adrenoceptor-selective dihydropyridine antagonists for the treatment of benign prostatic hyperplasia. J. Med. Chem. 41, 5320-5333. Packer, M., Bristow, M.R., Cohn, N.J. et al., 1996. Carvedilol heart failure study group. The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. N. Engl. J. Med. 334, 1349-1355. Patane, M.A., Scott, A.L., Broten, T.P., Chang, R.S.L., Ransom, R.W., DiSalvo, J., Forray, C., Bock, M.G., 1998. 4-Amino-2-[4-[1-(benzylo xycarbonyl)- 2( S)-[[ ( 1,1 -dimeth ylethyl)amino]carbonyl]-piperazin yl]6,7-dimethoxyquinazoline (L-765,314): a potent and selective alb adrenergic receptor antagonist. J. Med. Chem. 41, 1205-1208. Ramage, A.G., Wyllie, M.G., 1995. A comparison of the effects of doxazosin and terazosin on the spontaneous sympathetic drive to the bladder and related organs in anaesthetized cats. Eur. J. Pharmacol. 294, 645-650. Roesler, J.M., McCafferty, J.P., DeMarinis, R.M., Matthews, W.D., Hieble, J.P., 1986. Characterization of the antihypertensive activity of SK&F 86466, a selective a2-antagonist, in the rat. J. Pharmacol. Exp. Ther. 236, 1-7.

Rohrer, D.K., Kobilka, B.K., 1998. Insights from in vivo modification of adrenergic receptor gene expression (review). In: Cho, A.K., Blaschke, T.F., Ho, I.K., Loh, H.H. (Eds.), Annual Review of Pharmacology and Toxicology, Vol. 38, pp. 351-373. Rokosh, D.G., Stewart, A.F.R., Chang, K.C., Bailey, B.A., Karliner, J.A., Camacho, S.A., Long, C.S., Simpson, P.C., 1996. al-Adrenergic receptor subtype mRNAs are differentially regulated by al-adrenergic and other hypertrophic stimuli in cardiac myocytes in culture and in vivo. J. Biol. Chem. 271, 5839-5843. Sawyer, R., Warnock, P., Docherty, J.R., 1985. Role of vascular a 2adrenoceptors as targets for circulating catecholamines in the maintenance of blood pressure in anaesthetized spontaneously hypertensive rats. J. Cardiovasc. Pharmacol. 7, 809-812. Sennitt, M.V., Arch, J.R.S., Levy, A.L., Simson, D.L., Smith, S.Q., Cawthorne, M.A., 1985. Anti-hyperglycaemic action of BRL 26830, a novel fl-adrenoceptor agonist, in mice and rats. Biochem. Pharmacol. 34, 1279-1285. Sennitt, M.V., Kaumann, A.J., Molenaar, P., Beeley, L.J., Young, P.W., Kelly, J., Chapman, H., Henson, S.M., Berge, J.M., Dean, D.K., Kotecha, N.R., Morgan, H.K., Rami, H.K., Ward, R.W., Thompson, M., Wilson, S., Smith, S.A., Cawthorne, M.A., Stock, M.J., Arch, J.R., 1998. The contribution of classical (betal/2-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and fight atrial appendage contraction by novel beta3-adrenoceptor agonists of differing selectivities. J. Pharmacol. Exp. Ther. 285, 1084-1095. Starke, K., Montel, H., Gayk, W., Merker, R., 1974. Comparison of the effects of clonidine on pre- and postsynaptic adrenoceptors in the rabbit pulmonary artery. Naunyn-Schmiedeberg's Arch. Pharmacol. 285, 133-150. Takeda, M., Komeyama, T., Tsutsui, T., Hatano, A., Obara, K., Mizusawa, T., Koizumi, T., 1996. Molecular expression and pharmacological functions of beta-3 adrenoceptor in human urinary bladder detrusor. In: Barbalias, G.A. (Ed.), International Continence Society: 26th Annual Meeting. Monduzzi Editore, Bologna, pp. 153-156. Tamai, J., Hori, M., Kagiya, T., Iwakura, K., Iwai, K., Kitabatake, A., Watanabe, Y., Yoshida, H., Inoue, M., Kamada, T., 1989. Role of a~-adrenoceptor activity in progression of cardiac hypertrophy in guinea pig hearts with pressure overload. Cardiovasc. Res. 23, 315322. Tang, H.L., Zhu, Q.M., Eglen, R.M., Blue, D.R. Jr., 1997. Effect of prazosin, Ro 70-0004 and RS-100329 in an anesthetized dog prostate/blood pressure model. Pharmacologist 39, 45. Taniguchi, N., Hamada, K., Ogasawara, T., Ukai, Y., Yoshikuni, Y., Kimura, K., 1996. NS-49, an alpha 1A-adrenoceptor agonist, selectively increases intraurethral pressure in dogs. Eur. J. Pharmacol. 318, 117-122. Taniguchi, N., Ukai, Y., Tanaka, T., Yano, J., Kimura, K., Moriyama, N., Kawabe, K., 1997. Identification of alpha 1-adrenoceptor subtypes in the human prostatic urethra. Naunyn-Schmiedeberg's Arch. Pharmacol. 355, 412-416. Testa, R., Guarneri, L., Angelico, P., Poggesi, E., Taddei, C., Sironi, G., Colombi, D., Sulpizio, A.C., Naselsky, D.P., Hieble, J.P., Leonardi, A., 1997. Pharmacological characterization of the uroselective a lantagonist Rec 15/2739 (SB 216469): role of the alL-adrenoceptor in tissue selectivity: Part II. J. Pharmacol. Exp. Ther. 281, 1272-1283. Trendelenburg, A.U., Sutej, I., Wahl, C.A., Molderings, G.J., Rump, L.C., Starke, K., 1997. A re-investigation of questionable subclassifications of presynaptic alpha2-autoreceptors: rat vena cava, rat atria, human kidney and guinea-pig urethra. Naunyn-Schmiedeberg's Arch. Pharmacol. 356, 721-737. Tsunoo, M., Shishito, A., Soeishi, Y., 1990. Phase I clinical trial of YM617, a new a l-adrenoceptor antagonist ~ first report: a single oral dose of conventional formulation in healthy male subjects. Rinsho Iyaku 6, 2503-2528. Wilde, M.I., McTavish, D., 1996. Tamsulosin. Drugs 52, 883-898. Willette, R.N., Mitchell, M.P., Ohlstein, E.H., Lukas, M.A., Ruffolo,


R.R. Jr., 1998. Evaluation of intrinsic sympathomimetric activity of bucindolol and carvedilol in rat heart. Pharmacology 56, 30-36. Williams, T.J., Clarke, D.E., Ford, A.P.D.W., 1996. Whole cell radioligand binding assay reveals Cr~L adrenoceptor antagonist profile for the human cloned t21a adrenoceptor in Chinese hamster ovary cells. Br. J. Pharmacol. 119, 359P. Wilson, C., Wilson, S., Piercy, V., Sennitt, M.V., Arch, J.R.S., 1984. The

171

rat lipolytic /3-adrenoceptor: studies using novel fl-adrenoceptor agonists. Eur. J. Pharmacol. 100, 309-319. Yasutake, M., Avkiran, M., 1995. Exacerbation of reperfusion arrhythmias by o~1 adrenergic stimulation: a potential role for receptor mediated activation of sarcolemmal sodium-hydrogen exchange. Cardiovasc. Res. 29, 222-230.




The

O/.la

and oL b-adrenergic receptor subtypes" molecular mechanisms of receptor activation and of drug action

Susanna Cotecchia a'*, Olivier Rossier a Francesca Fanelli Pier G. De Benedetti b

b, A m e d e o

Leonardi ~

a Institut de Pharmacologie et Toxicologie, Universit~ de Lausanne, 27, Rue du Bugnon, Facult~ de Mddecine, 1005 Lausanne, Switzerland b Dipartimento di Chimica, Universith di Modena e Reggio Emilia, 41100 Modena, Italy c Pharmaceutical R&D Division, Recordati, 20148 Milan, Italy

Abstract In this chapter we summarize some aspects of the structure-functional relationship of the o~1a and ot lb-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the oLl a and oLlb-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies. 9 2000 Elsevier Science B.V. All rights reserved. Keywords:

Adrenergic receptors; G protein-coupled receptors; Constitutive activity; Inverse agonism; Molecular dynamics

1. The ~ 1-adrenergic receptor subtypes The receptors for a large number of hormones and neurotransmitters regulate cellular activity via the intermediary role of guanine nucleotide regulatory proteins (G proteins). Among these receptors, the adrenergic receptors (AR) mediate the effects of epinephrine and norepinephrine by coupling to several of the major signalling pathways modulated by G protein. The AR family includes nine different gene products: three [3 ([3 l, [32, [33), three OL2 (OL2A' OL2B' OL2C) and three oti (~ la, ot lb, OLl d) receptor subtypes. The ot ~-AR is present in many tissues including brain, heart, blood vessels, liver, kidney, prostate and spleen. In these tissues the ot~-ARs mediate a variety of physiological effects such as neurotransmission, vasoconstriction, cardiac inotropy and chronotropy, glycogenolysis (for reviews, see Michel et al., 1995; Graham et al., 1996). Radioligand binding studies in different rat tissues have demonstrated two classes of oL1-AR binding sites, " A " and " B " with high and low affinity for the oL-AR antagonists WB4101 and phentolamine, respectively. After large scale purification of the ot ~-AR from smooth muscle cells DDT1 MF-2 a first receptor was cloned, unequivocally


assigned to the pharmacological a 1B subtype and hence named ot lb-AR. On the other hand, the pharmacological OL1A subtype is mainly coded by the e~la-AR initially cloned from a bovine brain library and inappropriately named OL~c-AR. Finally, the cloned c~ld-AR (initially named o~IA-AR or o~1A/D-AR) represents a receptor subtype not clearly identified by previous pharmacological studies. Activation of the e~1-ARs causes polyphosphoinositide hydrolysis catalyzed by phospholipase C via pertussis toxin-insensitive G proteins in almost all tissues where this effect has been examined. Recent studies have shown that other signalling pathways can be activated upon oL1-AR stimulation such as phosphatidylcholine hydrolysis and phospholipase A2 (for review, see Graham et al., 1996). However, the comparison among different oL1-AR-mediated responses in various tissues has not allowed to assess any conclusive signalling differences among distinct oL1AR subtypes. In vivo studies aiming to assess a specificity of the functional responses mediated by distinct ot I-AR subtypes have been hampered by the fact that the subtype-selective drugs are only moderately selective and might interact with both adrenergic and non-adrenergic receptors. To contribute to the elucidation of the physiological role of the ot 1-AR subtypes in vivo we have used gene targeting to create knockout mice lacking the c~tb-AR (Cavalli et al.,

0031-6865/00/$- see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S003 1-6865(99)0003 1-X

174

S. Cotecchia et al. / Pharmaceutica Acta Helvetiae 74 (2000) 173-179

1997). Our findings provide strong evidence that the oLlbAR can be a mediator of the blood pressure response as well as of the aorta contractility induced by oL~-agonists. A full understanding of the functional implications of adrenergic receptor heterogeneity awaits the knock out of all AR subtypes as well as the intercross among different knock out models.

2. Structure-functional analysis of the c~1-AR subtypes Like all G protein-coupled receptors (GPCRs), the a 1AR subtypes share the presence of seven hydrophobic regions which form transmembrane (TM) oL-helices and are connected by alternating intracellular (i) and extracellu-

lar (e) hydrophilic loops (Fig. 1). Mutational analysis of several GPCRs has revealed that the a-helical bundle contributes to form the ligand binding site of the receptor, whereas amino acid sequences of the intracellular regions appear to mediate the interaction of the receptor with G proteins as well as with different signalling and regulatory proteins (Wess, 1997). Recent studies have focused on the molecular interactions of the endogenous catecholamines, epinephrine and norepinephrine, with the oLla and oLlb-AR subtypes (Cavalli et al., 1996; Hwa and Perez, 1996). Epinephrine and norepinephrine contain a protonated nitrogen atom separated from the aromatic catechol ring by a 6-hydroxylethyl chain. The molecular requirement for catecholamine binding to the AR should include the electrostatic interaction between the receptor and the amino group of the ligand,

9

E:I

:

g291

"5-

IE

1:t26

.t

S

1'tl22

Fig. 1. Average minimized structures of the wild type e~I-AR subtypes and of their constitutively active mutants. Views of the receptor structures are in a direction parallel to the membrane surface with the extracellular side at the bottom. The figure shows also the interactions involving some of the conserved polar amino acids including the arginine of the E / D R Y motif (R124 in the et la-AR and R143 in the oLlb-AR).

S. Cotecchia et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 173-179

hydrogen bonds between donor/acceptor sites of the receptor and the [3-hydroxyl as well as the catechol metaand para-hydroxyl groups of the ligand, and finally van der Waals attractive interactions. Mutagenesis studies of the oLlb-AR subtypes suggested that the amino group of the catecholamines as well as of the oL1-antagonists makes an electrostatic interaction with the carboxylate side chain of an aspartate D 125 on helix 3 which is highly conserved in all GPCR that bind amine ligands. On the other hand, there is evidence that the catechol meta- and para-hydroxyl groups of catecholamines interact with serine residues present in helix 5 which are also conserved in different catecholamine receptors. Our work on the oLlb-AR (Cavalli et al., 1996) suggested that $207 in helix 5 interacts with both catecholic hydroxy-groups of (-)-epinephrine. In contrast, in the oLla-AR of the two serines S 188 and S 192 present on helix 5, S188 seems to form one, but not two hydrogen bondings with the catechol ring (Hwa and Perez, 1996). Very little is known so far about the receptor amino acids which interact with different antagonists as well as about the structural basis underlying receptor selectivity for different ligands and pharmacological agents. Some information has been gained about the domains of the oLlb-AR involved in receptor coupling to the G protein. Studies with chimeric [32/oLlb-AR receptors indicated that a stretch of 27 amino acids (residues 233-259) derived from the N-terminal portion of the third intracellular loop of the oLlb-AR were sufficient to confer to the [32-AR the ability to activate phospholipase C (Cotecchia et al., 1992). Virtually nothing is known about the structural determinants of the other oL1-AR subtypes involved in Gq-mediated activation of phospholipase C.

3. Molecular modelling of the ~ 1-AR subtypes

To probe the structure-function relationships of GPCRs several useful molecular models of these membrane proteins were built using different methods. To investigate the potential intramolecular motions underlying different functional states of the oL1-AR subtypes, we combined 3-D model building of the receptor structure with computational simulation of receptor dynamics as recently described (Fanelli et al., 1998). The oL1-AR model was built using an iterative ab initio procedure started with a comparative molecular dynamics (MD) study on the helix bundle of seven GPCRs (OLlb-AR, ~2-AR, OLz-AR, dopamine D2, 5-HT1A, muscarinic M~ and the bovine rhodopsin not complexed with retinal). The arrangement of the helices was based on the structural constraints inferred from the analysis of a large number of GPCR sequences (Baldwin et al., 1997). The oLlb-AR model was progressively upgraded on the basis of the new structural information derived from the electron micrographs of 3-D frog rhodopsin crystals (Unger et al., 1997). In addition, the model was completed by adding the intracellular and

175

extracellular loops as shown in Fig. 1 (Scheer et al., 1996; Fanelli et al., 1998). Packing of the seven helices of the OLla-AR was achieved by using the last upgraded input structure of the oLlb-AR as a template. The seven helix bundle of the oLla-AR in the input arrangement was then completed with the extracellular and intracellular loops according to the procedure recently described (Fanelli et al., 1998, 1999). The analysis of the MD trajectories was used to compare the structural/dynamic features of functionally different receptor mutants with those of the wild type OL~a and Otlb-AR, to predict key residues the mutations of which would either activate or inactivate the receptor and to investigate the potential effects of mutations.

4. The activation process of the r la and ~ lb-AR subtypes

Agonist binding to a GPCR is believed to induce a conformational change of the receptor which results in its productive coupling to heterotrimeric G proteins thus leading to intracellular signalling events. Yet, very little is known on how binding of the agonist triggers receptor activation, i.e., the "conformational switch" of the receptor leading to productive receptor-G protein coupling. An important contribution to our understanding of receptor activation has been provided by the findings that point mutations in a GPCR could increase the constitutive (agonist-independent) activity of the receptor. In the oL~bAR, all possible amino acid substitutions of A293 in the C-terminal portion of the i3 induced variable levels of constitutive activity (Kjelsberg et al., 1992). Similar mutations were also found to increase the constitutive activity of the ~2-AR and oL2-AR (Ren et al., 1993; Samama et al., 1993a,b). A detailed analysis of the properties of the constitutively active AR mutants was instrumental to propose the "allosteric ternary complex" model to describe GPCR behaviour (Samama et al., 1993a,b). This model predicts that GPCRs can undergo an allosteric transition between at least two interconvertible states, R (inactive or ground state) and R* (active state). In the absence of the agonist, R predominates possibly because a structural constraint might prevent sequences of the intracellular loops to interact with the G proteins. On the other hand, agonists can trigger the equilibrium toward R* thus favouring its stabilization. Constitutively activating mutations might trigger the formation of R* releasing the structural constraint which keeps GPCR inactive. These findings led to the hypothesis that activating mutations mimic, at least to some extent, the conformational change triggered by agonist binding to GPCR. Thus, the identification of receptor sites susceptible to constitutive activation might contribute to elucidate the structural basis of the "conformational switch" underlying GPCR activation.


176

Two residues seem to play a crucial role in the activation process of the e~lb-AR subtype, D 142 belonging to the highly conserved E / D R Y motif lying at the end of helix 3 and A293 in the C-terminal portion of the 3i loop (Fig. 1). This is mainly supported by the findings that mutations of D142 as well as of A293 could markedly increase the constitutive activity of the o~lb-AR (Kjelsberg et al., 1992; Scheer et al., 1997). Recently, we have shown that in oLla-AR mutations of D123 into alanine and of A271 into glutamic acid (which are homologous to D142 and A293 of the oLlb-AR, respectively) could also increase the constitutive activity of the receptor (Fig. 2) (Rossier et al., 1999). Altogether these findings suggest that the E / D R Y motif as well as the C-terminal end of the 3i loop plays a role in the "conformational switch" underlying the activation process of both the OLla and OLlb-AR subtypes. The role played by these sequences in receptor activation is probably shared by other members of the rhodopsin family of GPCRs as supported by the fact that mutations in the homologous regions of other receptors can have marked effects on receptor-G protein coupling (Wess, 1997). The comparative MD analysis of the wild type oL1-AR subtypes and of a number of functionally different receptor mutants was instrumental to make hypothesis about the potential molecular mechanisms underlying the transition between the inactive (R) and active states (R*) of the receptor. Our findings highlighted a network of hydrogenbonding interactions among some conserved polar residues forming a "polar pocket" near the cytosol (Fig. 1) and the arginine of the E / D R Y motif. We suggested that this set of interactions constrains the receptor in its ground or inactive state (R) by controlling the degree of cytosolic exposure attainable by the arginine of the E / D R Y sequence. This hypothesis is supported by the observation that the charge reinforced H-bonding interaction between the conserved aspartate on helix 2 and the arginine of the E / D R Y sequence constitutes one of the constraining interactions in the receptor ground state. In contrast, a landmark of the constitutively active receptor mutants is the

A .r 25000.w_

I ~

basal

epinephrine

-I-

_

_~ 15oo0-

~--

breakage of this interaction and the shift of the arginine out of the "polar pocket" (Fig. 1). The results of the MD simulations indicate that the motions of the helix bundle triggered by different activating mutations in the a 1-AR subtypes induce the breakage of the interactions within the "polar pocket" and a rearrangement of the cytosolic domains which seem to form a site with docking complementarity with the G protein (Fig. 1) (Fanelli et al., 1999). Our findings suggest that the main role of the arginine of the E / D R Y motif is to mediate receptor activation either directly interacting with the G protein heterotrimer or allowing several cationic amino acids in i2 and i3 to attain the fight configuration for the formation of a site with docking complementarity with the G protein. This hypothesis is supported by the experimental findings showing that different mutations of R143 can almost entirely inactivate the oLlb-AR (Scheer et al., 1996).

5. Inverse agonism and neutral antagonism at the t~ la and ct lb-AR subtypes

Both selective and nonselective antagonists for different AR subtypes are widely used in a variety of pathological conditions including hypertension, heart failure, prostate hypertrophy as well as in mental diseases such as depression. Several studies have demonstrated that beta-blockers can behave either as neutral antagonists or inverse agonists at the wild type [32-AR or at a constitutively active [32-AR mutant (Samama et al., 1993a,b; Chidiac et al., 1994). On the other hand, inverse agonism at other AR subtypes has been less extensively investigated. It has been previously reported that a small range of alpha-blockers could inhibit the agonist-independent phospholipase C as well as phospholipase D responses mediated by constitutively active mutants of the et lb-AR (Lee et al., 1997). A recent study has demonstrated that some alpha-blockers can inhibit the spontaneous activity of the et ~d-AR subtype (Garcla-Sfiinz and Torres-Padilla, 1999).

~ ' 25000._=

basal

~

epinephrine

.2

E

E

o 10000M a.

o r a.

E

I

~= ,-.,

50000a la "AR

D123A

A271 E

v (x lb "AR

D142A

A293E

Fig. 2. Constitutivelyactive o~la-AR and oLlb-AR mutants. D123 and A271 of the ct la-AR as well as D142 and A293 of the ot lb-AR have been mutated into alanine and glutamic acid, respectively. Receptors expression in COS-7 cells ranged 1.5-2.5 pmol/mg of protein. Inositol phosphate ([3H]IP) accumulation was measured in cells expressing the wild type or mutated receptors after incubation in the absence (Basal) or presence of 10 - 4 M epinephrine for 45 min. Results are the mean+ S.E of three independent experiments.

177


We have recently characterized the pharmacological antagonism, i.e., neutral antagonism or inverse agonism, displayed by a number of alpha-blockers at two o~1-AR subtypes, the c~~a and o~~b-AR (Rossier et al., 1999). One strategy to identify inverse agonists is to screen drugs for their ability to inhibit the agonist-independent activity of the constitutively active receptor mutants. Thus, 24 alphablockers differing in their chemical structures were tested for their effect on the basal activity of the constitutively active A271E and A293E mutants expressed in COS-7 cells (Fig. 3). All the ligands used in this study, except REC 15/3039, were previously described for their structure, their binding affinity at recombinant as well as native o~~-AR subtypes and some of their pharmacological effects in different tissues (Michel et al., 1995; Leonardi et al., 1997; Testa et al., 1997). Our results show that the vast majority of alpha-blockers displayed inverse agonism as demonstrated by their

ability to decrease the basal activity of both CAMs. However, the various alpha-blockers differed in their negative efficacy and some of these differences depended on the o~~-AR subtype. Drugs with the highest negative efficacy (defined as > 70% inhibition of the basal activity) at both CAMs included WAY 100635, WB 4101, all the tested quinazolines (prazosin, terazosin, both ( + ) - and ( - ) cyclazosin, REC 15/2615 and alfuzosin), indoramin, corynanthine, spiperone and AH11 l l0A (Fig. 3). For the other drugs, their negative efficacy differed at the two CAMs. The most striking difference concerned some N-arylpiperazines which displayed modest negative efficacy (e.g., 5-methylurapidil, BMY 7378 and REC 15/2869) or neutral antagonism (e.g., REC 15/3039, REC 15/2739 and REC 15/3011) at the A271E mutant. On the other hand, the negative efficacy of these compounds was more pronounced at the A293E resulting in at least 45% inhibition of the receptor-mediated basal activity. For phentolamine,

~E'~-150oo1 oooo1

o-1

{--I

F-FI 00~

+

25000 20000-

~

§

j

A293E

, ooo l oooo-

,,

~

~

~

0

Fig. 3. Ligand-induced inhibition of the basal inositol phosphate response mediated by constitutively active o~I-AR mutants. Inositol phosphate ([3H]IP) accumulation was measured in COS-7 cells expressing the wild type (WT) o~la-AR and c~lb-AR and their respective mutants A271E and A293E. In cells expressing the receptor mutants, [3H]IP were measured in the absence (Basal) or presence of different ligands at a concentration of 10 -5 M (10 -4 M for REC 15/3039) for 45 min. Mock indicates cells not expressing the receptors. The ligands are grouped according to their structural similarities (from left to right the groups include: N-arylpiperazines; 1,4-dihydropyridines" imidazolines; benzodioxanes and phenylalkylamines; quinazolines; various structures). Results are the mean + S.E of three to six independent experiments.

178


BE 2254 and tamsulosin negative efficacy was also greater at the A293E than at the A271E. In contrast, for (S)-( +)niguldipine negative efficacy was greater at the A293E than at the A271E mutant. To assess whether the effect of the alpha-blockers observed on the CAMs reflected their behaviour at the wild type receptors, a selected number of drugs were tested for their effects in COS-7 cells expressing the wild type OLla and C~lb-AR subtypes. Overexpression of both oL1-AR subtypes resulted in a small, but measurable increase of agonist-independent accumulation of IP which was greater for the OLlb-AR than for the OLla-AR. The effect of the alpha-blockers in cells expressing the wild type oL~-AR subtypes displayed several similarities to that observed in cells expressing their CAMs. Altogether, our findings identify a group of N-arylpiperazines as alpha-blockers which display the most striking difference at the two o~~-AR subtypes being inverse agonists with profound negative efficacy at the wild type OLlb-AR, but not at the OLla-AR. In particular, REC 15/3039, REC 15/2739 and REC 15/3011 are the first alpha-blockers identified so far which do not display inverse agonism at one of the oL1-AR subtypes, namely, the O~la.

A preliminary structure-activity relationship analysis of the ligands suggests that the inhibitory effect observed for different ligands on the constitutive activity of the oLla-AR and, to a smaller extent, on that of the e~lb-AR is related to the structure of a defined portion of the ligands. Our results suggest that the geometry of the protonated nitrogen atom as well as that of the molecular moiety closest to this nitrogen might be responsible for the functional effect of the ligands tested (Rossier et al., 1999). The group of quinazolines including prazosin, terazosin and alfuzosin, which are among the most commonly used alpha-blockers, can display unwanted effects in vivo on the cardiovascular system such as orthostatic hypotension. In contrast, REC 15/2739, REC 15/2869 and REC 15/3011 are characterized by high selectivity for the urogenital tissues and seem to have less pronounced effects on the cardiovascular system in vivo (Testa et al., 1997). In future studies, it will be interesting to identify novel alpha-blockers which are neutral antagonists at one or more oL~-AR subtypes and to assess whether these compounds, including REC 15/3039, have fewer generalized cardiovascular effects as compared to full inverse agonists.

6. Conclusions. The results of our recent studies provide new information on the activation process of the OLla and OLlb-AR subtypes as well as on the mechanisms of action of drugs acting at these receptors. They might also provide generalities about the molecular mechanisms underlying the activation process of other GPCRs. The structural conservation

among different receptors suggests that the molecular mechanisms proposed for the OLla- and OLlb-AR subtypes might be generalized to the activation process of other members of the GPCR family. We propose that an interdisciplinary approach combining site-directed mutagenesis, molecular dynamics and thermodynamical analysis of receptor mutants is useful to elucidate the structural-dynamic properties of GPCRs.

Acknowledgements This work was supported by the Fonds National Suisse de la Recherche Scientifique (grant 31-51043.97) and by the European Community (grant BMH4-CT97-2152).

References Baldwin, J.M., Schertler, G.F., Unger, V.M., 1997. An a-carbon template for the transmembrane helices in the rhodopsin family of G proteincoupled receptors. J. Mol. Biol. 272, 144-164. Cavalli, A., Fanelli, F., Taddei, C., De Benedetti, P.G., Cotecchia, S., 1996. Amino acids involved in agonist binding: differences in docking catecholamines to receptor subtypes. FEBS Lett. 399, 9-13. Cavalli, A., Lattion, A.L., Hummler, E., Nenniger, M., Pedrazzini, T., Aubert, J.-F., Michel, M., Ming, Y., Lembo, G., Vecchione, C., Mostardini, M., Schmidt, A., Beermann, F., Cotecchia, S., 1997. Decreased blood pressure response in mice deficient of the C~lBadrenergic receptor. Proc. Natl. Acad. of Sci. USA 94, 11589. Chidiac, P., Hebert, T.E., Valiquette, M., Dennis, M., Bouvier, M., 1994. Inverse agonist activity of [3-adrenergic antagonists. Mol. Pharmacol. 45, 490-499. Cotecchia, S., Ostrowski, J., Kjelsberg, M.A., Caron, M.G., Lefkowitz, R.J., 1992. Discrete amino acid sequences of the OLl-adrenergic receptor determine the selectivity of coupling to phosphatidylinositol hydrolysis. J. Biol. Chem. 267, 1633-1639. Fanelli, F., Menziani, M.C., Scheer, A., Cotecchia, S., De Benedetti, P.G., 1998. Ab initio modeling and molecular dynamics simulation of the e~lb-adrenergic receptor activation. Methods Companion Methods Enzymol. 14, 302-317. Fanelli, F., Menziani, C., Scheer, A., Cotecchia, S., De Benedetti, P.G., 1999. Theoretical study on receptor/G protein recognition: new insights into the mechanism of the o~lb-adrenergic receptor activation. Int. J. Quantum Chem. 73, 71-83. Garcia-Sfiinz, J.A., Torres-Padilla, M.E., 1999. Modulation of basal intracellular calcium by inverse agonists and phorbol myristate acetate in rat-1 fibroblasts stably expressing OLld-adrenoceptors. FEBS Lett. 443, 277-281. Graham, R.M., Perez, D.M., Hwa, J., Piascik, M.T., 1996. ot 1-Adrenergic receptor subtypes, molecular structure, function and signalling. Circ. Res. 78, 737-749. Haw, J., Perez, D.M., 1996. The unique nature of the serine interactions for a l-adrenergic receptor agonist binding and activation. J. Biol. Chem. 271, 6322-6327. Kjelsberg, M.A., Cotecchia, S., Ostrowski, J., Caron, M.G., Lefkowitz, R.J., 1992. Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J. Biol. Chem. 267, 1430-1433. Lee, T.W., Cotecchia, S., Milligan, G., 1997. Up-regulation of the levels of expression and function of a constitutively active mutant of the hamster O~lB-adrenoceptor by ligands that act as inverse agonists. Biochem. J. 325, 733-739.


Leonardi, A., Hieble, J.P., Guarneri, L., Naselsky, D.P., Poggesi, E., Sironi, G., Sulpizio, A.C., Testa, R., 1997. Pharmacological characterization of the uroselective alpha-1 antagonist Rec 15/2739 (SB 216469): role of the alpha-1L adrenoceptor in tissue selectivity, part I. J. Pharmacol. Exp. Ther. 281, 1272-1283. Michel, M.C., Kenny, B., Schwinn, D.A., 1995. Classification of oLladrenoceptor subtypes. Naunyn-Schmiedeberg's Arch. Pharmacol. 352, 1-10. Ren, Q., Kurose, H., Lefkowitz, R.J., Cotecchia, S., 1993. Constitutively active mutants of the OLz-adrenergic receptor . J. Biol. Chem. 268, 16483-16487. Rossier, O., Abuin, L., Fanelli, F., Leonardi, A., Cotecchia, S., 1999. Inverse agonism and neutral antagonism at the O~la and oLlb adrenergic receptor subtypes. Mol. Pharmacol. (in press). Samama, P., Cotecchia, S., Costa, T., Lefkowitz, R.J., 1993a. A mutation-induced activated state of the [32-adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 268, 4625-4636.

179

Samama, P., Pei, G., Costa, T., Cotecchia, S., Lefkowitz, R.J., 1993b. Negative antagonists promote an inactive conformation of the [32adrenergic receptor. Mol. Pharmacol. 45, 390-394. Testa, R., Guarneri, L., Angelico, P., Poggesi, E., Taddei, C., Sironi, G., Colombo, D., Sulpizio, A.C., Naselsky, D.P., Hieble, J.P., Leonardi, A., 1997. Pharmacological characterization of the uroselective alpha-1 antagonist Rec 15/2739 (SB 216469): role of the alpha-lL adrenoceptor in tissue selectivity, part II. J. Pharmacol. Exp. Ther. 281, 1284-1293. Unger, V.M., Hargrave, P.A., Baldwin, J.M., Schertler, G.F., 1997. Arrangement of rhodopsin transmembrane alpha-helices. Nature 389, 203-206. Wess, J., 1997. G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J. 5, 346-354.


PHAl MACEUTICA ACTAHELVETIAE ELSEVIER

Pharmaceutica Acta Helvetiae 74 (2000) 181-190 www.elsevier.com/locate/ pharmactahelv

-Adrenoreceptor antagonists bearing a quinazoline or a benzodioxane moiety C. Melchiorre ~'*, P. Angeli b, M.L. Bolognesi ~, A. Chiarini a, D. Giardin?a b, U. Gulini b A. Leonardi c, G. Marucci b, A. Minarini ~, M. Pigini b W. Quaglia b, M. Rosini a V. Tumiatti ~ a Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy b Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy c Pharmaceutical R&D Division, Recordati, Via Civitali 1, 20148 Milan, Italy

Keywords: ~ l-Adrenoreceptor antagonists; Quinazoline; Benzodioxane moiety; Prazosin; WB 4101; Cystazosin; BMY-7378

1. Introduction In recent years, much effort has been directed towards characterization of receptor systems, which are composed usually of multiple subtypes, oL~-Adrenoreceptors do not represent an exception to the rule as they can be divided into at least three subtypes, namely oL1A (OL~a), OLJB (OL~b), and oL1D (OL~), with upper and lower case subscripts being used to designate native or recombinant receptor, respectively (Bylund et al., 1994; Faure et al., 1994; Ford et al., 1994; Hieble et al., 1995). However, some functional experiments indicate that an additional oL~-adrenoreceptor subtype may exist, which was named oL~c-adrenoreceptor (Docherty, 1998). Efforts to clone this receptor subtype have been unsuccessful so far (Testa et al., 1997), supporting the conclusion that it may represent a different affinity state of the oL~A-adrenoreceptor (Ford et al., 1997; Hieble and Ruffolo, 1997). The effort to design agents selective for each of the three oL~A-adrenoreceptor subtypes has been an active area of research. Whereas it has been demonstrated that oL1Aadrenoreceptor antagonists can be useful in the treatment of benign prostatic hyperplasia (Kenny et al., 1997; Matyus and Horvath, 1997), a potential therapeutic use for both oL~B- and OL~D-SUbtype antagonists has not been defined yet. Perhaps, the fact that only recently so-called selective oL~-adrenoreceptor antagonists have become available has prevented the physiological roles of oL~B and oL~D-adrenoreceptor subtypes in blood pressure control or other physiological functions from being revealed.

* Corresponding author.

A vast array of structurally unrelated compounds interacts with oLl-adrenoreceptor subtypes, which makes it inherently difficult to determine the structural requirements leading to receptor subtypes selectivity (Ruffolo et al., 1995; Leonardi et al., 1996; Kenny et al., 1997). The majority of oL~-adrenoreceptor antagonists displays a competitive mechanism of action and belongs to a variety of different structural classes such as yohimbanes, ergot alkaloids, quinazolines, N-arylpiperazines, imidazolines, phenylalkylamines, benzodioxanes, indoles, 1,4-dihydropyridines, hetero-fused 3-benzazepines, dibenzoquinolizines. Our research group has long been involved in designing new c~-adrenoreceptor antagonists structurally related to prazosin (Giardin?a et al., 1989, 1993, 1995, 1996a, 1997; Bolognesi et al., 1998; Minarini et al., 1998) and WB 4101 (Melchiorre et al., 1982, 1984; Giardin?a et al., 1984; Pigini et al., 1988; Quaglia et al., 1990, 1993, 1996, 1999), the prototypes of quinazoline- and benzodioxane-bearing compounds, respectively, with the goal of developing high-affinity, site-selective ligands for subtypes of the oL~adrenoreceptor. The aim of this short review is to update the knowledge on e~~-adrenoreceptor antagonists bearing a quinazoline or a benzodioxane moiety.

2. Prazosin-related antagonists Prazosin (1), the prototype of quinazoline-bearing compounds, is a selective oL1-adrenoreceptor antagonist widely used as a pharmacological tool for oL-adrenoreceptor subtypes characterization and as an effective agent in the

0031-6865/00/$- see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S003 1-6865(99)00049-7

182

C. Melchiorre et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 181-190

management of hypertension (Ruffolo et al., 1995; Kenny et al., 1997). For these reasons, prazosin represents a valid tool to explore et 1-adrenoreceptor binding site topography and a lead compound in developing new therapeutically useful agents. The role of piperazine ring of prazosin was investigated through its replacement by an oL,o~-alkanediamine chain (Giardin~t et al., 1989). It turned out that the piperazine ring may not be essential for activity at oL~-adrenoreceptors and that activity and selectivity depend on the length of alkane chain and N-methylation of both the amide and the 2-amino functions. The compound bearing an N,N'-dimethyl-l,6-hexanediamine moiety (2) was the most active of the series, being more potent than prazosin (Giardin?a et al., 1989). The chain length effect on potency allowed us to postulate that the rat v a s deferens OLl-adrenoreceptor incorporates a lipophilic area, located between the binding sites for the quinazoline and the furan rings of prazosin, which can optimally accommodate an hexane spacer. The finding that the affinity profile of prazosin-related quinazolines can depend on the type of moiety linking the two nitrogen atoms of the piperazine ring of prazosin, prompted us to further modify the structure of analogue 2. In particular, two types of structural modifications were performed on the structure of 2, that is, replacement of the 1,6-hexanediamine unit with 2,3-dialkylpiperazine, 1,2cyclohexanediamine or decahydroquinoxaline moieties, and replacement of the hexane spacer with a cystamine moiety as shown in Fig. 1. To obtain information about the size and possible stereochemical requirements of the lipophilic area, we designed a series of compounds in which the very flexible polymethylene chain of 2 is incorporated partially or totally into a constrained structure (Giardin~ et al., 1993). The objective of this structural modification was to allow compounds, in which the alkane moiety is forced, to assume a definite arrangement while keeping quinazoline and furan rings in a position likely similar to that of prazosin. It turned out that antagonist activity within c i s / t r a n s stereoisomeric compounds not only supported the presence of a lipophilic binding area on oL~-adrenoreceptor surface, but also suggested that the lipophilic pocket is endowed with a well-defined size and spatial orientation. Cyclazosin was the most potent and selective of the series with a significant selectivity for c~~B (oL~b)-adrenoreceptors with respect to the OL1A (OL la ) and OLld-SUbtypes, as well as an interesting long-lasting hypotensive effect, very similar to that of doxazosin (Giardin?a et al., 1993, 1996a). Since cyclazosin incorporates a decahydroquinoxaline nucleus in a cis relationship, which is responsible for the high affinity for oL1-adrenoreceptors, we have synthesized its enantiomers to investigated whether stereochemistry might increase the selectivity for oL1-adrenoreceptor subtypes (Giardin?~ et al., 1996b). The affinity profile displayed by the two enantiomers of cyclazosin at native oL1g-

MeO-

~.-

NH 2 / /1,.

0 P r a z o s i n (1): a l A = CtlB = O(lD

II v

eO. 'L 2: (3tlB = 0(ID = (~IA

NH,

NH 2 MeO.,. ~

JJ'N

i .o

o

( + ) - C y c l a z o s i n : ~ l b > Oqa = ~ l a

~

.~ C y s t a z o s i n (3): ~ld > ~ l a - ~ l b

Fig. 1. Design strategy for the synthesis of prazosin (1)-related compounds by replacing the piperazine ring of prazosin with an oL,to-alkanediamine chain, a decahydroquinoxaline system and a cystamine moiety, affording 2, cyclazosin and cystazosin (3), respectively.

and oL1B-' as well as at cloned oLla-, OLlb-, and oLld-adrenoreceptor subtypes, is reported in Table 1 and graphically shown in Fig. 2. (-)-Cyclazosin, although more potent than (+)-cyclazosin at all subtypes, was nearly devoid, like prazosin, of subtype selectivity with the exception of a 12-fold higher affinity at native CriB-relative to OL1gadrenoreceptors. On the contrary, (+)-cyclazosin displayed high affinity ( p K i - 9.16) at cloned C~lb-adrenoreceptors and a significantly lower potency at both oLla- and o~ld subtypes (p K i = 7.48 and 7.57, respectively). Furthermore, (+)-cyclazosin displayed selectivities of 1100-, 19 000-, and 12 000-fold in binding to oLlb-adrenoreceptors relative to etz-adrenoreceptors and 5-HT1A and D 2 receptors. Spiperone, which is considered a selective OL1Badrenoreceptor antagonist, showed high affinity for other receptors as well, namely 5-HT1A and D 2 receptors. Thus, (+)-cyclazosin emerges as a most interesting ligand of prazosin-related antagonists as it displayed high affinity, in the nanomolar range like prazosin, and an unprecedented selectivity for oLlb (O~ 1B)-adrenoreceptors, which is lacking in the antagonists presently available. Replacing the hexane chain of 2 by a cystamine moiety (Fig. 1) afforded cystazosin (3) which displayed an interesting selectivity profile in comparison with both ( + ) cyclazosin and the carbon analogue 2, owing to a significantly lower affinity for OL1A- and OL1B-adrenoreceptor subtypes relative to the ct 1D-Subtype (vide infra) (Minarini et al., 1998).

C. Melchiorre et al./Pharmaceutica Acta HeIvetiae 74 (2000) 181-190

183

Table 1 Affinity estimates, expressed as pK i , of the enantiomers of cyclazosin for native and cloned o~~-adrenoreceptor subtypes, native oL2-adrenoreceptors, and 5-HTIA and Dzreceptors in comparison to prazosin and reference compound spiperone a Compound

p K i, native receptors (rat) b

p K i, cloned receptors c

oLIA

oLIB

O~2

5-HT1A

D2

ot la

ot Ib

Otld

(+)-CycIazosin (-)-Cyclazosin ( +_)-Cyclazosin Prazosin Spiperone

7.73 8.77 8.41 9.03 7.42

9.68 9.85 9.57 9.44 8.81

6.13 5.86 6.17 6.83 6.86

4.89 5.21 5.16 5.63 7.60

5.08 < 5 < 5 < 5 9.24

7.48 8.62 8.18 9.14 7.87

9.16 9.51 9.23 9.34 8.15

7.57 9.24 9.28 8.96 7.66

a Data taken from Giardin?a et al. (1996b). bMembranes were from hippocampus + 10 ~M CEC (OL1A), liver (O~lB), cerebral cortex (o~2) , hippocampus (5-HTIA), and striatum (D2). CMembranes were from bovine brain (ot la), hamster smooth muscle (or lb), and rat brain (O~ld).

The finding that the selectivity for oL~-adrenoreceptor subtypes can be modulated by an appropriate spacer between the two nitrogen atoms of the piperazine ring of prazosin formed the basis for further structural modifications. We thought that increasing the number of contacts between a ligand and its receptor would increase hopefully also receptor subtype selectivity. To this end, we focused our attention on the furan ring of both cystazosin and the carbon analogue 2. The furan ring of 2 was replaced by a phenyl ring because it offered us the possibility to incorporate additional structural elements at different positions (Fig. 3). It is known that subtle and unpredictable differences in the binding pockets may account for selectivity; thus, incorporation of additional structural elements in the structure of a non-selective ligand may well lead to preferential recognition of a particular receptor subtype. Thus, a chloromethyl substituent was introduced in the aromatic ring because it can be easily functionalized affording compounds with different properties (Bolognesi et al., 1998). For example, an amine function can be protonated at physiological pH giving rise to a possible, additional interaction with a nucleophilic, complementary receptor group, which would increase the possibility to achieve receptor subtype selectivity. The replacement of the hexane spacer or the furan ring of 2 by a cystamine moiety or a phenyl group, affording cystazosin and the phenyl analogue 8, respectively, caused

pKi

(+)-Cyclazosin(-)-Cyclazosin (+_)-Cyclazosm Prazosin Fig. 2. Affinity estimates (p K i) of racemic cyclazosin and its enantiomers for cloned a j-adrenoreceptor subtypes (c~ la: bovine brain; c~lb: hamster smooth muscle; OLld: rat brain) in comparison to prazosin.

a dramatic effect in the affinity profile for oL1-adrenoreceptor subtypes as reported in Table 2 and graphically shown in Fig. 4. Clearly, 2 is a very potent oL~-adrenoreceptor antagonist but, at the same time, is not selective displaying only a slight preference for the oL1B-Subtype. Interestingly, the structural modifications performed on 2 did not improve affinity for ot ~-adrenoreceptor subtypes but, what is more important, gave rise to selectivity. Thus, cystazosin (3) proved to be a selective oL1D-adrenoreceptor antagonist owing to a slight (4.5-fold) decrease in affinity for the oL1D-subtype and a large drop in affinity (32- and 224-fold, respectively) for OtlA- and OtlB-Subtypes in comparison with 2. On the other hand, the phenyl analogue 8 displayed a significantly improved oLjB-selectivity (85- and 15-fold relative to ~ 1A- and oL~D-Subtypes, respectively), owing to a much larger decrease in affinity for both Ot~A- and e~~D-SUbtypes than for the ot 1B-Subtype in comparison with 2 (Fig. 4) (Bolognesi et al., 1998; Minarini et al., 1998). The insertion of a 5-chloromethyl or a 5-N,N-dimethylaminomethyl substituent on the furan ring of 3, affording 4 and 5, respectively, did not improve the selectivity profile. The same structural modification performed on 2 to afford 6 and 7 caused a marked decrease in affinity which was more pronounced for 5-N,N-dimethylaminomethyl group. However, compound 6 was slightly more potent than 2 at oL~D-adrenoreceptors while displaying a significantly lower affinity at OL1A- and OtlB-Subtypes as revealed by its p A 2 values (Ot lA, 8.17; OL1B, 8.97, OtlD, 9.39). Clearly, this finding suggests that appropriate substituents on the aromatic moiety may have a role in achieving receptor subtype selectivity. The insertion of a substituent on the benzene ring of 8, affording 9-20, affected differently, according to substituent type and position, the affinity and, as a consequence, the selectivity for oL~-adrenoreceptor subtypes (Table 2). A most intriguing finding was the observation that polyamines 11, 16 and 20 retained high affinity for oL~adrenoreceptor subtypes which suggests clearly that a 1,6diaminohexane chain on the benzene ring did not give rise to negative interactions with the receptor. This observation may have relevance for the development of new quinazo-

184


MeO~ ~

NH2 /~'N

X.X/J 2" X=CH 2 3:X-S

MeO. ~

NH2 "]'N

MeO. ~

MeO~N~x~.N ~N[ ~O O

R

NH~ ~ N

MeO~N(~N ~ ~ N ] ~O

R

Fig. 3. Design strategy for the synthesis of prazosin-related quinazolines by inserting a substituent on the furan ring of 2 or 3 or by replacing the furan ring of 2 with an (un)substituted phenyl ring. See Table 2 for structures.

lines bearing a polyamine backbone on which additional substituents can be mounted to improve selectivity for otl-adrenoreceptor subtypes. Clearly, the site where the terminal aromatic ring of 8 interacts does not seem to

Table 2 Antagonist affinities, expressed as pA 2 or pK B values, of 2 - 2 0 at a 1-adrenoreceptors on isolated tissue from the rat, namely prostatic vas deferens (alA), spleen ( a 1B), and thoracic aorta ( a 1D) in comparison to prazosin (1) and BMY-7378 a

MeO~A ~-~-

- -

Me

R

-~ MeO_A ~,~

O 2-7

No.

X

1 2 3 4 5 6 7 8 9 10 11 12 13 14

(prazosin) CH 2 S S S CH 2 CH 2

R

Me Me 8-20

R

O pA 2

alA alB aiD

15 16 17 18 19 20 BMY-7378

H H CHeC1 CH2NMe 2 CH2C1 CH 2 NMe2 H 2-CH 2C1 2-CH 2 NMe2 2-CH2 NH(CH 2)6 NH2 3-CH 2C1 3-CH2NH 2 3-CH 2NMe2 /"'S 3-CHaNx,~. j

8.60 9.04 7.53 7.78 6.70 8.17 7.41 7.42 6.38 7.04 7.97 7.10 7.32 7.26

8.99 9.84 7.49 7.37 7.27 8.97 8.62 9.35 8.73 7.88 9.17 7.18 8.61 7.43

8.91 9.19 8.54 8.03 8.22 9.39 8.23 8.16 8.55 8.26 8.50 7.29 8.69 8.26

PA2

7.82 8.03 8.51

3-CH 2 N(Me)(CH 2)6 NHMe 4-CH2C1 4-CH 2NMe2 ~S 4-CH2N,x..j]

8.05 7.71 7.84 7.11 9.15 7.71 7.14 8.53 7.86

4-CH 2N(Me)(CH 2)6 NHMe

6.72 9.21 8.46 6.94 7.55 8.34

aData taken from Bolognesi et al. (1998).

present steric hindrance and particularly stringent requirements. The results obtained in binding experiments with selected quinazolines (Table 3) did not show the same selectivity profile observed in functional assays (Bolognesi et al., 1998). While binding affinities of prazosin (1), 2 and BMY-7378 are qualitatively and quantitatively comparable with p A 2 values derived from functional experiments, those observed for 3 and 14 are not in agreement at all from both a qualitative and a quantitative point of view with functional affinities. Both compounds were devoid of selectivity for oL~-adrenoreceptor subtypes in binding assays owing to a marked increase in affinity of about two orders of magnitude for oLla- and oL~b-adrenoreceptors and of about one order of magnitude for the a ~d-subtype. As a matter of fact, the theory states that the affinity of an antagonist assessed in functional assays should not differ

1 7.23 8.01 8.17

2

3

Compounds Fig. 4. Affinity constants (pA 2) in rat prostatic vas deferens (alA) , spleen (alB), and aorta (ale)) a l-adrenoreceptor subtypes of cystazosin (3) and 8 in comparison with prazosin (1) and 2.

185


Table 3 Affinity constants (pK i) of 2, 3 and 14 (see Table 2 for structures) for cloned o~1-adrenoreceptor subtypes and 5-HT~A receptors in comparison with reference compounds a No.

p Ki, human cloned receptors b OLla

O~lb

Ot 1el

5-HT1A

1 2 3 14 BMY-7378

9.23 9.78 9.38 9.49 6.36

9.39 9.96 8.97 9.78 7.19

9.65 9.71 9.14 9.61 8.89

N R 1 / N R 2 B > N R 1 / N R 2 C > N R 1 / N R 2 D . This behaviour is also seen with MK-801, a compound which antagonises receptor activation by binding to a site within the associated ion channel. Its selectivity seems to be similar, but not quite equal to that of competitive NMDA antagonists. This observation is interesting since it has been generally assumed that because of its mechanism of action, it would be a non-selective inhibitor. The competitive glycine antagonists ACEA 1021 and GV 150526 both have a potency rank order of N R 1 / N R 2 A > N N R 1 / N R 2 C > NR1NR2B > NR2/NR2D. In the same way that agonists allosterically interact, an antagonist at one site, in theory, can also

Table 1 The selectivity of some agonists and antagonists for different combinations of NR1/NR2 subunits of the NMDA receptor. Data for Glutamate, MK-801, GCP39653 and ACEA 1021 were taken from Sucher et al. (1996) Agonist/antagonist

Rank order of potency against different combinationsof NMDA subunits

Glutamate NMDA Glycine MK-801 CGP 39653 ACEA 1021 GV 150526A

NR1/NR2B > NR1/NR2A > NR1/NR2D > NR1/NR2C NR1/NR2A = NR1/NR2B = NR1/NR2C = NR1/NR2D NR 1/NR2C > NR1/NR2B = NR1/NR2D > NR1/NR2A NR1/NR2A = NR1/NR2B > NR1/NR2C = NR1/NR2D NR1/NR2A > NR1/NR2B > NR1/NR2C > NR1/NR2D NR1/NR2A > NR1/NR2C > NR1/NR2B > NR1/NR2D NR1/NR2A > NR1/NR2C > NR1/NR2B > NRI/NR2D

D.G. Trist / Pharmaceutica Acta Helvetiae 74 (2000) 221-229

224

modify the behaviour of another antagonist (Corsi et al., 1996). Importantly, co-agonism predicts that a competitive antagonist against glycine will appear as a non-competitive antagonist against Glu. This is also seen in practice. Fig. 2 shows that a competitive glycine antagonist GV196771A exhibits the expected behaviour against increasing glycine concentrations (parallel displacement of the curve without an effect on the maximal response), but shows clear noncompetitively (reduced maximal response) against NMDA. Thus, in conditions of high Glu, the antagonism will be non-surmountable for a given fixed concentration of glycine. The relevance of this is discussed in more detail later in this article. NMDA receptor antagonists and channel blockers have a number of side effects, most of which are predictable from the physiological roles of NMDA receptors. Thus, learning impairment, ataxia, myorelaxation and sedation have all been reported (Dansysz and Parsons, 1998). In addition, psychomimetic side effects have also been seen (Albers et al., 1991). However, despite the ability of glycine antagonists to completely block Glu responses, the therapeutic window of these agents is considerably wider than that of the first two types of antagonists. This might be explained in terms of subunit composition and agonist preference. Experiments were carried out with the 2carboxy-indole glycine antagonist, GV 150526 (Fig. lb), against NR1/NR2 combinations. In each case, dissociation constants (K B values) and potencies (IC50 values) were measured against glycine/NMDA activation. It was found that differences in subunit selectivity as measured by potency, particularly between NR1/NR2A (plCs0 =

(a)

120 100 80

m

60 40

I

I

8 (b)

7 6 5 -log [glycine]

I

4

3

12(] 101]

~ m

8o 611 4O 2O

6

5 4 -log [NMDA]

3

Fig. 2. Antagonism by the selective glycine antagonist GV196771A (1 p~M, open circles) in embryonic rat hippocampal neurons of (a) a glycine concentration response curve in the presence of a maximal concentration of NMDA (100 IxM) and of (b) a NMDA concentration response curve in the presence of a maximal concentration of glycine (10 p~M). Modified from Quartaroli et al. (1999).

7.21) and NR1/NR2D (plCs0 = 5.25), were of greater magnitude than the relatively smaller changes in affinity (pK B = 8.1 and 7.1, respectively). Potency seemed to be inversely correlated to glycine potency for the subunit combinations (Table 1) suggesting that agonist preference may be the main driver of antagonist subunit selectivity.

3. Pharmacology of non-NMDA receptors

Non-NMDA ionotropic glutamate receptors exhibit none of the functional characteristics of the NMDA receptors (Barnard, 1997). The clear separation by selective agonists of these receptors from NMDA receptors was shown to have a molecular basis by cloning and expression experiments in Xenopus oocytes or in mammalian cell lines. Similar to NMDA receptors, subunits have been described for the non-NMDA types (GluR1-7 and KA1-2). Like NMDA receptors, non-NMDA receptors are postulated to be tetrameric or pentameric in nature (Mano and Teichberg, 1998; Rosenmund et al., 1998). The kainate binding subunits (GluR5-7, KA1-2) are widely distributed in the brain, but functional responses to kainate are only detected in a few cases (Chittajallin et al., 1999). Heteromeric combinations such as GluR6/KA2 have some AMPA sensitivity raising the question as to whether physiological combinations are normally sensitive to AMPA. AMPA receptor subunits traditionally comprise those from GluR1-4. Sequence homology between GluR5 and AMPA receptors (GluR1-4) is relatively low, ~ 40%. No agonists based on AMPA or on willardiine (an AMPA receptor agonist) have been able to show subunit selectivity for particular subunit combinations. (2S-4R)-4methylglutamate (SYM 2081) is 100-1000-fold selective for kainate receptors (Brauner-Osborne et al., 1997) and tert-butyl AMPA (ATPA) specifically activates homomeric GluR5 receptors with no affinity for homomeric GluR6 or GluR7 receptors (Clarke et al., 1997; Swanson et al., 1998). However, halogenated derivatives of willardiine do show different selectivities towards AMPA and kainate receptors in tissues. Thus, (S)-5-fluorowillardiine is a highly selective AMPA receptor agonist (ECs0 AMPA = 1.5 ~M vs. 70 p~M kainate), whereas (S)-5-iodowillardiine has good selectivity for kainate receptors (ECs0 kainate = 0.14 IxM vs. 19 txM AMPA) (Wong et al., 1994). The first generation of competitive antagonists at nonNMDA receptors was based on quinoxaline and quinoxalinedione structures. They showed poor selectivity between AMPA and kainate receptors. More selective compounds have been found with tetrazole-substituted decahydroisoquinolines, LY293558 and LY294486. These block homomeric GluR5 kainate receptors (1-10 txM) and are inactive on GluR6 receptors. LY294486 is between 10- and 100-fold less active on AMPA receptors (Clarke et al., 1997). Recently, it has been shown that the phosphono analogue

D.G. Trist/ Pharmaceutica Acta Helvetiae 74 (2000) 221-229

of AMPA, ATPO, is a competitive inhibitor of recombinant AMPA receptors (K B = 8 txM) (Wahl et al., 1998). Thus, although there are, as yet no really potent and selective compounds, there are sufficient indications for the development of better compounds.

4. Therapeutic potential of modulating glutamate receptors This section will deal with the major potential therapeutic applications of agonists and antagonists acting on glutamate receptors. Potential applications are listed in Table 2 together with the approaches being proposed. Evidences for some indications are stronger than others and, as mentioned above, this part of the review will mainly concentrate on the NMDA receptor as this is the receptor that has been studied the most. Where there are clear indications for other receptor types they will be discussed.

4.1. Acute neurodegeneration It has been known for many years that both Glu and aspartic acid are neurotoxins (Green and Greenamyre, 1996; Choi, 1998). This is especially true if their application is associated with a compromised energy supply. These observations have led to the hypothesis that neurodegeneration, associated with a variety of acute and chronic disorders (stroke, epilepsy, Parkinson's Disease, AIDS dementia) may in part be due to an over-activation of glutamate receptors. Of the possible neurodegenerative indications, stroke was the first to be considered for glutamate receptor antagonists. Animal studies of acute cerebral ischaemia have shown that both NMDA and AMPA receptor antagonists could reduce infarct size following artery occlusion (Meldrum and Garthwaite, 1990). Whilst, encouraging animal results have been obtained with AMPA receptor antagonists such as YM872 and a methylphosphonate derivative of noxalinedione (Turski et al., 1998), the main approach to stroke has been through the NMDA

Table 2 The therapeutic potential of either blocking or activating Glu receptors Therapeutic indication

Approach

Acute stroke

NMDA antagonists; glycine antagonists; kainate antagonists; mGlu 2/3 agonists Glycine and glycine partial agonists Glycine antagonist NMDA antagonists; glycine antagonists NMDA antagonists; rnGlu~ antagonists; mGlu2/3 agonists NMDA antagonists; glycine antagonists; kainate antagonists NMDA antagonists; glycine antagonists Glycine and glycine partial agonists

Alzheimer' s Disease Depression Drug dependency Epilepsy Pain Parkinson's Disease Schizophrenia

225

receptor. All three types of antagonist (channel blocker (MK-801)), competitive NMDA antagonist (CGS 19755), competitive glycine antagonist (ACEA 1021, GV 150526A) show marked efficacy in animal models and have been into man. In both animals and man, ischaemic conditions cause increases in both Glu and glycine as measured by microdialysis in different brain regions and by measuring serum levels (Globus et al., 199 l a; Castillo et al., 1997). In the CSF of patients with closed head injury, there are substantial (up to 8-fold) and long lasting (70 h) increases in glycine concentrations (Palmer et al., 1994b). It has been suggested that the ratio of (Glu • glycine) to GABA (the excitotoxic index) is a better predictor of neuronal damage than either Glu or glycine alone (Globus et al., 1991b). This suggestion fits well with the concept of co-agonism as described above. The rise in glycine levels in ischaemia on the whole seems to be smaller than that of Glu, but persists longer (Andine et al., 1991; Globus et al., 1991a). For these reasons, glycine antagonism in theory could be the preferred approach. Concentrations of a glycine antagonist might not have to be so high as for NMDA antagonists and hence a wider therapeutic window might be expected. Provided the pharmacokinetic profile was adequate, a better cover of protection might be predicted. In addition, the non-competitive nature of the glycine antagonism against Glu (see above) would mean that efficacy should be more consistent. In animals, GV 150526A has shown significant efficacy even when administered 6 h after the ischaemic insult (Bordi et al., 1997). In a rat, permanent distal MCAo model of focal ischaemia GV 150526A given at 3 m g / k g , i.v. was effective in stopping further advancement of infarct size when the progression was approximately halfcomplete (6 h post-occlusion). In the same model, neither MK-801 nor competitive NMDA antagonists have the same time window. Similar effects with GV 150526A have been seen in a transient ischaemia rat model, but slightly higher doses were needed. There seems to be consensus that full glycine antagonists such as GV 150526A do not have the side effects that have limited the clinical use of other NMDA inhibitors (Dansysz and Parsons, 1998). In particular, PCP-like effects, learning impairment, ataxia, myorelaxation and sedation have been seen with both channel blockers and classical NMDA competitive antagonists. Not only are these effects not wanted in a stroke compound, but they can severely reduce the likelihood of success in a clinical trial. Thus, the study with CGS 19755 was terminated early due to lack of efficacy at tolerable doses (Davis et al., 1997). GV 150526A is presently in Phase III clinical studies. A small MRI study in man with GV 150526A given by infusion (loading dose of 800 mg followed by 200 mg maintenance for 3 days) was carried out during Phase II. A trend to reduced infarct volume was seen which correlated with a trend in improvement of neurological function as measured by NIHSS scores (Warach, in press). These

226


results are encouraging that a glycine antagonist will be of use in acute stroke. 4.2. Pain Chronic pain is now thought to involve plastic changes resulting from sustained electrical activity. Peripheral tissue injury or inflammation induce a state of sensory hypersensitivity that manifests itself as allodynia (reduced pain threshold) and hyperalgesia (increased response to noxious stimuli). There is now a large amount of evidence that Glu receptors are involved in the development and the maintenance of pain hypersensitivity (Dougherty and Willis, 1991; Sluka and Westlund, 1992). In animals, NMDA receptor antagonists have suppressed formalin-induced pain behaviour, inflammation and hyperalgesia induced by chronic constriction injury (CCI) of the sciatic nerve (Haley et al., 1990; Davar et al., 1991; Ren et al., 1992). As in stroke, glycine antagonists have proven to be as effective as classical NMDA antagonists in blocking pain states. Thus, GV196771A (1-10 mg/kg, p.o.), a selective glycine antagonist (pKi = 7.6), structurally related to GV 150526A, both blocked the development of and reduced thermal hyperalgesia in the rat CCI model (Quartaroli et al., 1999). GV196771A also was active in other models where hyperalgesia was present. Thus, in the mouse formalin test, GV196771A dose-dependently (0.110 mg/kg, p.o.) reduced the duration of the licking time of the late phase of the test. This phase starts about 15 min after formalin is injected into the animal paw and follows an earlier initial pain response seen after 5 min which subsequently subsides. The first phase is thought to be a direct result of chemical activation of primary nociceptive afferent fibres, whilst the later phase is believed to reflect activation of processes within the spinal cord. This second phase has been shown to involve NMDA receptors and their involvement is consistent with the observed sensitization of spinal neurons within this phase. As might be expected, GV196771A and other NMDA receptor inhibitors do not have an effect on the first phase, whereas morphine will block both parts of the formalin response (Quartaroli et al., 1999). The animal data are supported by some clinical observations with medicines that are known to block NMDA receptor activation, even if they have other pharmacological activities. Thus, low-dose ketamine reduced chronic pain associated with spinal cord injury (Eide et al., 1995) and could reduce pain in patients with peripheral nerve injury and with peripheral vascular disease (Felsby et al., 1996; Eisenberg and Pud, 1998). Amantadine could significantly reduce pain in cancer patients and in those with trauma-induced neuropathic pain (Pud et al., 1998). Although it is not possible to identify that the analgesic action of amantadine and ketamine in chronic pain states is through an action on the NMDA receptor, the clinical data do support the hypothesis that the animal data have gener-

ated that blocking NMDA receptor activity will give potent anti-hyperalgesics. In addition to NMDA receptor inhibitors, the prominence of functional kainate receptors and the GluR5 subunit in dorsal root ganglia suggests that this might also be a target for chronic pain. A selective GluR5 antagonist LY382884 that has poor affinity for AMPA receptors and for GluR6 showed analgesic activity in the rat formalin model (Simmons et al., 1998). 4.3. Epilepsy Glu plays a crucial role in epileptogenesis. Overstimulation of ionotropic Glu receptors leads to convulsions and seizures. In fact, epilepsy was one of the early targets for NMDA receptor antagonists (Czuczwar and Meldrum, 1982). Despite being effective in some animal models, those NMDA receptor antagonists that have been tested in man have not been proven effective. On the one hand, side effects have seriously restricted the clinical trials of classical NMDA antagonists and, on the other hand, some doubts have been cast on the use of global seizure models induced by chemicals and sound as predictors of activity in partial seizures, the greatest population of epilepsy patients (Dansysz and Parsons, 1998). Kindled animals have been suggested as a better model (LiSscher and Schmidt, 1988). Some NMDA antagonists have been reported to interfere with kindling (LiSscher and Honack, 1993). Animal studies with glycine antagonists have been rather variable with no evidence that full antagonists might be a monotherapy in epilepsy. Paradoxically, the best pre-clinical data for modulating the glycine site have come from D-cycloserine, a partial agonist (LiSscher et al., 1994). mGlu 1 receptor activation facilitates depolarisation induced by NMDA or AMPA. Potassium channels are among the most common targets for modulation by mGlu receptors. The overall effect is to excite the postsynaptic membrane which can reduce the Mg 2+ block of the NMDA receptor (Davies et al., 1995; Gereau and Conn, 1995). mGlu 1 is the major subtype found postsynaptically and so antagonists of this receptor should have an effect on NMDA function. Injection of 1S,3R-ACPD, a relatively selective group I agonist, into the hippocampus or thalamus caused generalised limbic convulsions in rats and when given intracerebroventricularly potentiated NMDAinduced convulsions. Further, (S)-4C3HPG, an antagonist at group I receptors and an agonist at group II receptors had anticonvulsant effects in mice. 4.4. Other indications As can be seen in Table 2, there is a number of other potential therapeutic indications for Glu receptors. These are briefly summarised below. Other diseases where over-excitation has been postulated to lead to CNS damage are the chronic neurodegenerative diseases like Alzheimer's Disease, Parkinson's Dis-


ease and Huntington's Disease. Of these, Alzheimer's has created interest in that NMDA receptors seem to be implicated in the pathophysiology of dementia (Greenamyre and Young, 1989). D-cycloserine has shown positive effects in animal models of dementia and in human volunteers given scopolamine (Monahan et al., 1989b; Schuster and Schmidt, 1992; Sirvio et al., 1992; Mohr et al., 1995), but this effect was not reproduced in Alzheimer's patients (Mohr et al., 1995). It has been suggested that in Parkinson's Disease, neurodegeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc) involves excitotoxicity (Carlsson and Carlsson, 1990). In animals, NMDA receptor antagonists have protected against methamphetamine and MPTP-induced damage, models which give Parkinson's-like symptoms (Kupsch et al., 1992). Plastic changes in synaptic transmission have been postulated for a number of psychiatric indications. These include, drug addiction and depression. In drug addiction, there is a wealth of animal data suggesting that NMDA receptors are targets for blocking tolerance and withdrawal syndromes following opiate and cocaine cessation (Marek et al., 1991; Carey et al., 1995; Herman et al., 1995; Morrow et al., 1995). However, to date, these findings have not been tested in man. The data for depression are less robust and is mainly based on the observation that chronic treatment with antidepressants produces a change in glycine interaction with the NMDA receptor (Nowak et al., 1993; Paul et al., 1993; Altamura et al., 1995). This is possibly due to an alteration in subunit composition. The relevance of this observation is still unclear. High affinity NMDA channel blockers, such as PCP, mimic both positive and negative symptoms of schizophrenia in humans. This has supported the hypothesis that there is a glutamatergic basis to this disease and that there might be a deficit in glutamate drive (Carlsson and Carlsson, 1990). Thus, it has been suggested that NMDA agonists might be beneficial. However, the possible neurotoxicity and proconvulsive activity of such an approach makes it less than ideal. However, it has been suggested that the strychnine-insensitive site might offer an alternative approach. Experiments with glycine in animals has shown that PCP-like activity can be decreased (Toth and Lajtha, 1986) and D-serine attenuated stereotypic behaviour produced by NMDA channel blockers such as (+)MK-801 in rats (Contreras, 1990). In man, glycine has been reported to be beneficial in alleviating negative symptoms of schizophrenia (Waziri, 1988) and in enhancing the efficacy of neuroleptics (Rosse et al., 1989). Studies with Dcycloserine were less positive and seemed to have a bellshaped dose-response curve (Cascella et al., 1994).

5. Conclusions

Glu has been proposed to be the major excitatory amino acid of the CNS. As such, it can become excitotoxic in

227

conditions such as ischaemia. In addition, it has been shown to play an important role in plastic changes that occur following repeated stimulation that is seen in chronic pain states, in drug abuse and following constant stress. Glu activates a large number of specific receptors. However, to date, most information has come through studying the NMDA subtype. The fact that this receptor has two co-agonists that are required to be present in order to obtain a response allows a number of approaches to be followed which seem not to suffer from the side effects that might be predicted from the physiological role that these receptors play. A number of therapeutic indications have been suggested for an intervention at the NMDA receptor, but the most likely to be successful are in the stroke, pain and perhaps the epilepsy areas.

References Albers, G.W., Saenz, R.E., Moses, J.A., Choi, D.W., 1991. Safety and tolerance of oral Dextromethorpan in patients at risk for brain ischemia. Stroke 22, 1075-1077. Altamura, C., Maes, M., Dai, J., Meltzer, H.Y., 1995. Plasma concentrations of excitatory amino acids, serine, glycine, taurine and histidine in major depression. Eur. Neuropsychopharmacol. 5, 71-75. Andine, P., Sandberg, M., Bagenholm, R., Lehmann, A., Hagberg, H., 1991. Intra- and extracellular changes of amino acids in the cerebral cortex of the neonatal rat during hypoxic-ischemia. Dev. Brain Res. 64, 115-120. Barnard, E.A., 1997. Ionotropic glutamate receptors: new types and new concepts. TIPS 18, 141-148. Bordi, F., Pietra, C., Ziviani, L., Reggiani, A., 1997. The glycine antagonist GV150526 protects somatosensory evoked potentials and reduces the infarct area in the MCAo model of focal ischemia in the rat. Exp. Neurol. 145, 425-433. Brauner-Osborne, H., Nielsen, B., Stensbol, T.B., Johansen, T.N., Skjaerbaek, N., Krogsgaard-Larsen, P., 1997. Molecular pharmacology of 4-substituted glutamic acid analogues at ionotropic and metabotropic excitatory amino acid receptors. Eur. J. Pharmacol. 335, R1-R3. Brose, N., Gasic, G.P., Vetter, D.E., Sullivan, J.M., Heinemann, S.F., 1993. Protein chemical characterisation and immunocytochemical localisation of the NMDA receptor subunit NMDA R1. J. Biol. Chem. 268, 22663-22671. Carey, R.J., Dai, H.L., Krost, M., Huston, J.P., 1995. The NMDA receptor and cocaine: evidence that MK-801 can induce behavioral sensitization effects. Pharmacol. Biochem. Behav. 51,901-908. Carlsson, M., Carlsson, A., 1990. Interaction between glutamatergic and monoaminergic systems within the basal ganglia - - implications for schizophrenia and Parkinson's disease. Trends Neurosci. 13, 272-276. Cascella, N.G., Macciardi, F., Cavallini, C., Smeraldi, E., 1994. cycloserine adjuvant therapy to conventional neuroleptic treatment in schizophrenia - - an open-label study. J. Neural Transm. 95, 105-111. Castillo, J., Davalos, A., Noya, M., 1997. Progression of ischaemic stroke and excitotoxic amino acids. Lancet 349, 79-83. Chittajallin, R., Braithwaite, S.P., Clarke, V.R.J., Henley, J.M., 1999. Kainate receptors: subunits, synaptic localization and function. TIPS 20, 26-35. Choi, D., 1998. Antagonizing excitotoxicity: a therapeutic strategy for stroke?. Mt. Sinai J. Med. 65, 133-138. Ciabarra, A.M., Sullivan, J.M., Gahn, L.G., Pecht, G., Heinemann, S., Sevarino, K.A., 1995. Cloning and characterisation of chi-l: a developmentally regulated member of a novel class of the ionotropic glutamate receptor family. J. Neurosci. 15, 6498-6508.

228


Clarke, V.R.J., Ballyk, B.A., Hoo, K.H., Mandelzys, A., Pellizzari, A., Bath, C.P., Thomas, J., Sharpe, E.F., Davies, C.H., Ornstein, P.L., Schoepp, D.D., Kamboj, R.K., Collingridge, G.L., Lodge, D., Bleakman, D., 1997. A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission. Nature (London) 389, 599-603. Conn, P.J., Pin, J.-P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205-237. Contreras, P.C., 1990. ~Serine antagonized phencyclidine-induced and MK-801-induced stereotyped behavior and ataxia. Neuropharmacology 29, 291-293. Corsi, M., Fina, P., Trist, D.G., 1996. Co-agonism in drug-receptor interaction: illustrated by the NMDA receptors. TIPS 17, 220-222. Czuczwar, S.J., Meldrum, B.S., 1982. Protection against chemically induced seizures by 2-amino-7-phosphonoheptanoic acid. Eur. J. Pharmacol. 83, 335-338. Dansysz, W., Parsons, C.G., 1998. Glycine, NMDA receptors - - physiological significance and possible therapeutic applications. Pharmacological Reviews 50, 597-664. Davar, G., Hama, A., Deykin, A., Vos, B., Maciewicz, R., 1991. MK-801 blocks the development of thermal hyperalgesia in a rat model of experimental painful neuropathy. Brain Res. 553, 327-330. Davies, C.H., Clarke, V.R.J., Jane, D.E., Collingridge, G.L., 1995. Pharmacology of postsynaptic metabotropic glutamate receptors in rat hippocampal CA1 pyramidal neurons. Br. J. Pharmacol. 116, 18591869. Davis, S.M., Albers, G.W., Diener, H.-C., Lees, K.R., Norris, J., 1997. Termination of acute stroke studies involving selfotel treatment. Lancet 349, 32. Dingledine, R., Borges, K., Bowie, D., Traynelis, S.F., 1999. The glutamate receptor ion channels. Pharmacological Reviews 51, 7-61. Dougherty, P.M., Willis, W.D., 1991. Modification of the responses of primate spinothalamic neurons to mechanical stimulation by excitatory amino acids and an N-methyl-o-aspartate antagonist. Brain Res. 542, 15-22, Suppl. 1. Doyle, D.A., Cabral, J.M., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., MacKinnon, R., 1998. The structure of the potassium channel: molecular basis of K + conduction and selectivity. Science (Washington, D. C.) 280, 69-77. Eide, K., Stubhaug, A., Oye, I., Breivik, H., 1995. Continuous subcutaneous administration of the N-methyl-~aspartic acid (NMDA) receptor antagonist ketamine in the treatment of post-herpetic neuralgia. Pain 61,221-228. Eisenberg, E., Pud, D., 1998. Can patients with chronic neuropathic pain be cured by acute administration of the NMDA receptor antagonist amantadine?. Pain 74, 337-339. Fadda, E.D.W., Wroblewski, J.T., Costa, E., 1988. Glycine and D-serine increase the affinity of the N-methyl-D-aspartate sensitive glutamate binding sites in rat brain synaptic membranes. Neuropharmacology 27, 1183-1185. Felsby, S., Nielsen, J., Arendt-Nielsen, L., Jensen, T.S., 1996. NMDA receptor blockade in chronic neuropathic pain: a comparison of ketamine and magnesium chloride. Pain 64, 283-291. Gereau, R.W., Conn, P.J., 1995. Roles of specific metabotropic glutamate receptor subtypes in regulation of hippocampal CA1 pyramidal cell excitability. J. Neurophysiol. 74, 122-129. Globus, M.Y.T., Busto, R., Martinez, E., Valdes, I., Dietrich, W.D., Ginsberg, M.D., 1991a. Comparative effect of transient global ischemia on extracellular levels of glutamate, glycine and gammaaminobutyric acid in vulnerable and nonvulnerable brain regions in the rat. J. Neurochem. 57, 470-478. Globus, M.Y.T., Ginsberg, M.D., Busto, R., 199 lb. Excitotoxic index B a biochemical marker of selective vulnerability. Neurosci. Lett. 127, 39-42. Green, J.G., Greenamyre, J.T., 1996. Bioenergetics and glutamate excitotoxicity. Prog. Neurobiol. 48, 613-634. Greenamyre, J.Y., Young, A.B., 1989. Excitatory amino acids and Alzheimer's disease. Neurobiol. Aging 10, 593-602.

Haley, J.E., Sullivan, A.F., Dickenson, A.H., 1990. Evidence for spinal N-methyl-~aspartate receptor involvement in prolonged chemical nociception in the rat. Brain Res. 518, 218-226. Herman, B.H., Vocci, F., Bridge, P., 1995. The effects of NMDA receptor antagonists and nitric oxide synthase inhibitors on opioid tolerance and withdrawal - - medication development issues for opiate addiction. Neuropsychopharmacology 13, 269-293. Johnson, J.W., Ascher, P., 1987. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325, 529-531. Kleckner, N.W., Dingledine, R., 1988. Requirement for glycine in activation of NMDA receptors expressed in Xenopus oocytes. Science 214, 843-848. Kupsch, A., Loschmann, P.-A., Sauer, H., Arnold, G., Renner, P., Pufal, D., Burg, M., Wavhtel, H., Ten Bruggencate, G., Oertel, W.H., 1992. Do NMDA receptor antagonists protect against MPIP-toxicity? Biochemical and immunocytochemical analyses in black mice. Brain Res. 181, 1-10. Laube, B., Hirozaku, H., Sturgess, M., Betz, H., Kuhse, J., 1997. Molecular determinants of agonist discrimination by NMDA receptor subunits: analysis of the glutamate binding site on the NR2B subunit. Neuron 18, 493-503. Laube, B., Kushe, J., Betz, H., 1998. Evidence for a tetrameric structure of recombinant NMDA receptors. J. Neurosci. 18, 2954-2961. LiSscher, W., Honack, D., 1993. Effects of the competitive NMDA receptor antagonist, CGP-37849, on anticonvulsant activity and adverse effects of valproate in amygdala-kindled rats. Eur. J. Pharmacol. 234, 237-245. LiSscher, W., Schmidt, D., 1988. Which animal models should be used in the search for new antiepileptic drugs? A proposal based on experimental and clinical findings. Epilepsy Res. 2, 145-181. LiSscher, W., Wlaz, P., Rundfeldt, C., Baran, H., Honack, D., 1994. Anticonvulsant effects of the glycine/NMDA receptor ligands dcycloserine and d-serine but not R(+)-HA-966 in amygdala-kindled rats. Br. J. Pharmacol. 112, 97-106. Mano, I., Teichberg, V.I., 1998. A tetrameric subunit stoichiometry for a glutamate receptor-channel complex. NeuroReport 9, 327-331. Marek, P., Beneliyahu, S., Gold, M., Liebeskind, J.C., 1991. Excitatory amino acid antagonist (kynurenic acid and MK-801) attenuate the development of morphine tolerance in the rat. Brain Res. 547, 77-81. Meldrum, B.S., Garthwaite, J., 1990. Excitatory amino acid neurotoxicity and neurodegenerative disease. TIPS 11,379-387. Mohr, E., Knott, V., Sampson, M., Wesnes, K., Hefting, R., Mendis, T., 1995. Cognitive and quantified electroencephalographic correlates of cycloserine treatment in Alzheimer's disease. Clin. Neuropharmacol. 18, 28-38. Monahan, J.B., Hanadelmann, G.E., Hood, W.F., Cordi, A.A., 1989b. ~cycloserine, a positive modulator of the N-methyl-o-aspartate receptor, enhances performance of learning tasks in rats. Pharmacol. Biochem. Behav. 34, 647-653. Morrow, B.A., Taylor, J.R., Roth, R.H., 1995. R-( + )-HA-966, an antagonist for the glycine NMDA receptor, prevents locomotor sensitization to repeated cocaine exposures. Brain Res. 673, 165-169. Nakanishi, N., Shneider, N.A., Axel, R., 1990. A family of glutamate receptor genes - - evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron 5, 569-582. Nguyen, L., Monaghan, D.T., Cotman, C.W., 1987. Glycine binding sites reciprocally interact with glutamate binding sites at NMDA receptor complex. Soc. Neurosci. Abstr. 13, 209.16. Nowak, G., Trullas, R., Layer, R.T., Skolnick, P., Paul, I.A., 1993. Adaptive changes in the N-methyl-D-aspartate receptor complex after chronic treatment with imipramine and 1-aminocyclopropanecarboxylic acid. J. Pharmacol. Exp. Ther. 265, 1380-1386. Palmer, A.M., Marion, D.W., Botscheller, M.L., Bowen, D.M., Dekosky, S.T., 1994b. Increased transmitter amino acid concentration in human ventricular CSF after brain trauma. NeuroReport 6, 153-156. Paul, I.A., Layer, R.T., Skolnick, P., Nowak, G., 1993. Adaptation of the NMDA receptor in rat cortex following chronic electroconvulsive


shock or imipramine. Eur. J. Pharmacol., Mol. Pharmacol. 247, 305-311. Pud, D., Eisenberg, E., Spitzer, A., Adler, R., Fried, G., Yarnitsky, D., 1998. NMDA receptor antagonist amantadine reduces surgical neuropathic pain in cancer patients: a double blind, randomized, placebo controlled trial. Pain 75, 349-354. Quartaroli, M., Carignani, C., Dal Forno, G., Mugnaini, M., Ugolini, A., Arban, R., Bettelini, L., Maraia, G., Belardetti, F., Reggiani, A., Trist, D.G., Ratti, E., Di Fabio, R., Corsi, M., 1999. Potent antihyperalgesic activity without tolerance produced by glycine site antagonist of N-methyl-I>aspartate receptor GV196771A. J. Pharmacol. Exp. Ther. 290, 158-169. Ren, K., Hylden, J.L.K., Williams, G., Ruda, M.A., Dubner, R., 1992. The intrathecal administration of excitatory amino acid receptor antagonists selectively attenuated carrageenan-induced behavioural hyperalgesia in rats. Eur. J. Pharmacol. 219, 235-243. Rosenmund, C., Stern-Bach, Y., Stevens, C.F., 1998. The tetrameric structure of a glutamate receptor channel. Science (Washington, D. C.) 280, 1596-1599. Rosse, R.B., Theut, S.K., Banay-Schwartz, M., Leighton, M., Scarcella, E., Cohen, C.G., Deutsch, S.I., 1989. Glycine adjuvant therapy to conventional neuroleptic treatment in schizophrenia: an open-label, pilot study. Clin. Neuropharmacol. 12, 416-424. Schuster, G.M., Schmidt, W.J., 1992. I>cycloserine reverses the working memory impairment of hippocampal-lesioned rats in a spatial learning task. Eur. J. Pharmacol. 224, 97-98. Simmons, R.M.A., Li, D.L., Hoo, K.H., Deverill, M., Ornstein, P.L., Iyengar, S., 1998. Kainate GluR5 receptor subtype mediates the nociceptive response to Formalin in the rat. Neuropharmacology 37, 25-36. Sirvio, J., Ekonsalo, T., Riekkinen, P., Lahtinen, H., Riekkinen, P., 1992. D-cycloserine, a modulator of the N-methyl-D-aspartate receptor, im-

229

proves spatial learning in rats treated with muscarinic antagonist. Neurosci. Lett. 146, 215-218. Sluka, K.A., Westlund, K.N., 1992. An experimental arthritis in rats: dorsal horn aspartate, glutamate increases. Neurosci. Lett. 145, 141144, Suppl. 2. Sucher, N.J., Awobuluyi, M., Choi, Y.B., Lipton, S.A., 1996. NMDA receptors: from genes to channels. TIPS 17, 348-355. Swanson, G.T., Green, T., Heinemann, S.F., 1998. Kainate receptors exhibit differential sensitivities to (S)-5-Iodowillardiine. Mol. Pharmacol. 53, 942-949. Toth, E., Lajtha, A., 1986. Antagonism of phencyclidine-induced hyperactivity by glycine in mice. Neurochem. Int. 11,393-400. Turski, L., Huth, A., Sheardown, M., McDonald, F., Neuhaus, R., Schneider, H., Dirnagl, U., Wiegand, F., Jacobsen, P., Ottow, E., 1998. ZK20075: a phosphonate quinoxalinedione/(AMPA) antagonist for neuroprotection in stroke and trauma. Proc. Natl. Acad. Sci. U. S. A. 95, 10960-10965. Wahl, P., Anker, C., Traynelis, S.F., Egebjerg, J., Rasmussen, J.S., Krosgaard-Larsen, P., Madsen, U., 1998. Antagonist properties of a phosphono isoxazole amino acid at glutamate R1-4 (R,S)-2-Amino3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid receptor subtypes. Mol. Pharmacol. 53, 590-596. Waziri, R., 1988. Glycine therapy of schizophrenia. Biol. Psychiatry 23, 209-214. Williams, K., Chao, J., Kashiwagi, K., Masuko, T., Igarashi, K., 1996. Activation of N-methyl-~aspartate receptors by glycine: role of an aspartate residue in the M3-M4 loop of the NR1 subunit. Mol. Pharmacol. 50, 701-708. Wong, L.A., Mayer, M.L., Jane, D.E., Watkins, J.C., 1994. Willardiines differentiate agonist binding sites for kainate-versus AMPA-preferring glutamate receptors in DRG and hippocampal neurons. J. Neurosci. 14, 3881-3897.




Metabotropic glutamate receptors" a structural view point Roberto Pellicciari *, Gabriele Costantino, Antonio Macchiarulo Istituto di Chimica e Tecnologia del Farmaco, Universitb degli Studi di Perugia, Via del Liceo, 1, 06123 Perugia, Italy

Keywords: Metabotropic glutamate receptors; 3D-homology model; Neuroprotective agents; Amino terminal domain

1. Introduction Most neurons and glia in the mammalian central nervous system express multiple receptor subtypes activated by L-Glutamic acid (L-Glu, 1). These receptors have been classified into two main families, termed ionotropic glutamate (iGlu) receptors and metabotropic glutamate (mGlu) receptors (Fig. 1). The ionotropic glutamate receptors are integral membrane-spanning ion channels, formed by different stoichiometric arrangements of hetero-subunits which determine the cation selectivity (Fig. 2a). They are pharmacologically classified as NMDA, AMPA and KA receptors, and are involved in the control of the fast (AMPA) and slow (NMDA) component of excitatory postsynaptic currents. Metabotropic glutamate receptors, first discovered in 1985 (Sladeczek et al., 1985; Nicoletti et al., 1986), constitute a heterogeneous family of GTP-binding proteins (Fig. 2b). So far, eight mGlu receptor subtypes (and several splice variants) have been identified and classified intro three groups according to sequence homology, transduction mechanisms and agonist pharmacology. Group I includes mGluR1 and mGluR5 which are coupled, when expressed in heterologous systems to the activity of phospholipase C. Group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6-mGluR8) are negatively coupled to adenylyl cyclase (AC) but are endowed with a completely different localization and pharmacology (Conn and Pin, 1997; Pellicciari et al., 1999). In addition to the cloned mGlu receptor subtypes, a metabotropic glutamate receptors directly coupled to the activity of phospholipase D (PLD) has been pharmacologically identified (Albani Torregrossa et al., 1999).

Over the last decade, mGlu receptors have become the object of an intense research activity, aimed at the discovery of new therapeutic agents that may help to control malfunctioning of glutamatergic pathways in a clinically useful way. Indeed, the molecular diversity, the modulatory properties and the peculiar synaptic localization of metabotropic glutamate receptors make them an especially attractive targets for the developing of neuroprotective agents devoid of the severe side effects associated with ionotropic glutamate receptor modulators. In particular, it is currently accepted that antagonists of the group I receptor subtypes and agonists of the group II/III have neuroprotective properties following ischemic insult in several models of brain ischemia (Strasser et al., 1998; Bruno et al., 1999). There are, however, indications that this paradigm is a relatively simplistic one, and that the understanding of the precise involvement of individual subtypes requires that selective and potent ligands become available. Central to the design and synthesis of selective ligands is the comprehension of the structural factors that regulate the interaction between ligands and the receptor sites of individual subtypes. The increasing knowledge on the molecular and structural biology of metabotropic glutamate receptors has recently provided the rational basis upon which functional profile, selectivity and potency of available ligands can be understood. In this chapter, we will present our recent results on the molecular modeling and synthesis of selective mGlu receptor ligands, with a major emphasis on group I mGlu receptors.

2. Architecture of glutamate receptors 2.1. I o n o t r o p i c r e c e p t o r s

* Corresponding author. Tel.: +0039-075-46640; fax: +0039-075-5855124; e-mail: [email protected]

In 1990, Seeburg et al. reported the first cloning of a gene expresing a functional ionotropic glutamate receptor


232

R. Pellicciari et al. / Pharmaceutica Acta Helvetiae 74 (2000) 231-237

NRI NMDA

NR2

~ AMPA non-NMDA

KA

~

Ionotropic Glutamate Receptors

I~:~1ooo NRlool NR1olo NR1o11 NR11oo NR11ol NR111o NR1111 NR2-A NR2-B NR2-C NR2-D GluR1 GIuR2 GIuR3 GIuR4

LIVBP-like domain (Unknown Function) LAOBP-like

S1

GluR5 GIuR6 Low Affinity GluR7 KA-1 High Affinity KA-2

~_~

mGlu6 mGlu7 mGlu8 mGlu4

mGluRs

I I

mGlu3 mGlu2 mGlul mGlu5

GroupIII

GroupII Group I

Fig. 1. Classification of glutamate receptors.

(Keinanen et al., 1990). Since then, the family of ionotropic glutamate receptors has continuously grown up, and now at least 14 several spliced variant subunits have been molecularly characterized and shown to assemble into functionally active NMDA, AMPA or KA receptors. Ionotropic glutamate receptors are oligomeric assembly of four or five subunits which form the channel pore and exist into a resting (close), activated (open) and desensitized (closed) state, cooperatively determined by allosteric interaction of different subunits. The macro-architecture of the oligomeric channels and the specific architecture of individual subunits participating to oligomeric complex has been object of an intense debate (for a recent review see Paas, 1998). The currently accepted model for an individual subunits consists of three transmembrane domain (M1, M3, M4), a membrane re-entering segment, two citoplasmatic loops, one extracellular loop and an amino and a carboxy terminus domain (Fig. 3). Glutamate Receptors

.~iiiii~ ..~. '

O

~i~i~ '~i~i~iti!

Ionotropic Receptors a

~i!~i~!!~!~-!!i~iii;i~iii~T M N H 2

Metabotropic Receptors b

-k~UUl-!

Fig. 2. Membrane architecture of glutamate receptors. Ionotropic receptors are integral membrane spanning ion channels formed by the assembly of four or five subunits. Metabotropic glutamate receptors are G-pro-

tein coupled receptors characterized by an unusually extended amino terminal domain (ATD).

$2

domains

(Contains the Binding Site)

COOH

Fig. 3. Architecture of ionotropic glutamate receptors. A single subunit is formed by three transmembrane domains, a membrane-reentering loop and an extended amino terminal domain (ATD). The N-terminal portion of ATD is homologous to LIVBP. The C-terminal portion of ATD and the $2 segment of the extracellular loop are homologous to LAOBP and constitute the ligand-binding domain.

Primary sequence alignment studies, performed by Nakanishi et al. (1990) and by O'Hara et al. (1993) have provided important clues on the structure and localization of the putative neurotransmitter binding site. Of particular interest was the discovery that some elements of either AMPA, NMDA or KA subunits share significant sequence homology with members of the family of Periplasmic Binding Proteins (PBPs). PBPs are bacterial soluble receptors involved in the transport of nutrients (amino acids, sugars, ions) across the cell membrane (Adams and Oxender, 1989). All the PBPs so far crystallized, although often unrelated in sequence, display a common 3D folding patterns and a peculiar mechanism of substrate recognition and processing. Briefly, PBPs are soluble proteins of ellipsoid shape, constituted by two globular domains (lobes) connected by two or three hinge filaments. PBPs exist in equilibrium between an open form, where the two lobes are separated each other by a depth interdomain cleft, and a closed, functionally active, form, where the two lobes are collapsed one on the top of the other upon a rigid body movement around the hinge region. The substrate is thought to initially bind to one lobe of the open form and to induce the conformational change leading to the closed one (Olah et al., 1993) (Fig. 4). Sequence alignment studies demonstrated that the first ~ 400 amino acids of the Amino Terminal Domain (ATD) of ionotropic receptors are homologous to L e u / I s o / V a l Binding Protein (LIVBP), the last ~ 150 amino acids of the ATD (S1 segment) and the $2 segment of the first extracellular loop are homologous to L y s / A r g / O r n Binding Protein (LAOBP). In subsequent years, site-directed mutagenesis experiments, coupled with molecular modeling studies, have permitted to conclude that the L-Glu (1) binding site is localized between the S1 and $2 domains, which fold similarly to LAOBP and keep the same mechanism of domain closure (Stern-Bach et al., 1994; Laube et al., 1997). More recently, the X-ray structure of a KA-bi-

233


riding protein, complexed with kainate has experimentally confirmed this hypothesis (Armstrong et al., 1998).

2.2. Metabotropic glutamate receptors

PBPs exist as a conformationai equilibrium between an open and a closed form

0 k3

-91

k_2

LIVBP-like domain (Contains the Binding Site) Cysteine Rich

Since the first cloning of a metabotropic glutamate receptor in 1991 (Masu et al., 1991), it was clear that this family of receptors owned all the structural features common to the superfamily of G-protein coupled receptors (GPCRs), namely seven transmembrane domains, a cytoplasmatic carboxy terminus which regulates the interaction with G-proteins and an extracellular amino terminal domain. However, several indications pointed out that metabotropic glutamate receptors belong to a different family of GPCRs than the "classical" ones. In particular, the seven transmembrane domains do not share any sequence homology with classical GPCRs and the amino terminal domain (ATD) is unusually long, more than 500 amino acids in mGluR1. Several elegant studies have dealt with the task of clarifying the role of the ATD of mGlu receptors (O'Hara et al., 1993; Takahashi et al., 1993; Shigemoto et al., 1994) and nowadays it is generally accepted that it contains the neurotransmitter binding site and is directly involved in the first step of signal transduction mechanism. A milestone in the understanding of the role of the mGlu receptor's ATD was the discovery, by O'Hara et al. (1993) that the ATD of mGlul, as ionotropic glutamate receptors, share a low but significant sequence homology with members of the family of periplasmic binding proteins (PBP). In particular, the last ~ 400 amino acids of the ATD of mGluR1 are homologous to LIVBP. Based on this sequence homology, O'Hara et al. have identified two amino acids, Ser165 and Thr188, which can be directly involved in the binding of L-Glu (1). Site-directed mutagenesis have confirmed this hypothesis (Fig. 5). More recently, it was recognized that the same architecture is not limited to mGluR1 subtype, but also shared with all the other mGlu receptor subtypes and with the GAB A B and Ca2+-sensing receptors. Taken together, all these re-

IP-

Metabotropic Glutamate Receptors

r

k.3

Unliganded Closed UnligandedOpen LigandedOpen

LigandedClosed

Fig. 4. Mechanism of action of periplasmic binding proteins. In absence of substrate, PBPs exist in equilibrium between an open and closed form. Substrate binds the open form and causes the closure of the two globular domains.

G-Protein Coupling

COOH

Fig. 5. Architecture of metabotropic glutamate receptors. The first 400 amino acids of the ATD are homologous to LIVBP and include the ligand binding domain. The C-terminal portion of the ATD contains a cysteinerich region which is responsible for the homodimerization of the receptor.

ceptors constitute the type 3 family of GPCRs (Galvez et al., 1999).

3. Role of the amino terminal domain in mGluR1. A medicinal chemistry perspective Starting from the initial findings of O'Hara et al. on the sequence homology of mGluR1 with PBPs, we engaged ourselves in a research program aimed at the construction of heuristic models of the ATD of mGluR1 that may help in disclosing those structural features which account for the observed ligand potency and selectivity. By taking advantage of the known 3D-structure of the open form of LIVBP, we reported in 1996 a three-dimensional model of the ATD of mGluR1 (Costantino and Pellicciari, 1996). The model was recently revised by adopting a different alignment strategy that minimizes the occurrence of long gap in the core of the protein. The resulting model displays the overall architecture of the open form of PBPs, with two globular domains (lobes) connected by a hinge regions constituted by three [3-sheets. In our model, the two amino acids identified by site-directed mutagenesis to be involved in L-Glu (1) binding (Ser165 and Thr188) are localized on the surface of one lobe, faced to the interdomain cleft and easily accessible to the neurotransmitter. We thought that, in order to be of some help in the design of novel ligands, the three-dimensional model of ATD of mGluR1 should respond at least to the following characteristics: (i) It should be in agreement with the known structureactivity relationships of mGlul receptor agonists and antagonists. (ii) It should be able to provide mechanistic explanation for agonism and antagonism. (iii) It should be able to provide mechanistic details for the domain closure and signal transduction.

234


The above points will be addressed in the following paragraphs.

4. Agonist binding modes According to the site-directed mutagenesis experiments, the Ser165 and Thr188 residues must be directly implicated in the binding of L-Glu (1) and other agonists. Once having in our hands the 3D-structure of the ATD of mGluR1, we performed a manual docking experiment and we fitted L-Glu (1) in the putative binding site. The amino acidic moiety was accommodated in the close proximity of Ser165 and Thr188 in such a way to form hydrogen bonds with the side chain hydroxy groups. The distal carboxylate group was projected towards the interior of the interdomain cleft, in the same way as the side chain of Leucine does in the crystal structure of LIVBP. It was of great interest to realize that in the homology model of the ATD, a arginine residue (Arg 106) replaces the hydrophobic pocket that forms the binding site for hydrophobic amino acids in the LIVBP crystal structure (Olah et al., 1993). The side chain of such arginine residues may form productive charge-reinforced hydrogen bonds with the distal carboxylate group of mGlul receptor agonists. (Fig. 3, top) We and others have reported mGlul agonist pharmacophores by a ligand-based approach (Costantino et al., 1999; Jullian et al., 1999) The topological features of the agonist pharmacophore(s) are in excellent agreement with the proposed architecture of the ATD binding site. Indeed, the five pharmacophoric points perfectly match Ser165 (which makes hydrogen bonds with the a-amino acidic carboxylate), Thr188 (which makes hydrogen bond with the a-amino acidic amino group) and Arg 106 which interacts with the distal carboxylate. The pharmacophore modeling also pointed out the presence of excluded volume regions. When projected into the 3D-binding sites, many of these regions clearly overlap with a number of amino acids that provide the environment for agonist selectivity. One forbidden region was identified in the mGluR1 agonist pharmacophore which cannot be identified in the 3D-model. This region is localized in the upper part of agonists and is directly exposed to the interdomain cleft. The consequence of this observation will be discussed below.

5. Antagonist binding mode: the interdomain binding hypothesis According to the classical definition, mGlu receptor antagonists are chemical entities that cause the receptor to be in a functionally inactive state. Given the operational model of domain closure for the ATD of mGlu receptors, it can be hypothesized that antagonists may either block

the ATD in the open form or prevents the transduction of the signal after the closed form is achieved. Antagonists of mGluR1 generally belong to the class of carboxyphenylglycine (Chart II) (Eaton et al., 1993). CPGs are considerably longer than L-Glu (1) or other mGluR1 agonists and do not have a conformational flexibility which could allow them to reach a glutamate-like disposition. The attempts to superimpose the pharmacophoric groups of mGluR1 agonists and antagonists into an unique scheme have failed. It can therefore hypothesized that mGluR1 agonists and antagonists adopt different mode of binding inside the receptor cavity. In a medicinal chemistry approach devoted to the understanding of the structure-activity relationships in the class of CPG, we have designed and synthesized AIDA (3) (Pellicciari et al., 1995), a conformationally restricted analog of 4-carboxyphenylglycine (2), which is endowed with a moderate potency but a very high selectivity for mGluR1 and S-CBPG (4), a novel derivative in which the phenyl ring of CPGs is substituted by a propellane moiety (Pellicciari et al., 1996). The propellane moiety has a physicochemical environment very different than the phenyl ring, but still has the property to keep the oL-amino acidic and the distal carboxylate groups in a linear, coplanar disposition. The high affinity and selectivity of (S)-CBPG (4) has a mGluR1 antagonist demonstrated that the linear disposition of pharmacophoric groups is a key property for mGluR1 antagonism. Based on these findings, we have docked mGluR1 antagonists, including 4-CPG (2), AIDA (3) and S-CBPG (4) into the 3D-homology model of the ATD of mGluR1. As expected, antagonists cannot be accommodated in the same binding pocket of agonists, but, rather, they can simultaneously interact with the two lobes of the open form. The oL-amino acidic moiety can form hydrogen bond with Ser165 and Thr188, thus accounting for the competitive antagonism, whereas the distal carboxylate is projected, through the interdomain cleft, towards the second lobe, where a salt-bridge with Lys 341 is formed (Fig. 6). This peculiar mode of binding of antagonists is in agreement with the proposed mechanism of action of the ATD of mGluR1. The simultaneous interaction of antagonists, with the two lobes "freezes" the ATD in the open, functionally inactive, conformation thus preventing the subsequent collapse into the closed, functionally active, form. From another point of view, the mechanistic hypothesis for antagonist binding that we propose is able to explain the absolute requirement for a rigid, coplanar and linear disposition of pharmacophoric groups in mGluR1 antagonists.

6. Comparison between mGluR1 and mGluR5. Structural origin of antagonist selectivity The two mGlu receptor subtypes belonging to group I, namely mGluR1 and mGluR5, share more than 90% of


235

Fig. 6. Proposed mode of binding of agonists and antagonists to the ATD of mGluR1. Agonists (top) bind one lobe. The charged amino group of the agonists represents the trigger for domain (bottom) bind the two lobes simultaneously thus preventing the domain closure.

sequence homology. It is therefore expectable that they will also share a common 3D-folding and that the methodological protocol used to build up the homology model of the ATD of mGluR1 can also be applied to the ATD of mGluR5. Thus, we have engaged ourselves in the task of constructing the 3D-model of the ATD of mGluR5 with the aim of obtaining structural information upon which selectivity among the two subtypes can be discussed. Indeed, there are a very few example of ligands that selectively act on mGluR1 with no effect at mGluR5. Among these, the first reported was AIDA (3) which, despite a only moderate potency (214 txM), selectively antagonizes mGluR1 over mGluR5. More recently, another relatively simple CPG derivatives, LY367385 (5) has been shown to selectively antagonize mGluR1 with a higher potency (8 txM) than AIDA (3). The visual inspection of these two derivatives suggests that very subtle differences in the active site of the two subtypes must be responsible for the observed selectivity. The 3D-model of the ATD of mGluR5 was thus built up (Pellicciari et al. unpublished results) according to the methodological protocol previously described. The binding site region of mGluR5 was

then compared with the binding site region of mGluR1 and it came out that there is an extremely high residue conservation in a sphere of 10 A centered around Ser165 (mGluR1 numbering). The only non conserved residue is a proline (Pro 369) in mGluR1 which is substituted by a glutamine (Gln356) in mGluR5. Can this only substitution account for the observed selectivity? Although neither Pro369 in mGluR1 nor Gln 356 in mGluR5 are directly involved in ligand binding, the inspection of the 3D-models reveals that this substitution affects the overall topology of the active sites. Indeed, Gln356 interacts with Arg96 (homologous to Argl06 in mGluR1), and modifies the orientation of the side chain, by projecting it directly towards the ligand binding pocket, thus making the active site of mGluR5 less sterically accessible than the active site of mGluR1. Docking experiments of the selective mGluR1 antagonists confirmed this hypothesis. The 2-methyl group of LY367386 (5) and the 2-methylene group of AIDA (3) are easily accommodated in the active site of mGluR1 but form steric bumps with Arg 96 in the active site of mGluR5 (Fig. 7). In conclusion, the homology models of the two group I subtypes have allowed us to generate

236


/"

/

!

Thr175 QO2H

mGluR1

~ ) LY-367385

H3C" .,.~ HO2C'"' ~NH2

mGluR5

,,,,,,,,,,,,,,,

Fig. 7. The selective mGluR1 antagonist LY367385 docked into the active site of mGluR1 and mGluR5. In the mGluR5 there is a severe Van deer Waals contact between the 2-methyl group and the side chain of Arg96.

plausible hypothesis concerning the molecular basis of antagonist selectivity.

7. Agonist-mediated domain closure: a mechanistic hypothesis According to the molecular mechanism domain closure of PBPs, the ATD of mGlul receptors should exist in equilibrium between an open and a closed form, both in their ligand-bound or unbound state. The closed, ligandbound, state is the functionally active one. The shift of the equilibrium towards the closed, ligand-bound, state that takes place upon agonist binding to the open form of the ATD must be driven by some modification of the chemical environment of the two globular domains. In order to gain insight into the molecular basis of domain closure, we first analysed the electrostatic potential of the surfaces of the two lobes. It appears evident that the binding lobe, which contains the Ser165 and Thr188 key residues, is endowed with a positive potential, whereas the second lobe is characterized by an overall negative potential, mainly resulting by the presence of a cluster of acidic amino acids. We thought that this electrostatic complementarity between the two lobes must be the principal driving force leading to domain closure. The electrostatic complementarity alone, however, cannot explain how the agonist binding

triggers the domain closure. A possible mechanistic explanation is that in the open unbound form, the interdomain cleft is filled by ordered water molecules that shield the electrostatic interaction between the two lobes. The destruction of the ordered water by the agonist has a twofold effect. The first one is to contribute by an entropic component to the free energy required to the domain closure, the second one is to strengthen the electrostatic attraction between the two lobes. Central to this effect is the role of the oL-amino acidic amino group. Indeed, when docked into the binding site, all the agonists have their positively charged amino group oriented towards the cluster of acidic amino acids in the complementary lobe. The strong, long range interaction between the amino group of agonists and the side-chain carboxylate groups on the second lobe must therefore be seen as the main driving force leading to domain closure. It is interesting to note that this mechanism may account for the somewhat surprising observation that the zwitterionic amino acid group of agonists is not directly involved in electrostatic interactions with charged residues.

8. Conclusions Advances in the molecular biology of metabotropic glutamate receptors, coupled with the application of homology modeling techniques, allow to gain insights into


the molecular mechanism responsible for ligand interaction and signal transduction. The availability of homology models of the ATD of mGluR1, in particular, has permitted us to postulate a molecular mechanism for the action of agonists and antagonists. The information thereby obtained can be usefully instrumental in the design of new, potent and selective ligands.

References Adams, M.D., Oxender, D.L., 1989. Bacterial periplasmic binding protein tertiary structures. J. Biol. Chem. 264, 15739-15742. Albani Torregrossa, S., Attucci, S., Marinozzi, M., Pellicciari, R., Moroni, F., Pellegrini-Giampietro, D.E., 1999. Antagonist pharmacology of metabotropic glutamate receptors coupled to phospholipase D activation in adult rat hippocampus: focus on (2R,I'S,2'R,3'S)-2-(2'carboxy-3'-phenylcyclopropyl)glycine versus 2,3-dihydroxyphenylglycine. Mol. Pharmacol. 55, 699-707. Armstrong, N., Sun, Y., Chen, G.Q., Gouaux, E., 1998. Structure of a glutamate-receptor ligand-binding core in complex with kainate. Nature 395, 913-917. Bruno, V., Battaglia, G., Kingston, A., O'Neill, M.J., Catania, M.V., Di Grezia, R., Nicoletti, F., 1999. Neuroprotective activity of the potent and selective mGlula metabotropic glutamate receptor antagonist, (+)-2-methyl-4 carboxyphenylglycine (LY367385): comparison with LY357366, a broader spectrum antagonist with equal affinity for mGlula and mGlu5 receptors. Neuropharmacology 38, 199-207. Conn, P.J., Pin, J.-P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205-237. Costantino, G., Pellicciari, R., 1996. Homology modeling of metabotropic glutamate receptors (mGluRs). Structural motifs affecting binding modes and pharmacological profile of mGluR1 agonists and competitive antagonists. J. Med. Chem. 39, 3998-4006. Costantino, G., Macchiarulo, A., Pellicciari, R., 1999. Pharmacophore models of group I and group II metabotropic glutamate receptor agonists. Analysis of conformational, steric, and topological parameters affecting potency and selectivity. J. Med. Chem. 42, 2816-2827. Eaton, S.A., Jane, D.E., Jones, P.L., Porter, R.H., Pook, P.C., Sunter, D.C., Udvarhelyi, P.M., Roberts, P.J., Salt, T.E., Watkins, J.C., 1993. Competitive antagonism at metabotropic glutamate receptors by (S)4-carboxyphenylglycine and (RS)-alpha-methyl-4-carboxyphenylglycine. Eur. J. Pharmacol. 244, 195-197. Galvez, T., Parmentier, M.-L., Joly, C., Malitischek, B., Kaupmann, K., Kuhn, R., Bittiger, H., Froestl, W., Bettler, B., Pin, J.-P., 1999. Mutagenesis and modeling the GABA B receptor extracellular domain support a Venus' Flytrap mechanism for ligand binding. J. Biol. Chem. 274, 13362-13369. Jullian, N., Brabet, I., Pin, J.-P., Acher, F.C., 1999. Agonist selectivity of mGluR1 and mGluR2 metabotropic receptors: a different environment but similar recognition of an extended glutamate conformation. J. Med. Chem. 42, 1546-1555. Laube, B., Hirai, H., Sturgess, M., Betz, H., Kuhse, J., 1997. Molecular determinants of agonist discrimination by NMDA receptor subunits:

237

analysis of the glutamate binding site on the NR2B subunit. Neuron 18, 493-503. Keinanen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoorn, T.A., Sakmann, B., Seeburg, P.H., 1990. A family of AMPA-selective glutamate receptors. Science 249, 556-560. Masu, M., Tanabe, Y., Tsuchida, K., Shigemoto, R., Nakanishi, S., 1991. Sequence and expression of a metabotropic glutamate receptor. Nature 349, 760-765. Nakanishi, N., Schneider, N.A., Axel, R., 1990. A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron 5, 569-581. Nicoletti, F., Meek, J.L., Iadarola, M.J., Chuang, D.M., Roth, B.L., Costa, E., 1986. Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippocampus. J. Neurochem. 46, 40-46. O'Hara, P.J., Sheppard, P.O., Thogersen, H., Venezia, D., Haldeman, B.A., McGrane, V., Houamed, K.M., Thomsen, C., Gilbert, T.L., Mulvihill, E.R., 1993. The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins. Neuron 11, 41-52. Olah, G.A., Trakhanov, S., Trewhella, J., Quiocho, F.A., 1993. Leucine/isoleucine/valine-binding protein contracts upon binding of ligand. J. Biol. Chem. 268, 16241-16247. Paas, Y., 1998. The macro- and microarchitectures of the ligand binding domains of glutamate receptors. Trends Neurosci. 21, 117-122. Pellicciari, R., Luneia, R., Costantino, G., Marinozzi, M., Natalini, B., Jakobsen, P., Kanstrup, A., Lombardi, G., Moroni, F., Thomsen, C., 1995. 1-Aminoindan-l,5-dicarboxylic acid: a novel antagonist at phospholipase C-linked metabotropic glutamate receptors. J. Med. Chem. 38, 3717-3719. Pellicciari, R., Raimondo, M., Marinozzi, M., Natalini, B., Costantino, G., Thomsen, C., 1996. (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)glycine, a structurally new group I metabotropic glutamate receptor antagonist. J. Med. Chem. 39, 2874-2876. Pellicciari, R., Marinozzi, M., Costantino, G., Natalini, B., PellegriniGiampietro, D.E., Moroni, F., 1999. (2R, l'S,2'R,3'S)-2-(2'-carboxy3'-phenylcyclopropyl)glycine (PCCG-13), the first potent and selective competitive antagonist of PLD-Coupled metabotropic glutamate receptors. Asymmetric synthesis and preliminary biological properties. J. Med. Chem. 42, 2716-2720. Shigemoto, R., Abe, T., Nomura, S., Nakanishi, S., Hirano, T., 1994. Antibodies inactivating mGluR1 metabotropic glutamate receptor block long-term depression in cultured Purkinje cells. Neuron 12, 1245-1255. Sladeczek, F., Pin, J.-P., Recasens, M., Bockaert, J., Weiss, S., 1985. Glutamate stimulates inositol phosphate formation in striatal neurones. Nature 317, 717-719. Stern-Bach, Y., Bettler, B., Hartley, M., Sheppard, P.O., O'Hara, P.J., Heinemann, S.F., 1994. Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins. Neuron 13, 1345-1357. Strasser, U., Lobner, D., Behrens, M.M., Canzoniero, L.M., Choi, D.W., 1998. Antagonists for group I mGluRs attenuate excitotoxic neuronal death in cortical cultures. Eur. J. Neurosci. 10, 2848-2855. Takahashi, K., Tsuchida, K., Tanabe, Y., Masu, M., Nakanishi, S., 1993. Role of the large extracellular domain of metabotropic glutamate receptors in agonist selectivity determination. J. Biol. Chem. 268, 19341-19345.




Synthesis and pharmacological properties of novel glycine antagonists D. Donati *, R. Di Fabio Glaxo Wellcome, Medicines Research Centre Via A. Fleming, 4, 37100 Verona, Italy

Abstract

The NMDA receptor is an ionotropic receptor complex widely distributed in the central nervous system and its activation, particularly in hypoxic conditions such as stroke, traumatic head injury and hypoglycemia, results in a massive influx of calcium ions into the post-synaptic neurones, leading to cell death through the activation of several neurotoxic cascades. The NMDA receptor is a unique ionotropic receptor complex because its activation requires the simultaneous binding of glutamate and glycine and selective antagonists at the glycine binding site are endowed with a better side-effect profile than competitive NMDA antagonists. Then, considerable efforts have been devoted to find potent and selective ligands, resulting in the identification of several classes of glycine antagonists. The research at Glaxo Wellcome has been aimed at the identification of novel in vivo active glycine antagonists, and led to the synthesis and pharmacological characterization of a number of novel, potent and systemically active compounds belonging to different chemical classes. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: NMDA; Glycine antagonists; Stroke; Indole-2-carboxylates;Benzoazepines; Pyrido[2,3-b]pyrazines

1. Introduction

In the recent past, the role of excitatory amino acids (EAAs) in the physiology and pathophysiology of the central nervous system has been widely investigated and elucidated (Meldrum and Garthwaite, 1990; McCullogh, 1992) and since the discovery of the neurotoxic properties of glutamate, the most important excitatory neurotransmitter in mammalian brain, a significant body of evidence have associated, among others, the glutamatergic hypothesis of acute neurodegeneration with stroke (Choi, 1988). Glutamate acts through the activation of either the ionotropic (NMDA, AMPA and kainic acid subtypes) or the metabotropic receptors (Hollman and Hwinemann, 1994). A great attention has been concentrated on the activation of NMDA receptors as a key event in triggering excitotoxicity and therefore in studying their role in major brain physiological and pathological conditions, such as stroke, traumatic head injury and hypoglycemia. Stroke is generally defined as an abrupt onset of neurological functions caused by a sudden reduction of cerebral blood flow, which is due in turn to either an ischaemic occlusion or a haemorrhagic episode. This event is one of the leading causes of death worldwide and the first cause of long-term

* Tel.: +0039-045-9218653; fax: +0039-045-9218196; e-mail: [email protected]

disability with a high health-care cost for society. Although it is possible to reduce the factors of risk for stroke, no effective neuroprotective agents, able to block the progression of the cerebral damage, are currently available. Therefore, neuroprotection after stroke is clearly an area of unmet need in current medicine. In ischaemic conditions, the reduction of the oxygenenergy supply to the cells causes a significant increase of glutamate within the synaptic clefts (Cotman et al., 1981; Watchins and Evans, 1981; Choi and Rothman, 1991). This event is responsible for the overstimulation of the glutamatergic receptor (Fig. 1). In particular, the activation of the ion channel associated with the NMDA receptor results in a massive influx of Ca 2+ into the post-synaptic neurones, leading to cell death through the activation of different neurotoxic cascades (excitotoxicity) (Di Fabio et al., 1998a). Therefore, a pharmacological intervention with competitive and non-competitive NMDA antagonists, blocking the abnormal influx of Ca 2+ within the post-synaptic neurones, has been suggested to have a potential therapeutic benefit (Meldrum, 1991; Collingridge and Watkins, 1994). The molecular structure of the NMDA receptor is not fully elucidated yet. By analogy with other receptor gated ion channels (e.g., nicotinic receptor), it is believed that NMDA receptor is formed (Fig. 2) by the assembly of five different subunits named NR1 and NR2A_B_c_D, respectively (Nakanishi, 1992; Mori and

0031-6865/00/$ - see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S0031-6865(99)00059-X

D. Donati, R. Di Fabio / Pharmaceutica Acta Helvetiae 74 (2000) 239-245

240

ISCHEMIA ~

~ / Depol

~

+ AXP, glucose, O 2 ,It @ Glutamate 41, Activation of NMDAreceptors ~, ~, /~r" Intracellular Ca ++ ~

Activation of intracellularenzymes (proteases, lipasesetc.) Free-radicals[ ~ production

Cellswelling ~

t

CELL NECROSIS Fig. 1. Involvementof NMDAreceptors in the excitotoxiccascade.

Mishina, 1995) which are encoded by separate genes and several splice variants for each gene have been described (Danysz and Parsons, 1998). On the basis of morphological studies, it has been shown that NMDA receptors are probably heteromeric assemblies of at least four subunits, with their composition depending from the regional expression in different brain areas (Danysz and Parsons, 1998). Such a conclusion is also supported by electrophysiological and pharmacological studies showing that the pharmacology of NMDA receptor ligands (agonist and antagonists) is strongly influenced by the receptor composition. The pathophysiological implication of these differences is still unknown, but this remains a fascinating area of research interest. In addition to the molecular/structural complexity described above, the NMDA receptor is also characterized by a higher level of complexity which is given by the different regulatory sites which control its function. Among the different binding sites present within the NMDA receptor, a special interest has been attracted by the strychnine-insensitive glycine site (glycine receptor). The conductance through the channel associated with the NMDA receptor is indeed strongly influenced by the presence of glycine. Some years ago, it has been reported (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988) that in cultured cerebral neurones, the frequency of the opening of this ion channel was markedly affected by the presence of glycine. This event was so relevant that in the absence of glycine, glutamate failed to activate the NMDA receptor. Based on

these results, glycine was defined as co-agonist of glutamate. In other words, glutamate and glycine co-operate together to promote the transition of the NMDA ion channel associated from the closed to the open state (Corsi et al., 1996; Danysz and Parsons, 1998). Therefore, the glycinergic site associated to the NMDA receptor has gained considerable interest and, during the last decade, it was perceived as a unique target for medicinal chemistry in terms of potential therapeutic intervention (Iversen and Kemp, 1994; Leeson and Iversen, 1994; Di Fabio et al., 1996), since the observation that one of the first glycine antagonists identified, the 7-chloro kynurenic acid (7-C1 KA) (1) depicted in Fig. 3, was endowed with a greater therapeutic index compared to different series of competitive and non-competitive NMDA antagonists (Chiamulera et al., 1990). Then, considerable efforts have been devoted to find potent and selective ligands, resulting in the identification of several classes of glycine antagonists (e.g., tetrahydroquinolines, indoles, quinoxalines, etc.) as exemplified in Fig. 3. However, despite their high in vitro affinity at the glycine binding site, many of the aforementioned compounds showed poor in vivo activity in a variety of animal models, probably because of their insufficient brain penetration (Carling et al., 1993; Moore et al., 1993). As a result of our efforts aimed at the identification of novel in vivo active glycine antagonists as potential antistroke agents, a number of new templates were selected for further exploration, and, among others, we focused our attention on pyrido[2,3-b]pyrazines (7) (Micheli et al., 1996, 1997) indole-2-carboxylates (8) (see for example, Di Fabio et al., 1998a) and benzoazepines (9) (Di Fabio et al., 1999a) (Fig. 4). The synthesis and pharmacological characterization of both indoles and benzoazepines will be described in detail in the following sections. Even though the

o

CI~COOH I H 1

glycine site

H z N " " ~ N''OH O 2

~

Y

g lutarnate site

cl NA'N o

polyamlne site extra

R

IN H

c site

O

CI

3

I H

""COOH 4

R

hosp horylation site MK-801

Ion channel sites

Fig. 2. Schematicrepresentationof the NMDAreceptor.

CI

COOH H 5

CI" v

"N- ~0 HI 6

Fig. 3. Classes of glycine antagonists.

241

D. Donati, R. Di Fabio / Pharmaceutica Acta Helvetiae 74 (2000) 239-245 H

R

R ~

R'

R" COOH

R'

R

H

(7)

(8)

H

0

(9)

Fig. 4. General structures of pyrido[2,3-b]pyrazines (7), indole-2carboxylates (8) and benzoazepines (9).

following discussion will be centered on the discovery and characterization of novel glycine antagonists as potential antistroke agents, it is worth mentioning that increasing evidences support the hypothesis that the NMDA receptors play a pivotal role in the development and maintenance of pain hypersensitivity (Woolf, 1983; Haley et al., 1990; Woolf, 1995; Harris et al., 1996). Following such a hypothesis, glycine antagonists, blocking the overactivation of the NMDA receptor and restoring the baseline level of nociceptive transmission should be effective for the treatment of the chronic pain. The identification of novel glycine antagonists as potential and effective antihyperalgesic agents was therefore considered an attractive and further objective for our project. The results obtained in this field will however be reported and discussed in detail elsewhere (Di Fabio et al., 1998b; Quartaroli et al., 1999).


2.1. Biological evaluation All the new chemical entities (NCEs) were evaluated, both in vitro and in vivo, according to the following screening cascade: (a) binding assay to evaluate the affinity for the glycine site (Kishimoto et al., 1981) and selectivity for glutamate receptors (Honor~ et al., 1986; Giberti et al., 1991; Van Amsterdam et al., 1992); (b) in vitro functional antagonism studies to evaluate potency and activity (Kloog et al., 1988; Ratti et al., 1990); (c) in vivo anticonvulsant activity (Chiamulera et al., 1990); and (d) the most interesting compounds in the NMDA-induced convulsions model, were then submitted for in vivo models of stroke in rats (MCAo) both pre- and post-ischaemia (Tamura et al., 1981).

2.2. Indole-2-carboxylates Indole-2-carboxylates of general structure (5) represent a class of glycine antagonists widely explored in the last

years by a number of different research groups (see for example, Huettner, 1989; Salituro et al., 1990; Rowley et al., 1992) following the preliminary evidence that the substituent at the C-3 position of the indole nucleus could modulate the affinity of these compounds for the strychnine-insensitive glycine binding site. However, glycine antagonists identified within this class were devoid of significant in vivo activity probably because of their poor ability to penetrate the brain. Therefore, we became interested in the synthesis of novel indole-2-carboxylates exhibiting the desired in vivo profile. This activity led to the synthesis of indole-2-carboxylates substituted at the C-3 position with suitable oL,13-unsaturated side chains, which showed nanomolar affinity for the glycine binding site coupled with both high receptor selectivity and high in vivo potency in the NMDA-induced convulsions model in mice (i.v. an p.o. route). Among the compound initially synthesized, GV150526 (gavestinel) (10), currently undergoing clinical studies in man, is the most promising glycine antagonist identified to date (Di Fabio et al., 1997; Di Fabio et al., 1998a). The most relevant characteristics of GV150526 are summarized in the Table 1. The ability of GV150526 to cross the blood-brain barrier results in a substantial neuroprotective effect in a rat model of Middle Cerebral Artery occlusion (MCAo) when given both preischaemia (70% of maximal protection at 3 m g / k g i.v.) and post-ischaemia up to 6 h from the induction of the cerebral damage. As shown in Fig. 5, GV150526, given 6 h after the occlusion, was able to completely stop the damage progression.

O NHPh

N (10) GV150526 (gavestinel)

These results support the view that the blockade of NMDA receptor could be of therapeutic benefit, and, in this respect, glycine antagonists appear to be extremely effective. Furthermore, GV150526 lacks the adverse behavioral effects in rats observed for both competitive NMDA antagonists and NMDA channel blockers: neither

Table 1 Pharmacological profile of GV150526 Type of study

Assay

Results

Notes

Affinity Selectivity Systemic activity MCAo model

[3H]-glycine binding Receptogram (40 brain receptors) NMDA induced convulsion in mice, i.v. Rat, i.v.

PKi= 8.49 _+0.02 No displacement at 10 lxM EDs0 = 0.06 mg/kg EDs0 = 0.8 mg/kg pre-ischaemia EDs0 = 3.0 mg/kg post-ischaemia

Rat cortical membranes Reversed by glycine agonist (I>serine) Single fully protective dose given 6 h after occlusion

242

D. D o n a t i , R. D i F a b i o / P h a r m a c e u t i c a

100

- -

~

80--

~

60--

~

40--

v

~

A c t a H e l v e t i a e 74 ( 2 0 0 0 ) 2 3 9 - 2 4 5

o

0

_____~---OH

I

Cl

2-Aldrithiol

Cl"" ~

surgery

~

~" ~ ~ ~ "~ "

~ ~" "~ "~ _ _

~H N

cooR

C

. . . . .

l .~

PPh3

CI/ ~

(12)

vehicle

\">~-S

N

/~

~L ?--COOR ~N H

(13)

20-OX'~-NHAr

0-d

~ 10 li5 2}) time after occlusion( hrs )

215

30

ArNH2 DMF

Fig. 5. Neuroprotective effect of GV 150526A after post-ischaemia administration (6 h).

O

#~~

ci....j~/-coo

ClaN"

.

.

.

.

H (14)

c~~.

j.--...

NHAr

J

ClaN"

H (15)

Scheme 1. Synthesis of substituted indole-2-carboxylates.

ataxic effects (rotarod) nor impairment of performance (passive avoidance) were observed in mice up to a dose of 30 m g / k g administered i.v. (500-fold the ED50 observed in inhibition of NMDA-induced convulsions), confirming the wider therapeutic window observed with glycine antagonists (Di Fabio et al., 1997; Di Fabio et al., 1998a and references cited therein). These results also confirm the view that our indole 2-carboxylate template could be useful to generate novel and effective compounds, strengthening the need of continuing the chemical exploration of such a class (Di Fabio et al., 1998a). This further chemical exploration allowed identification of a number of analogues of GV150526, and among them, GV228869 (11) exhibited high in vitro affinity to the glycine binding site (Table 2) and promising physico-chemical and pharmacological profile (Di Fabio et al., 1999b). In particular, it was found to be effective in blocking NMDA induced convulsions and it showed a significantly better neuroprotective activity in the MCAo model with respect to GV150526 when given post-ischaemia up to 6 h after occlusion of the middle cerebral artery.

'~NH2 ~NH

The aforementioned indoles and their analogues can be prepared according to different reported methods (Di Fabio et al., 1997, 1999b; Giacobbe et al., 1999 and references cited therein), and in particular, following the general synthetic route described in Scheme 1 (Di Fabio et al., 1999b and references cited therein). The key intermediate (12), which is now available in large-scale as previously described (Di Fabio et al., 1997) was transformed into the amido derivative (14) by using different methods of activation of the carboxyl group. The best results were obtained via the formation of the corresponding 2-pyridyl thioester (13) by using a stoichiometric amount of 2,2'-dipyridyldisulphide and triphenylphosphine. In general, (13) is stable enough to be purified by classical chromatographic procedures; then, it can be reacted with the proper arylamines to give the amides (14) (the same products can be also obtained by a "one-pot" procedure, without the isolation of (13)). The amides (14) can then be transformed into the final indole-2-carboxylates (15) in fairly good overall yields by removal of the various protecting groups according to standard methods.

2.3. Benzoazepines

C!

To discover novel non-indole classes of glycine antagonists as potential back-up to GV150526A, new templates were designed by receptor mapping techniques based on the pharmacophore model of the glycine binding site (Di

H (11) GV228869

Table 2 Summary of the pharmacological profile of GV228869 compared to GV150526 Type of study

GV150526

GV228869

Affinity Ki Receptor selectivity IC 50 NMDA-induced convulsions ED50 MCAo model pre-ischaemia (rat, i.v.) Max. protection

3 nM

2 nM

> 10 IxM (70 receptors) 0.06 mg/kg (mice, i.v.) EDs0 = 0.8 mg/kg 70% at 3 mg/kg i.v.

> 10 ixM (70 receptors) 0.07 mg/kg (mice, i.v.) EDs0 = 0.2 mg/kg 70% at 3 mg/kg i.v.

243


Table 4 Summary of the pharmacological profile of GV224029 compared to GV150526 Type of study GV150526 GV224029 CI

R

Affinity K i Receptor selectivity ICs0

(16)

Fig. 6. Benzoazepine derivatives.

NMDA-induced

convulsions EDs0

3 nM > 10 ~M (70 receptors) 0.06 mg/kg (mice, i.v.)

32 nM > 10 IxM (70 receptors) 0.07 mg/kg (mice, i.v.)

ED50 = 0.8 mg/kg

ED50 = 0.6 mg/kg

ED50 = 3 mg/kg

ED50 = 2.5 mg/kg

MCAo model

Fabio et al., 1997 and references cited therein). Among others, a novel series of benzoazepine derivatives of general structure (16), bearing the same oL,[3-unsaturated side chain as GV150526 at the position C-5 (Fig. 6) and different substituent at C-3 was proposed. A number of different compounds was then prepared and evaluated and results are reported in Table 3 (Di Fabio et al., 1999a). As expected, the unsubstituted derivative was devoid of any in vitro activity, while the most active compound of such a series was the 3-carboxylic derivative GV224029 (Table 3, entry 3). Also, 3-carboxyamides (entries 4 and 5) have shown an interesting affinity, comparable to GV224029. The last one was further characterized from a pharmacological point of view and results are reported in Table 4 below. In particular, it is worth underlying that in spite of its 10-fold lower affinity for the glycine binding site, GV224029 exhibited a similar neuroprotective activity with respect to GV150526 in the MCAo test both pre- and post-ischaemia. The synthesis of benzoazepine derivatives was performed following different synthetic routes, according to the nature of the substituent at C-3. For the sake of simplicity, only the synthesis of the C-3 carboxylic derivatives GV224029 is reported (Scheme 2). Thus, 4-chloro2-nitro-iodobenzene (18) was obtained via Sandmeyer reaction from 4-chloro-2-nitroaniline (17), then reduced to the corresponding aniline derivative (19) in high yield using Fe in AcOH/EtOH. Reaction with p-methoxybenzyl chloride and sodium iodide in DMF, gave the protected aniline (20), which was reacted with methyl malonylchloride to give the amide (21) in high yield after proper deprotection and esterification with t-butyl alcohol (the protection as t-butyl ester in place of the methyl ester was

Pre-ischaemia (rat, i.v.) Post-ischaemia (rat, i.v.)

required to avoid the double bond isomerization during deprotections in the last steps). When the malonamide (21) was reacted with 3-bromo-N-phenylacrylamide under basic conditions, we obtained the open intermediate (22) which was then cyclized, in the presence of palladium tetrakis(triphenylphosphine) to give the protected benzoazepine (23). The final removal of both the N-1 and C-3 carboxylic protecting groups was smoothly accomplished treating (23) first with anisole in H zSO4/TFA and then with base to give the final compound GV224029 (24).

i) NaNOz I H*

NH2 CI~

v

-NO 2

C!

(17)

Entry

R

K i (nM) a

1

H

>

2 3 (GV224029) 4 5 6 7

OH COOH CONH2 CONHPh COOCH3 CN

a[3H]-glycine displacement.

105 1288 32 39 72 916 339

02

CI

H2

(18)

(19)

0

0

i)CI~OM

CI

HPMB

/I

e

ii) LiOH / THF iii) t-BuOH I EDC-CI

OR

PMB

(20)

0 (21)

(21a) R = Me (21b) R = H (21c) R = t-Bu

".~",2y,,

Table 3 Affinity of benzoazepine derivatives for the strychnine-insensitive binding site

Fe I AcOH I EtOH

ii) KI

, cv

v

oo-t-~,

cr

U ' % PMB

v

F'M~

O (22)

(23)

i) HzSO41 anisole

ii) NaoH or Me3SiONa

H

O

GV224029 (24)

Scheme 2. Synthesis of GV224029.

~oo.,..~

,N-'% O

244


3. Conclusions In spite of great efforts by many research groups, there are still no effective drugs available to treat stroke in humans. Among the number of different approaches that are currently under investigation, the search for novel and effective antagonists at the strychnine-insensitive binding site remains probably the most exciting in the field of neuroprotection. Our search in this context has provided new and further evidences of the validity of such an approach and the discovery of new templates has represented a clear step forward. The synthesis and pharmacological characterization of novel indole-2-carboxylates bearing a suitable oL,[3-unsaturated side chain at position C-3 led to the identification of a number of potent, selective and systemically active glycine antagonists and a member of this class, GV150526 (gavestinel) is currently in phase III clinical trials. In this respect, the importance of the oL,[3-unsaturated side chain in the interaction with the glycine binding site has been recently supported with the synthesis and the pharmacological characterization of pyrrole-2-carboxylic acids bearing the same side chain as GV150526 and analogues (Tarzia et al., 1998; Tarzia et al., 1999). At the same time, the identification of novel antagonists belonging to the benzoazepine series has allowed us to further elaborate the existing pharmacophoric model concerning the interaction with the glycine binding site and GV224029 represents a valid prototype for a further progression of the relevant studies.

Acknowledgements I wish to thank all my colleagues who gave a strong enthusiastic contribution to the very important results reported above; their names are reported in the references below.

References Carling, R.W., Leeson, P.D., Moseley, A.M., Smith, J.D., Saywell, K., Tricklebank, M.D., Kemp, J.A., Marshall, J.R., Foster, A.C., Grimwood, S., 1993. Anticonvulsant activity of glycine-site NMDA antagonists: 2. Trans 2-carboxy-4-substituted tetrahydroquinolines. Bioorg. Med. Chem. Lett. 3, 65-70. Chiamulera, C., Costa, S., Reggiani, A., 1990. Effect of NMDA and strychnine-insensitive glycine site antagonists on NMDA-mediated convulsions and learning. Psychopharmacology 102, 551. Choi, D.W., 1988. Neuron 1,623. Choi, D.W., Rothman, S.M., 1991. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu. Rev. Pharmacol. Toxicol. 31, 171-182. Collingridge, G.L., Watkins, J.C., 1994. The NMDA Receptor, 2nd edn. IRL Press, Oxford. Corsi, M., Fina, P., Trist, D.G., 1996. Co-agonism in drug receptor activation: illustrated by the NMDA receptor. Trends Pharmacol. Sci. 17, 220-222. Cotman, C.W., Foster, A.C., Lanthorn, T.H., 1981. An overview of

glutamate as a neurotransmitter. Adv. Biochem. Biopharmacol. 27, 1-27. Danysz, W., Parsons, C.G., 1998. Glycine and NMDA receptors-physiological significance and possible therapeutic applications. Pharmacol. Rev. 50, 597-664. Di Fabio, R., Gaviraghi, G., Reggiani, A., 1996. Strychnine-insensitive glycine binding site and the NMDA receptor. La Chimica e l'Industria 78, 283-289. Di Fabio, R., Capelli, A.M., Conti, N., Cugola, A., Donati, D., Feriani, A., Gastaldi, P., Gaviraghi, G., Hewkin, C.T., Micheli, F., Missio, A., Mugnaini, M., Pecunioso, A., Quaglia, A.M., Ratti, E., Rossi, L., Tedesco, G., Trist, D.G., Reggiani, A., 1997. Substituted indole-2carboxylates as in vivo potent antagonists acting at the strychnine-insensitive glycine binding site. J. Med. Chem. 40, 841-850. Di Fabio, R., Cugola, A., Donati, D., Feriani, A., Gaviraghi, G., Ratti, E., Trist, D.G., Reggiani, A., 1998a. Identification and pharmacological characterization of GV150526, a novel glycine antagonist as a potent neuroprotective agent. Drugs Future 23, 61-69. Di Fabio, R., Corsi, M., Donati, D., Gaviraghi, G., Quartaroli, M., Ratti, E., Reggiani, A., Trist, D.G., 1998. Glycine antagonists as potent analgesic compounds, XVth EFMC International Symposium on Medicinal Chemistry. Book of Abstract, D3. Di Fabio, R., Antolini, M., Bertani, B., Conti, N., Feriani, A., Messeri, T., Missio, A., Pasquarello, A., Pentassuglia, G., Quaglia, A.M., Reggiani, A., Sabbatini, F.M., 1999. SAR and neuroprotective activity of a novel class of glycine antagonists. Book of Abstracts 217th ACS National Meeting, Anaheim, USA, no. 278. Di Fabio, R., Barnaby, R.J., Conti, N., De Magistris, E., Feriani, A., Provera, S., Sabbatini, F.M., Reggiani, A., Rovatti, L., 1999b. Substituted analogues of GV150526 as potent glycine binding site antagonists in animal models of cerebral ischaemia. J. Med. Chem. 42, in press. Giacobbe, S., Di Fabio, R., Baraldi, D., Cugola, A., Donati, D., 1999. Synthesis of substituted indole-2-carboxylates: versatile introduction of a carbamoyl moiety at the C-position. Synth. Commun. 29, 31253135. Giberti, A., Ratti, E., Gaviraghi, G., van Amsterdam, F.Th.M., 1991. Binding of DL-[3H]-oL-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) to rat cortex membranes reveals two sites or affinity states. J. Recept. Res. 11 (5), 727-741. Haley, J.E., Sullivam, A.F., Dickenson, A.H., 1990. Evidence for spinal N-methy-D-aspartate receptor involvement in prolonged chemical nociception in the rat. Brain Res. 518, 218-226. Harris, J.A., Corsi, M., Quartaroli, M., Arban, R., Bentivoglio, M., 1996. Up-regulation of spinal glutamate receptors in chronic pain. Neuroscience 74, 7-12. Hollman, M., Hwinemann, S., 1994. Cloned glutamate receptors. Annu. Rev. Neurosci. 17, 31-108. Honor6, T., Drejer, J., Nielsen, M., 1986. Calcium discriminates two [3H]-kainite binding sites with different molecular targets sizes in rat cortex. Neurosci. Lett. 65, 47-52. Huettner, J.E., 1989. Indole-2-carboxylic acid: a competitive antagonist of potentiation by glycine at the NMDA receptor. Science 243, 1611-1613. Iversen, L.L., Kemp, J.A., 1994. Noncompetitive NMDA antagonists as drug. In: Collingridge, G.L., Watkins, J.C. (Eds.), The NMDA Receptor, 2nd edn. IRL Press, Oxford, pp. 469-486. Johnson, J.W., Ascher, P., 1987. Glycine potentiates the NMDA receptor reponse in cultured brain mouse neurones. Nature 325, 529-531. Kishimoto, H., Simon, J.R., Aprison, M.H., 1981. Determination of the equilibrium dissociation constant at a number of glycine binding site in several areas of the rat central nervous system, using a Na-independent system. J. Neurochem. 37, 1015-1024. Kleckner, N.W., Dingledine, R., 1988. Requirements for glycine in activation of NMDA receptors expressed in xenopus oocytes. Science 241,835-837. Kloog, Y., Haring, R., Sokolovsky, M., 1988. Kinetic characterization of


the phenylcyclidine-N-methyl-~aspartate receptor interaction: evidence for steric blockade of the channel. Biochemistry 27, 843-848. Leeson, P.D., Iversen, L.L., 1994. The glycine site on the NMDA receptor: structure-activity relationships and therapeutic potential. J. Med. Chem. 37, 4053-4067. McCullogh, J., 1992. Excitatory amino acid antagonists and their potential for the treatment of ischemic brain damage in man. Br. J. Clin. Pharmacol. 34, 106-114. Meldrum, B., 1991. Excitatory amino acid antagonists. Blackwell, Oxford. Meldrum, B., Garthwaite, J., 1990. Excitatory amino acid neurotoxicity and neurodegenerative diseases. Trends Pharmacol. 11, 379-387. Micheli, F., Cugola, A., Donati, D., Guarneri, M., Missio, A., Pecunioso, A., Reggiani, A., Tarzia, G., Zanirato, V., 1996. Synthesis and biological evaluation of pyrido[2,3-b]pyrazines and pyrido[2,3b]pyrazine-N-oxide as selective glycine antagonists. Bioorg. Med. Chem. Lett. 6, 2749-2754. Micheli, F., Cugola, A., Donati, D., Missio, A., Pecunioso, A., Reggiani, A., Tarzia, G., 1997.2,3-Dihydro-6,7-dichloro-pyrido[2,3-b]pyrazine8-oxide as selective glycine antagonist with in vivo activity. Bioorg. Med. Chem. 5, 2129-2132. Moore, K.W., Leeson, P.D., Carling, R.W., Tricklebank, M.D., Singh, L., 1993. Anticonvulsant activity of glycine-site NMDA antagonists: 1. 2-Carboxyl prodrugs of 5,7-dichlorokynurenic acid. Bioorg. Med. Chem. Lett. 3, 61-64. Mori, H., Mishina, M., 1995. Structure and function of the NMDA receptor channel. Neuropharmacology 34, 1219-1237. Nakanishi, S., 1992. Molecular diversity of glutamate receptors and implications for brain function. Science 258, 597-603. Quartaroli, M., Carignani, C., Dal Forno, G., Mugnaini, M., Ugolini, A., Arban, R., Bettelini, L., Maraia, G., Belardetti, F., Reggiani, A., Trist, D.G., Ratti, E., Di Fabio, R., Corsi, M., 1999. Potent antihyperalgesic activity without tolerance produced by glycine site antagonist of N-methyl-~aspartate receptor GV196771A. J. Pharmacol. Exp. Ther. 290, 158-169.

245

Ratti, E., Tacconi, S., Graziani, F., Gaviraghi, G., 1990. Requirement of the glycine for the glutamate activity at the NMDA receptor site. Eur. J. Pharmacol. 183, 1665. Rowley, M., Leeson, P.D., Grimwood, S., Foster, A., Saywell, K., 1992. 2-Carboxy-indolines and indoles as potential glycine/NMDA antagonists: effect of five-membered ring conformation of affinity. Bioorg. Med. Chem. Lett. 2, 1627-1630. Salituro, F.G., Harrison, B.L., Baron, B.M., Nyce, P.M., Stewart, K.T., McDonald, I.A., 1990. Indole derivatives: antagonists of the stychnine-insensitive glycine receptor associated with the N-methyl-~ aspartate receptor complex. J. Med. Chem. 33, 2944-2946. Tamura, A., Graham, D.I., McCullogh, J., Teasdale, G.M., 1981. Focal cerebral ischaemia in rat: 1. Description of techniques and early neuropathological consequences. J. Cereb. Blood Flow Metab. 1, 53-60. Tarzia, G., Balsamini, C., Bedini, A., Diamantini, G., Spadoni, G., Tontini, A., Di Fabio, R., Feriani, A., Reggiani, A., Tedesco, G., Valigi, R., 1998. (E)-3-(2-(N-Phenylcarbamoyl)vinyl)pyrrole-2carboxylic acid derivatives: a novel class of glycine antagonists. J. Med. Chem. 41,808-820. Tarzia, G., Balsamini, C., Bedini, A., Diamantini, G., Spadoni, G., Tontini, A., Di Fabio, R., Donati, D., 1999. 3-(2-Carbamoylvinyl)4,5-dimethylpyrrole-2-carboxylic acids as ligands at the NMDA glycine-binding site: a study on the 2-carbamoylvinyI chain modification. I1 Farmaco 54, 101-111. Van Amsterdam, F.Th.M., Giberti, A., Mugnaini, M., Ratti, E., 1992. 3-[( + )-2-Carboxypiperazin-4-yl]propyl-1-phosphonic acid recognizes two N-methyl-~aspartate binding sites in rat cerebral cortex membranes. J. Neurochem. 59, 1850-1855. Watchins, J.C., Evans, R.H., 1981. Excitatory amino acids transmitters. Annu. Rev. Pharmacol. Toxicol. 21, 165-204. Woolf, C.J., 1983. Evidence for a central component of post-injury hypersensitivity. Nature 306, 686-688. Woolf, C.J., 1995. An overview of the mechanism of hyperalgesia. Pulm. Pharmacol. 8, 161-167.



Pharmaceutica Acta Helvetiae 74 (2000) 247-251 www.elsevier.com/locate /pharmactahelv Introduction

Receptors in neurodegenerative diseases W o l f g a n g Froestl * NOVARTISPharma, Therapeutic Area Nervous System, WKL-136.5.25,CH-4002 Basel, Switzerland

Abstract The ability of trophic factors to regulate developmental neuronal survival and adult nervous system plasticity suggests the use of these molecules to treat neurodegeneration associated with human diseases, such as Alzheimer's, Huntington's and Parkinson's disease, of amyotrophic lateral sclerosis and peripheral sensory neuropathies. Recent biological data on the neutrotrophins NGF and BDNF, on GDNF, CNTF and IGF-I are discussed together with first results from clinical trials. Literature is presented on the three-dimensional structures of these trophic factors and on models proposed for ligand-receptor interactions. Substantial progress has been made in the understanding of the mechanisms of apoptosis. The cascade consisting of interaction of apoptosis-inducing ligands with death receptors, the coupling of this complex to adaptor proteins via death domains, the further recruitment of procaspases via death effector or caspase recruitment domains and the execution of cell death via the effector caspases is briefly outlined. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: Nerve growth factor (NGF); Brain-derived neurotrophic factor (BDNF); Glial cell line-derived neurotrophic factor (GNTF); Ciliary neurotrophic factor (CNTF); Insulin-like growth factor-I (IGF-1); Death receptors

1. Introduction In the last few years we witnessed a spectacular increase of knowledge in the field of receptor research. Previously, research dealt mainly with small molecule neurotransmitter receptors from acetylcholine to serotonin m followed by intense work on neuropeptide receptors, such as cholecystokinin, somatostatin or the tachykinins. More recently, much effort has been devoted to the better understanding of trophic factors and their receptors. Although research on some of these topics has started already decades ago, e.g., as Levi-Montalcini and Hamburger (1953), winners of the Nobel Prize 1986, showed that a soluble factor - - the nerve growth factor was responsible for neuronal growth or as Kerr et al. (1972) drew a clear distinction between the morphology of apoptosis and necrosis. The ability of trophic factors to regulate developmental neuronal survival and adult nervous system plasticity suggests the use of these molecules (or of small, brain-penetrating mimetics) to treat neurodegeneration associated with human diseases (Hefti, 1994). However, only the recent development of powerful tools of molecular biology allowed new insights into the complex

* Corresponding author. Tel.: +41-61-696-21-82; fax: +41-61-696-8676; e-mail: [email protected].

architecture of trophic factors, their receptors (or receptor systems) and their complicated signalling cascades.

2. Nerve growth factor and brain-derived neurotrophic factor One of the best investigated groups of neurotrophic factors, defined by Hefti et al. (1993) as "endogenous soluble proteins regulating survival, growth, morphological plasticity or synthesis of proteins for differentiated functions of neurons", is the group of the neurotrophins. Nerve growth factor (NGF) mediates its effects via the high affinity receptor, the tyrosine kinase Trk A, brain-derived neurotrophic factor (BDNF) and the neurotrophins 4 and 5 ( N T - 4 / 5 ) via Trk B, NT-3 via Trk C, while all neurotrophins interact, albeit at different binding sites, with the low affinity receptor p75 NTR (Chao and Hempstead, 1995). p75 NTR belongs to the tumor necrosis factor-related cytokine-receptor family and also plays a role in apoptosis (Springer and Kitzman, 1998). During the last years, a large body of information was collected on the involvement of the neurotrophins in diverse biological and pathological processes. Several studies in rats showed that the supply of hippocampal and neocortical neurons with BDNF is a prerequisite to elicit


248

W. Froestl / Pharmaceutica Acta Helvetiae 74 (2000) 247-251

long-term potentiation (LTP) (Thoenen, 1995; Altar and DiStefano, 1998; Lessmann, 1998). BDNF mRNA levels are increased in rat hippocampus after treatment with antidepressants acting via different mechanisms of action (Altar, 1999). Gene expression and protein synthesis for the neurotrophins is significantly changed after epileptic, hypoglycaemic, ischemic or traumatic brain insults (Lindvall et al., 1994; Elm6r et al., 1998). As earlier studies have shown that NGF prevents cholinergic neuron atrophy and ameliorates spatial memory impairment in rats (Fischer et al., 1987; Hefti, 1994), several clinical trials were started administering recombinant human NGF to Alzheimer's disease patients via intracerebroventricular infusion. It seems that the optimistic expectations (Olson, 1994) were premature. The long-term intracranial administration caused severe negative side effects, which outweighed the potentially beneficial effects (JiSnhagen et al., 1998). Clinical trials with recombinant human BDNF in patients with amyotrophic lateral sclerosis (ALS) are still underway (Amgen-Regeneron, 1998). Detailed information is available on the three-dimensional structure of the NGF dimer arranged in a head-tohead conformation (McDonald et al., 1991; see also the excellent review by Ibfifiez, 1998), whereas structural information on the NGF-Trk A receptor complex is not available yet, which would allow the rational drug design of small brain penetrating neurotrophin mimetics. However, some molecules have been identified, which induce the synthesis of NGF and potentiate its actions, such as the 5-HT1A receptor agonist SR-57746A (currently in Phase 3 clinical trials), the hypoxanthine AIT-082 (Phase 2) and glycosyl phosphatidylinositol GPI-1046 originally derived from immunophilins (Wood, 1998; see Fig. 1). Disappointingly, an independent analysis of the neurotrophic effects of GPI-1046 (Harper et al., 1999) failed

to obtain evidence for nerve regeneration in vivo as was claimed earlier (Steiner et al., 1997).

3. Glial cell line-derived neurotrophic factor (GDNF) GDNF, a survival factor for dopaminergic neurons and spinal motoneurons, is a member of the transforming growth factor-J3 (TGF-[3) superfamily (Cass et al., 1998). Very elegant work of several groups showed that GDNF utilizes a multicomponent receptor system for its signalling cascade. GDNF first binds to its receptor, GDNFR-oL (now called GFRoL-1), an extracellular protein tethered to the plasma membrane by a glycosyl phosphatidylinositol (GPI) anchor. The GDNF-GFRoL-1 complex is then recognized by the transmembrane receptor tyrosine kinase RET. After formation of the stable tripartite complex, RET undergoes tyrosine phosphorylation starting the signalling cascade (Mason, 1996; Robertson and Mason, 1997). The X-ray structure of rat GDNF has been refined to 1.9 A resolution showing that the dimer is arranged in a head-to-tail orientation (Eigenbrot and Gerber, 1997). Jing et al. (1996) showed that the stoichiometry of the ligand receptor complex is (GFRoL-1)2(RET)2GDNF. Taking into account the distribution of positively and negatively charged regions a GDNF-receptor model has been proposed with two GFRoL-1 molecules binding GDNF at the two ends of the molecule through contacts with negatively charged residues in the loops, whereas RET interacts with the positively charged central region of GDNF (Ibfifiez, 1998).

4. Ciliary neurotrophic factor Signalling via a four-component receptor system has been shown for the cytokine CNTF, which promotes the

o

H--CI

SR-57746A (Sanofi)

H

AIT-082 (NeoTherapeutics)

GP1-1046 (Guilford) o

Fig. 1. Structuresof small molecules,which induce the synthesisof NGF and potentiate its actions.


survival of embryonic chick ciliary neurons and of cultured hippocampal and sensory neurons. CNTF first binds to the extracellular protein CNTFRoL anchored to the cell surface via a GPI linkage. This complex then interacts with a 130 kDa transmembrane glycoprotein, the [31 signal transducing receptor component gpl30. In the next step, the tripartite complex recruits the leukemia inhibiting factor receptor (LIFR[3), the [32 signal transducing receptor component. The [31 component is preassociated with a member of the Janus (Jak/Tyk) kinase family. Only on recruitment of the [32 receptor component (leading to a [3 component heterodimerization) activation of the J a k / T y k kinases occurs leading to tyrosine phosphorylation of the Jak kinases and of the [3 receptor components. The phosphotyrosines serve as binding sites for a variety of signal transduction molecules, such as the signal transducers and activators of transcription (STATs; see: Lamb et al., 1998). Phosphorylation of the STATs causes them to form dimers, translocate to the nucleus and bind to specific DNA sequences present in the promotors of cytokine-responsive genes and activate gene expression (Stahl and Yancopoulos, 1994). First clinical investigations using intrathecal administration of CNTF for the treatment of ALS were carried out (Penn et al., 1997). When CNTF was delivered via genetically modified cells encapsulated in porous membranes protection of neurons in a primate model of Huntington's disease was observed (Emerich et al., 1997). The three-dimensional structure of CNTF revealed a four helix bundle motif (helices A to D; McDonald et al., 1995). Site directed mutagenesis studies on CNTF allowed to identify candidate regions for the interaction of CNTF on helix A with gpl30 and on the N-terminus of helix D with LIFR[3 (Di Marco et al., 1996).

5. Insulin-like growth factor-I (IGF-I) An intensely investigated trophic factor is IGF-I, a pleiotropic protein important for normal development and maintenance of central and peripheral nervous system tissues. It interacts with the tyrosine kinase IGF-IR consisting of two extracellular a-subunits linked via disulfide bonds and two [3-subunits, which are largely cytoplasmic and contain the tyrosine kinase domains. When activated by its substrate, IGF-I the IGF-IR signals to its major substrates, i.e., insulin receptor substrate-1 and -2 (IRS-1, IRS-2) and to Shc and from there on via the Ras/Raf pathway, via MEK and MAPK to the nucleus (Rubin and Baserga, 1995). IGF-I supports the survival of cholinergic neurons and upregulates the expression of CHAT. The group of R6mi Quirion showed that IGF-I can protect hippocampal neurons against amyloid A~l_42 induced toxicity and is able to rescue hippocampal neurons pre-exposed to A[3 pep-

249

tides up to 4-5 days (Dor6 et al., 1997a,b). No low molecular IGF-I mimetics are known yet.

6. Death receptors During the last few years, substantial progress has been made in the understanding of the mechanisms of apoptosis or programmed cell death. The reason for this intense work is obvious. Too little apoptosis may cause cancer or autoimmune diseases, whereas neurodegeneration may be a consequence of excessive apoptosis. Apoptosis signalling is mediated by death receptors, a subgroup of the tumor necrosis factor-related cytokine-receptor superfamily (Ashkenazi and Dixit, 1998; Ware et al., 1998), such as the TNF-receptor 1, the Fas receptor (or CD95), TRAMP (TNF-receptor-related apoptosis-mediating protein), and TRAIL (TNF-related apoptosis-inducing ligand) receptors 1 and 2 and others, after interaction with their respective ligands (TNF, Fas ligand or CD95L and TRAIL). By binding of the ligand to the death receptor, homotrimerization of the receptor is induced. In the intracellular region of the death receptors "death domains" (DD) of 80-90 amino acids were identified, which can bind to DDs of adaptor proteins, such as FADD, RAIDD, TRADD or RIP. FADD (Fas-associated DD) is preferentially recruited to the Fas receptor (CD95) via a D D - D D interaction, which in turn recruits procaspase-8 via binding of its N-terminal "death effector domain" (DED) to the corresponding DED of procaspase-8, after which the long N-terminal prodomain of procaspase-8 is cleaved to caspase-8. The formation of this death-inducing signalling complex (DISC) was shown to occur in less than 1 second (Tschopp et al., 1998). An alternative pathway involves the adaptor protein RAIDD (RIP-associated ICH/CED-3-homologous protein with a death domain), which is recruited by the Fas receptor via its C-terminal DD. At its N-terminus RAIDD contains a "caspase recruitment domain" (CARD) that is also present in procaspase-2. The adaptor protein TRADD (TNFR-associated death domain), most effectively bound following ligation of TNFR-1, functions as a platform that recruits several signalling molecules, such as TRAF-2 (the TNFR-associated factor-2), RIP (the receptor-interacting protein), both of which stimulate activation of NF-KB (the nuclear factor KB) and also FADD, which mediates activation of apoptosis (Ashkenazi and Dixit, 1998). TRAMP has been reported to bind TRADD, TRAF-2, FADD and caspase-8. TRAIL can bind either to a TRAIL-R1 trimer or to a TRAIL-R2 trimer, which recruit FADD and caspase-8 or caspase-10 (Schneider et al., 1997). Novel pathways include apoptotic protease-activating factor-1 (APAF-1) with an N-terminal caspase recruitment domain (CARD) recruiting caspase-9, or the RIP-like kinase CARDIAK specifically interacting with the CARD of caspase-1 (Thome et al., 1998). The overall structure of the

250


three protein-protein interacting domains DD, DED and CARD is similar as all consist of six oL-helices (Kumar and Colussi, 1999). The caspases, cysteine proteases, which cleave their substrates after a P1 Asp residue, are the key effector molecules of the apoptosis machinery (Aravind et al., 1999). So far, 13 caspases have been identified. In apoptosis they are involved as upstream initiators, such as caspase-2, -8, -9, and -10 or as downstream effectors directly responsible for the proteolytic cleavage that leads to cell disassembly (caspases-3, -6 or-7). The effector caspases are activated via proteolytic processing by the initiator caspases (Thornberry and Lazebnik, 1998). Death occurs, when the inhibitory subunit of caspase-activated Dnase (ICAD) is cleaved by e.g., caspase-3, which allows Dnase to translocate to the nucleus and to degrade DNA (Schulze-Osthoff et al., 1998). The recruitment of procaspases by adaptor proteins, however, is only one pathway resulting in apoptosis. The second pathway is mediated by members of the bcl-2 family of proteins, either anti-apoptotic (bcl-2 subfamily) or pro-apoptotic proteins (bax subfamily) working in conjunction with distinct Ap-ATPases (Adams and Cory, 1998). It is to be expected that the rapidly growing knowledge on apoptosis will open up new avenues for pharmacological intervention and the development of pro- and antiapoptotic drugs (Kinloch et al., 1999).

References Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival (Review). Science 281, 1322-1326. Altar, C.A., 1999. Neurotrophins and depression (Review). Trends Pharmacol. Sci. 20, 59-61. Altar, C.A., DiStefano, P.S., 1998. Neurotrophin trafficking by anterograde transport (Review). Trends Neurosci. 21,433-437. Amgen-Regeneron, 1998. Brain-derived neurotrophic factor (BDNF, rhBDNF). Drugs Future 23, 1031-1033. Aravind, L., Dixit, V.M., Koonin, E.V., 1999. The domains of death: evolution of the apoptosis machinery. Trends Biochem. Sci. 24, 47-53. Ashkenazi, A., Dixit, V.M., 1998. Death receptors: signaling and modulation (Review). Science 281, 1305-1308. Cass, W.A., Kearns, C.M., Gash, D.M., 1998. Protective and regenerative properties of GDNF in the central nervous system. In: Mattson, M.P. (Ed.), Neuroprotective Signal Transduction. Humana Press, Totowa, pp. 145-161. Chao, M.V., Hempstead, B.L., 1995. p75 and Trk: a two-receptor system (Review). Trends Neurosci. 18, 321-326. Di Marco, A., Gloaguen, I., Graziani, R., Paonessa, G., Saggio, I., Hudson, K.R., Laufer, R., 1996. Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists. Proc. Natl. Acad. Sci. U.S.A. 93, 9247-9252. Dor6, S., Kar, S., Quirion, R., 1997a. Insulin-like growth factor I protects and rescues hippocampal neurons against [3-amyloid- and human amylin-induced toxicity. Proc. Natl. Acad. Sci. U.S.A. 94, 4772-4777.

Dor6, S., Kar, S., Quirion, R., 1997b. Rediscovering an old friend, IGF-I: potential use in the treatment of neurodegenerative diseases (Review). Trends Neurosci. 20, 326-331. Eigenbrot, C., Gerber, N., 1997. X-ray structure of glial cell-derived neurotrophic factor at 1.9 A resolution and implications for receptor binding. Nature Struct. Biol. 4, 435-438. Elm6r, E., Kokaia, Z., Kokaia, M., Carnahan, J., Nawa, H., Lindvall, O., 1998. Dynamic changes of brain-derived neurotrophic factor protein levels in the rat forebrain after single and recurring kindling-induced seizures. Neuroscience 83, 351-362. Emerich, D.F., Winn, S.R., Hantraye, P.M., Peschanski, M., Chen, E.-Y., Chu, Y., McDermott, P., Baetge, E.E., Kordower, J.H., 1997. Protective effect of encapsulated cells producing neurotrophic factor CNTF in a monkey model of Huntington's disease. Nature 386, 395-399. Fischer, W., Wictorin, K., Bj6rklund, A., Williams, L.R., Varon, S., Gage, F.H., 1987. Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor. Nature 329, 65-68. Harper, S., Bilsland, J., Young, L., Bristow, L., Boyce, S., Mason, G., Rigby, M., Hewson, L., Smith, D., O'Donnell, R., O'Connor, D., Hill, R.G., Evans, D., Swain, C., Williams, B., Hefti, F., 1999. Analysis of the neurotrophic effects of GPI-1046 on neuron survival and regeneration in culture and in vivo. Neuroscience 88, 257-267. Hefti, F., 1994. Neurotrophic factor therapy for nervous system degenerative diseases (Review). J. Neurobiol. 25, 1418-1435. Hefti, F., Denton, T.L., Knusel, B., Lapchak, P.A., 1993. Neurotrophic factors: what are they and what are they doing? In: Loughlin, S.E., Fallow, J.H. (Eds.), Neurotrophic Factors. Academic Press, San Diego, pp. 25-49. Ib~fiez, C.F., 1998. Emerging themes in structural biology of neurotrophic factors (Review). Trends Neurosci. 21,438-444. Jing, S., Wen, D., Yu, Y., Hoist, P.L., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., Cupples, R., Louis, J.-C., Hu, S., Altrock, B.W., Fox, G.M., 1996. GDNF-induced activation of the Ret protein tyrosine kinase is mediated by GDNFR-oL, a novel receptor for GDNF. Cell 85, 1113-1124. JiSnhagen, M.E., Nordberg, A., Amberla, K., Biickman, L., Ebendal, T., Meyerson, B., Olson, L., Seiger, A., Shigeta, M., Theodorsson, E., Viitanen, M., Winblad, B., Wahlund, L.-O., 1998. Intracerebroventricular infusion of nerve growth factor in three patients with Alzheimer's disease. Dementia Geriatr. Cognit. Disord. 9, 246-257. Kerr, J.F.R., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257. Kinloch, R.A., Treherne, J.M., Furness, L.M., Hajimohamadreza, I., 1999. The pharmacology of apoptosis (Review). Trends Pharmacol. Sci. 20, 35-42. Kumar, S., Colussi, P.A., 1999. Prodomains-adaptors-oligomerization: the pursuit of caspase activation in apoptosis. Trend Biochem. Sci. 24, 1-4. Lamb, P., Tapley, P., Rosen, J., 1998. Biochemical approaches to discovering modulators of the JAK-STAT pathway. Drug Discovery Today 3, 122-130. Lessmann, V., 1998. Neurotrophin-dependent modulation of glutamatergic synaptic transmission in the mammalian CNS (Review). Gen. Pharmacol. 31,667-674. Levi-Montalcini, R., Hamburger, V., 1953. A diffusible agent of mouse sarcoma, producing hyperplasia of sympathetic ganglia and hyperneurotization of viscera in the chick embryo. J. Exp. Zool. 123, 233-287. Lindvall, O., Kokaia, Z., Bengzon, J., Elm6r, E., Kokaia, M., 1994. Neurotrophins and brain insults (Review). Trends Neurosci. 17, 490496. Mason, I., 1996. The GDNF receptor: recent progress and unanswered questions (Review). Mol. Cell. Neurosci. 8, 112-119. McDonald, N.Q., Lapatto, R., Murray-Rust, J., Gunning, J., Wlodawer, A., Blundell, T.L., 1991. New protein fold revealed by a 2.3

W. Froestl/ Pharmaceutica Acta Helvetiae 74 (2000) 247-251

resolution crystal structure of nerve growth factor. Nature 354, 411414. McDonald, N.Q., Panayotatos, N., Hendrickson, W.A., 1995. Crystal structure of dimeric human ciliary neurotrophic factor determined by MAD phasing. EMBO J. 14, 2689-2699. Olson, L., 1994. Neurotrophins in neurodegenerative disease: theoretical issues and clinical trials (Commentary). Neurochem. Int. 25, 1-3. Penn, R.D., Kroin, J.S., York, M.M., Cedarbaum, J.M., 1997. Intrathecal ciliary neurotrophic factor delivery for treatment of amyotropic lateral sclerosis (Phase I trial). Neurosurgery 40, 94-100. Robertson, K., Mason, I., 1997. The GDNF-RET signalling partnership (Comment). Trends Genet. 13, 1-3. Rubin, R., Baserga, R., 1995. Insulin-like growth factor-I receptor. Laboratory Investigation 73, 311-331. Schneider, P., Thome, M., Burns, K., Bodmer, J.-L., Hofmann, K., Kataoka, T., Holler, N., Tschopp, J., 1997. TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-KB. Immunity 7, 831-836. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S., Peter, M.E., 1998. Apoptosis signaling by death receptors (Review). Eur. J. Biochem. 254, 439-459. Springer, J.E., Kitzman, P.H., 1998. Neuroprotective strategies involving the neurotrophins and their signaling pathways. In: Mattson, M.P. (Ed.), Neuroprotective Signal Transduction. Humana Press, Totowa, NJ, pp. 1-21.

251

Stahl, N., Yancopoulos, G.D., 1994. The tripartite CNTF receptor complex: activation and signaling involves components shared with other cytokines. J. Neurobiol. 25, 1454-1466. Steiner, J.P., Hamilton, G.S., Ross, D.T., Valentine, H.L., Guo, H., Connolly, M.A., Liang, S., Ramsey, C., Li, J.-H.J., Huang, W., Howorth, P., Soni, R., Fuller, M., Sauer, H., Nowotnik, A.C., Suzdak, P.D., 1997. Neurotrophic immunophilin ligands stimulate structural and functional recovery in neurodegenerative animal models. Proc. Natl. Acad. Sci. U.S.A. 94, 2019-2024. Thoenen, H., 1995. Neurotrophins and neuronal plasticity. Science 270, 593-598. Thome, M., Hofmann, K., Burns, K., Martinon, F., Bodmer, J.-L., Mattmann, C., Tschopp, J., 1998. Identification of CARDIAK, a RIP-like kinase that associates with caspase-1. Current Biology 8, 885-888. Thornberry, N.A., Lazebnik, Y., 1998. Caspases: enemies within (Review). Science 281, 1312-1316. Tschopp, J., Irmler, M., Thome, M., 1998. Inhibition of Fas death signals by FLIPs. Curr. Opin. Immunol. 10, 552-558. Ware, C.F., Santee, S., Glass, A., 1998. Tumor necrosis factor-related ligands and receptors. In: Thomson, A.W. (Ed.), The Cytokine Handbook, 3rd edn. Academic Press, San Diego, pp. 549-592. Wood, P.L., 1998. Small molecule inducers of neurotrophic function (Review). IDrugs 1,452-455.


PHARMA( ,EUTICA ACTAHELVETIAE ELSEVIER


Neurotrophin receptor structure and interactions Hiroko Yano, M o s e s V. Chao * Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine Department of Cell Biology Department of Physiology and Neuroscience New York UniL,ersitySchool of Medicine 540 First Avenue New York, NY 10016, USA

Keywords: Neurotrophins; Brain derived neurotrophic factor (BDNF); p75 receptor; NGF; neurotrophin-3 (NT-3); neurotrophin-4/5 (NT-4)

1. Introduction Neurotrophins represent a family of survival and differentiation factors that exert profound effects in the central and peripheral nervous systems (Levi-Montalcini, 1987; Barde, 1989; Thoenen, 1995). Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4) associate as non-covalent homodimers in a biologically active form. These target-derived trophic factors are active on distinct sets of embryonic neurons, whose dependence is restricted in duration during development (Davies, 1994). Neurotrophins have been proposed as therapeutic agents for the treatment of neurodegenerative disorders and nerve injury, either individually or in combination with other trophic factors, such as ciliary neurotrophic factor (CNTF), insulin-like growth factor and fibroblast growth factor (Lindsay et al., 1994; Nishi, 1994). Recent clinical efforts have met with disappointing results. Improvements in overcoming difficulties of delivery and pharmacokinetics in the central nervous system will provide more impetus for the application of neurotrophins for neurodegenerative diseases. An understanding of the mechanisms of neurotrophin action will provide insights into the use of this proteins therapeutically. Responsiveness of neurons to neurotrophins is governed by the expression of two classes of cell surface receptors (Chao, 1992a). For NGF, they are p75 NTR (p75) and the TrkA receptor tyrosine kinase. Three vertebrate trk receptor genes have been isolated, including numerous variants of trk structure (Barbacid, 1994). The related TrkB recep-

* Corresponding author. Tel.: +1-212-263-0761; fax: +1-212-2630723; e-mail: chao-saturn.med.nyu.edu

tor tyrosine kinase binds both BDNF and NT-4, the most closely related neurotrophins from phylogenetic analysis, while TrkC receptor binds only NT-3. NT-3 can also bind to the TrkA receptor. The p75 receptor binds to all neurotrophins with similar affinities, but different kinetics (Rodriguez-Tebar et al., 1992). Hence, the neurotrophins can engage two separate receptors, which can act independently, or interact with each other. The mechanism of action of neurotrophins has provided a challenging and formidable problem in signal transduction. In addition to promoting cell differentiation and survival, neurotrophins can induce cell death under certain conditions. Several mechanisms have been proposed to explain how NGF might act as a trophic factor and as a cell killer (Casaccia-Bonnefil et al., 1998). The survival and cell death properties of the receptors are dependent upon the relative ratio of receptors and the duration of the signalling events. Here we provide evidence that neurotrophin receptors exist as preformed multi-subunit complexes. Such complex formation by transmembrane receptors is likely to dictate biological responsiveness to neurotrophins.

2. Neurotrophin structure Neurotrophins are produced as precursor proteins, which are cleaved at dibasic amino acids to form a mature form of 118-120 amino acids (Angeletti and Bradshaw, 1971; Leibrock et al., 1989; Maisonpierre et al., 1990). The X-ray crystal structure of NGF has been solved and provides a structural model for this family (McDonald et al., 1991). The conservation of structural features indicates that the neurotrophins will adopt similar conformations to that of NGF.

0031-6865/00/$ - see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S003 1 - 6 8 6 5 ( 9 9 ) 0 0 0 3 6 - 9

254

H. Yano, M. V. Chao / Pharmaceutica Acta Helvetiae 74 (2000) 253-260

The dimeric NGF possesses a novel tertiary fold that results in a flat asymmetric molecule with dimensions of 60 A by 25 * by 30 A (McDonald et al., 1991). Each NGF subunit is characterized by two pairs of anti-parallel [3strands that contribute to the molecule's flat, elongated shape. These [3-strands are connected at one end of the neurotrophin by three short loops. These loops are known to be highly flexible and represent the regions in the neurotrophin structure where many amino acid differences exist between the neurotrophins. The three disulfide bridges in each neurotrophin are clustered at the one end of molecule and provides rigidity to the structure. The arrangement of the disulfide linkages is unusual. The disulfide bridges and their connecting residues form a ring structure and a tightly packed cystine knot motif (McDonald and Hendrickson, 1993). This cystine knot allows the two pairs of [3-strands from each neurotrophin to pack against each other, generating an extensive subunit interface. The interface has a largely hydrophobic character comprised primarily of aromatic residues, consistent with the tight association constant (10 ~3 M) measured for NGF. The NGF tertiary fold and "cystine knot" motif have been identified in structures of transforming growth factor [3 (TGF-[3), platelet-derived growth factor (PDGF) and more recently in human chorionic gonadotrophin (McDonald and Hendrickson, 1993). Members of this diverse structural superfamily of ligands typically form homo- or hetero-dimeric species. The structural features of the neurotrophin family, in particular the dimer interface, are highly conserved, as evidenced in the ability of these members to form dimers

Trk receptor

in vitro (Arakawa et al., 1994; Jungbluth et al., 1994; Radziewjewski and Robinson, 1993). These heterodimeric proteins give functional activity in many cases, indicating there is overall compatibility of these structures.

3. Neurotrophin receptors The neurotrophins are unusual among polypeptide growth factors in that two different transmembrane proteins serve as receptors for each neurotrophin. The structural features of Trk tyrosine kinases and the p75 neurotrophin receptor are displayed in Fig. 1. The Trk subfamily of receptor tyrosine kinases is distinguished by immunoglobulin-C2 domains and repeats rich in leucine and cysteine residues in the extracellular domain and a tyrosine kinase domain with a small interruption and a short cytoplasmic tail. The p75 receptor contains four negatively charged cysteine-rich extracellular repeats, and a unique cytoplasmic domain which is highly conserved among species. There are no sequence similarities between the Trk and p75 receptors, in either ligand binding or cytoplasmic domains. The p75 receptor is the founding member of a superfamily of receptors, exemplified by tumor necrosis factor (TNF) and the Fas receptor (Smith et al., 1994)..The TNF family of receptors is defined by canonical cysteine-rich domains in the extracellular region. Each cysteine-rich domain folds independently and is stabilized by extensive intrachain disulfide bonding. Other members include the lymphoid

p75 receptor

i ~

I

Ceramide

i

1

9

m

Fig. 1. Transmembrane receptors for NGF. Whereas the TrkA receptor is a member of tyrosine kinase family of receptor, the p75 neurotrophin receptor belongs to the TNF superfamily of receptors. A schematic representation of the TrkA and the p75 neurotrophin receptor is shown, together with their substrates and adaptor proteins Trk adaptor proteins include FRS II (Kouhara et al., 1997) and SH2B (Qian et al., 1998). Signal transduction by the neurotrophins involves recruitment of enzymatic activities, such as phosphatidylinositol 3' kinase (PI3-K) and phospholipase C-~/ for TrkA (Kaplan and Stephens, 1994) and TRAF6 for p75 (Khursigara et al., 1999).


cell-specific receptor CD30, CD40, CD27, and other diverse members, such as DR-3, DR-4, CAR-1 and GITR (Smith et al., 1994; Brojatsch et al., 1996; Chinnaiyan et al., 1996; Montgomery et al., 1996; Pan et al., 1997). All of these transmembrane proteins share in their extracellular domains a cysteine motif that spans 40 amino acids and is repeated two to six times. Each class of receptors undergoes ligand-induced dimerization (Grob et al., 1985; Jing et al., 1992) that activates multiple signal transduction pathways. Neurotrophin binding to Trk family members produces biological responses through activation of the tyrosine kinase domain resulting in a rapid increase in the phosphorylation of selected effector enzymes, such as phospholipase C-7 and phosphatidylinositol 3'-kinase, PI3-K (Fig. 1). Increased r a s activity, a common signal from all tyrosine kinase receptors, results from the stimulation of guanine nucleotide exchange factors coupled to SHC adaptor proteins which directly interact with Trk after ligand binding (Kaplan and Stephens, 1994). The p75 receptor signals via pathways involved with activation of sphingomyelinase activities (Dobrowsky et al., 1994), NFKB (Carter et al., 1996) and c-jun N-terminal Kinase, JNK (Casaccia-Bonnefil et al., 1996). More crucially, expression of p75 modulates Trk signalling activity at low concentration of neurotrophins (Barker and Shooter, 1994; Hantzopoulos et al., 1994; Verdi et al., 1994). The substrates for neurotrophin Trk receptors-phospholipase C-~/, PI3-K SHC and Grb2 adaptor proteins-are utilized by many tyrosine kinase receptors. This raises the question of how these phosphorylation events lead to different biological outcomes (Chao, 1992b). There are several possibilities. First, the strength and duration of the receptor autophosphorylation events may determine downstream signalling outcomes. Second, differential signalling may be controlled by specific dephosphorylation events. Third, there may be unique second messengers or substrates which determine the specific nature of the response. For neurotrophin factors, the timing of signalling has provided an important criteria for differential signalling. In PC12 cells, NGF induces a prolonged activation of Ras and MAP kinase activity, lasting for several hours, while EGF-mediated MAP kinase activation is transient in nature (Qui and Green, 1991, 1992). The duration signalling is one of the major differences that accounts for the differentiation program elicited by NGF vs. the action of other mitogenic growth factors, such as EGF. However, the magnitude of receptor signalling is not sufficient. Liganddependent autophosphorylation of the EGF receptor is higher than that of the TrkA NGF receptor in PC12 cells (Berg et al., 1992). Since EGF does not elicit neuronal differentiation as NGF, the level of receptor activation cannot solely account for the difference in trophic factor action. Another mechanism is the recruitment of receptor substrates such as docking or adaptor proteins. FRS-2/SNT is

255

an highly phosphorylated protein that contains an NH 2terminal myristylation site, a phophotyrosine binding (PTB) domain and binding sites for SH2 and SH3 containing proteins (Kouhara et al., 1997). Other adaptor protein, SH2-B/rAPS, bind to Grb2 and promotes NGF-dependent activation of Ras and MAP kinases. These proteins interact with the catalytic domain of the TrkA receptor (Qian et al., 1998) and mediate signalling in a variety of neuronal populations. The large number of adaptor proteins that associate with Trk receptors suggest that there are multiple pathways for signal transduction.

4. Binding properties of Trk and p75 receptors Two classes of NGF binding sites exist on the surface of responsive neurons (Sutter et al., 1979). These sites differ 100-fold in equilibrium binding constants, which can be further distinguished by the rates of ligand association and dissociation. The proteins responsible for the high affinity NGF binding site were a subject of considerable debate, since a small percentage of high affinity sites were detected for the TrkA receptor (Jing et al., 1992). However, both p75 and TrkA receptors have been shown to contribute to the high affinity NGF binding site. Whereas p75 displays fast rates of association and dissociation with NGF, TrkA interacts with much slower on- and off-rates (Mahadeo et al., 1994). Due to its unusually slow on-rate, NGF binding to TrkA results in a relative low affinity K d of 10-9-10 -l~ M. A similar affinity has been determined for B DNF binding to TrkB receptors (Dechant et al., 1993). These affinities are lower than the high affinity binding site, K d = 10 -11 M, measured in sensory neurons (Dechant et al., 1993; Sutter et al., 1979). When TrkA and p75 receptors are co-expressed, the on-rate is accelerated 25-fold, creating a new kinetic site whose features are consistent with the high affinity NGF binding site ( K d - - 10 -~1 M). This site requires an excess ratio of p75 to TrkA (Chao, 1994). Hence, one function of the p75 receptor is to increase the binding affinity of NGF for TrkA. It is therefore not correct to refer to p75 and Trk receptors as low and high affinity receptors, since TrkA and TrkB displayed predominantly low affinity values (Hempstead et al., 1991; Dechant et al., 1993) and p75 receptors can exist in a high affinity state (Dechant and Barde, 1997). It is more correct to use the terms high and low affinity to refer to binding sites. Other receptor systems exhibit similar behavior. At least two functional receptor subunits for IL-2, IL-6, and CNTF are required for high affinity ligand binding, with each subunit independently binding the ligand with a lower affinity. A difference of nearly a 100-fold in the equilibrium binding constants can be contributed by the existence

256

H. Yano, M.V. Chao / Pharmaceutica Acta Helvetiae 74 (2000) 253-260

of a multisubunit receptor system (Nicola and Metcalf, 1992; Mahadeo et al., 1994). After binding neurotrophins, the ligand-receptor complex is internalized and retrogradely transported in the axon to the cell body (Distefano et al., 1992). Both Trk and p75 receptors play a role in the retrograde transport of neurotrophins. For example, in the isthmo-optic nucleus, B DNF is taken up at the axon terminal and transported to the cell body by both p75 and TrkB receptors (von Bartheld et al., 1995). Neurotrophin binding to Trk receptors requires the two extracellular immunoglobulin (Ig)-like domains, which may also be necessary for dimerization (Perez et al., 1995; Urfer et al., 1995). The identification of an immunoglobulin-like domain for neurotrophin binding is a recurrent theme for ligand-receptor signalling by receptor tyrosine kinase, such as FGF and c-kit receptors. Besides the IgC2-1ike domains, the region between the transmembrane and the Ig domain nearest to the membrane may be also critical to binding of neurotrophins (McDonald and Meakin, 1996). The localization of neurotrophin binding sites on their receptors will ultimately lead to the generation of small molecule agonists.

5. Interactions between Trk and p75 receptors The ratio of p75 and Trk receptors dictates responsiveness to individual neurotrophins and their respective equilibrium binding properties (Benedetti et al., 1993; Wyatt and Davies, 1993; Verdi et al., 1994). The properties exhibited by neurotrophins for Trk tyrosine kinase receptors result in different consequences in the presence of p75, or other Trk isoforms. Direct interactions between p75 and Trk receptors have been difficult to document biochemicaUy. However, immunoprecipitation experiments carried out in crosslinked spinal cord and brain tissues with ~25I-NGF suggest that an association between the TrkA and p75 may take place (Huber and Chao, 1995). Photobleaching experiments following a fluorescently tagged p75 receptor have also revealed a potential physical interaction with TrkA receptors (Wolf et al., 1995). Based upon these previous experiments, it is conceivable that the two receptors exist as part of a complex. The relative low abundance of the receptors found in most cells has made this difficult to assess. To test these possibility, TrkA and p75 receptor cDNAs have been transfected in human embryonic kidney 293T cells, subjected to immunoprecipitation with anti-Trk antibodies, followed by 9immunoblot analysis. After high level expression of the receptors in 293T cells, an association between the two receptors could be easily observed (Fig. 2). The results with p75 and TrkA receptors have also been detected using other cell expression systems and epitope-tagged versions of the receptors (Gargano et al., 1997; Bibel et al., 1999).

,,

130 IP: Trk

Blot: p75

IP: Trk

Blot: Trk

p75 ,

,,

,,,,,,,,,,,,,,,,,

_ TrkA

77

205 tt3o i 77

p75

/i~iiii7)iii~

130 Lysate Blot: p75

77

Fig. 2. TrkA associates with p75 receptor in 293T cells. 293T cells (~ 1• 106) were co-transfected with p75NTR and either TrkA or empty vector. 36 h after transfection, the cells were harvested into TNE buffer [10 mM Tris (pH 8.0), 150 mM NaC1, 1 mM EDTA, and 1% NP40]. The lysates were immunoprecipitated with anti-pan Trk antibody (C14, Santa Cruz) which recognizes the C-terminusof Trk and subsequentlysubjected to Western blot analysis with an anti-p75NTR antiserum (9992), or anti-pan Trk antiserum. The amount of p75 protein for each transfection is assayedin the bottompanel. The protein visualized in both lanes of the top panel reflects reactivity of the heavy chain of IgG. In addition to binding, signal transduction by TrkA can be influenced by p75 (Berg et al., 1991; Hantzopoulos et al., 1994; Verdi et al., 1994). Cell culture experiments indicate that p75 is capable of enhancing TrkA autophosphorylation (Barker and Shooter, 1994; Verdi et al., 1994). A potential function of the p75 receptor may be to increase the effective concentration of neurotrophin at the cell surface in order to enhance trkA binding. This is consistent with the limited amounts of neurotrophin available to competing neurons during development. Another model is that an altered conformation of Trk may be formed in the presence of p75 which facilitates ligand binding and subsequent signalling functions (Mahadeo et al., 1994). An unexpected property that has emerged is that neurotrophins can, under certain circumstances, promote apoptotic cell death, in addition to a survival function (Casaccia-Bonnefil et al., 1996; Frade et al., 1996; Bamji et al., 1998). The similarity in the intracellular domains of the p75 with other family members, such as the Fas antigen and the p55 TNF receptor (Liepinsh et al., 1997), suggested that p75 might function as a cell death molecule. The Fas and TNF receptor share significant homology within their intracellular domains - - an 80 amino acid region called the death domain which has been shown to be required for the apoptosis-promoting activities of Fas


and TNF receptors. The death domain is a novel proteinprotein association motif found in several pro-apoptotic proteins (Feinstein et al., 1995). The first report of a cell death activity for p75 was made in immortalized cerebellar neuronal cell lines (Rabizadeh et al., 1993). There are only a few examples in which neurotrophins have been directly shown to be responsible for apoptotic cell death. Many cell types express p75, but do not undergo apoptosis as a result of neurotrophin treatment. This suggests that cell context and history are important determinants and that p75 alone is not sufficient for this activity. The effect of p75 on cell death is highly dependent on the developmental stage of the cells and by their differentiative state in culture (Casaccia-Bonnefil et al., 1998). Apoptosis mediated by p75 requires specific conditions, with regard to cell type, cell cycle stage, developmental stage, injury or stress. This could be due to time in culture, metabolic impairment, hypoxia, or other types of "stress" signals, such as nerve injury or trauma (Gu et al., 1999). Sensitive cells may be highly susceptible to injury and inflammation and may become more reactive to released growth factors and cytokines by upregulating receptor expression at the site of injury.

NGF(min)

An important consequence is that Trk tyrosine kinase receptors can negate the signalling properties of p75, with respect to programmed cell death activities (Yoon et al., 1998). When both NGF receptors are co-expressed, as in the case of PC12 cells, TrkA exerts a suppressive effect upon p75, as assayed by ceramide production (Dobrowsky et al., 1995). Introduction of TrkA receptors into oligodendrocyte susceptible to cell death through p75 activation resulted in a reduction in JNK activation and NGF-induced apoptosis (Yoon et al., 1998). In the absence of TrkA expression, binding of NGF to p75 receptors can increase intracellular ceramide levels through increased sphingomyelin hydrolysis. Therefore, TrkA signalling can selectively down-regulate responses elicited from p75 activation. It should be emphasized that p75 co-expression is ordinarily associated with the ability to enhance NGF binding to TrkA (Hempstead et al., 1991; Barker and Shooter, 1994; Mahadeo et al., 1994). To address the state of endogenous TrkA and p75 receptors in PC12 cells, co-immunoprecipitation experiments have been carried out in PC12 cells. A subline of PC12 (615) cells that expressed higher levels of TrkA receptors (Hempstead et al., 1992) was employed. The PC12 615 cell line was subjected to NGF treatment for 10

0

IP: otTrk

'I'L,LLL

77

Blot: pY

+

=.i_

Lysate ........

iI~:'!:''~ "i!i :i#ii~ii~l!ii: ~ii

......

ii::

-- p 7 5 N T R

........

I TrkA

130 -

77

-

0 10

i~;~i~!~!~i~ :'ii!~ii ~jE:~i'~ ~i.~i

205 -

Blot: Trk

10

,

.

§

-

.......#i~

Blot:

257

i 84

:!!i!i!

...........................

i!:

,,~,

205

'

.......

13 0

pY-TrkA

..... .........

Fig. 3. Association of TrkA receptor with p75 in PC12 cells. TrkA-overexpressingPC12 (615) cells were treated with or without NGF (50 ng/ml, 10 min) and then harvested in TNE buffer. Cell lysates (18 mg of protein) were immunoprecipitated with either anti-pan Trk IgG or normal IgG as a negative control (as described - or - on top of the panel). The resulting immunocomplexes were analyzed by immunoblotting with anti-p75NTR antiserum (9992). The immunoprecipitation of TrkA was confirmed by immunoblotting of a portion of the immunocomplex with anti-pan Trk serum and reprobed with anti-phosphotyrosine antibody (pY99, Santa Cruz). The amount of p75 protein in each lysate was analyzed by western blot analysis on the two rightmost lanes of the top panel.

258


min and lysates were prepared. Interestingly, an association between the two receptors could be detected without NGF treatment. After addition of NGF, a similar interaction between the two receptors could be observed (Fig. 3). These results suggest a preformed complex of neurotrophin receptors exists at the cell membrane. A direct interaction between the two receptors has been difficult to observe. Recent studies indicate that both extracellular and intracellular domains of p75 and Trk receptors may be responsible for the co-immunoprecipitation results (Gargano et al., 1997; Bibel et al., 1999). These observations may be explained by additional cytoplasmic proteins that mediate the association of neurotrophin receptors. There are several ways to account for an association between receptors. For the Trk receptors, SH2-containing proteins serve as adaptor molecules that link to enzymatic functions. For p75 receptors, signalling intermediates used by TNF and Fas receptors, such as FADD and TRADD proteins, downstream effectors, such as caspase-8 and the F L I C E / M A C H interacting enzyme (Cleveland and Ihle, 1995) may be functional. It remains to be seen whether p75 utilize these adaptor proteins, or other interacting proteins of different specificity. A family of cytokine associated signaling intermediates, the TNF receptor-associated factors (TRAFs), have been described. Originally discovered as molecules associated with the p75 TNF receptor (Rothe et al., 1994), TRAF proteins represent a family of molecules that signal through activation of JNK and NFKB activities. Moreover, these proteins have been found to be associated with receptors required for different biological activities. These receptors include CD30, CD40, and lymphotoxin 13 (Gedrich et al., 1996; Nakano et al., 1996; Rothe et al., 1995) and the p55 TNF receptor (Hsu et al., 1996). TRAF6 has been found to bind to p75 receptors and to modulate NFKB responses in Schwann cells (Khursigara et al., 1999). The TRAF6 protein contains several protein-protein binding motifs, such as a RING finger and TRAF domain, that may be instrumental in linking the p75 receptor to other cell surface proteins. Another protein that may be involved in linking Trk receptors with the p75 receptor is caveolin-1 (Bilderback et al., 1999). Caveolin-1 is localized to invaginations in the plasma membrane which are frequently sites where signaling molecules are found. A mutual interaction of caveolin-1 protein with an N G F / p 7 5 / T r k A complex may target neurotrophin responses locally in specialized membrane domains.

6. Summary Although ligand-induced dimerization or oligomerization of receptors is a well established mechanism of growth factor signaling, increasing evidence indicates that biological responses are often mediated by receptor trans-signaling mechanisms involving two or more receptor systems. These include G protein-coupled receptors, cytokine,

growth factor and trophic factor receptors. Greater flexibility is provided when different signaling pathways are merged through multiple receptor signaling systems. Trophic factors exemplified by NGF and its family members, ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) all utilize increased tyrosine phosphorylation of cellular substrates to mediate neuronal cell survival. Actions of the NGF family of neurotrophins are not only dictated by ras activation through the Trk family of receptor tyrosine kinases, but also a survival pathway defined by phosphatidylinositol-3kinase activity (Yao and Cooper, 1995), which gives rise to phosphoinositide intermediates that activate the serine/ threonine kinase Akt/PKB (Dudek et al., 1997). Induction of the serine-threonine kinase activity is critical for cell survival, as well as cell proliferation. Hence, for many trophic factors, multiple proteins constitute a functional multisubunit receptor complex that activates r a s - d e p e n dent and r a s - i n d e p e n d e n t intracellular signaling. The NGF receptors provide an example of bidirectional crosstalk. In the presence of TrkA receptors, p75 can participate in the formation of high affinity binding sites and enhanced neurotrophin responsiveness leading to a survival or differentiation signal. In the absence of TrkA receptors, p75 can generate, in only specific cell populations, a death signal. These activities include the induction of NFKB (Carter et al., 1996); the hydrolysis of sphingomyelin to ceramide (Dobrowsky et al., 1995); and the pro-apoptotic functions attributed to p75. Receptors are generally drawn and viewed as isolated integral membrane proteins which span the lipid bilayer, with signal transduction proceeding in a linear step-wise fashion. There are now numerous examples which indicate that each receptor acts not only in a linear, independent manner, but can also influence the activity of other cell surface receptors, either directly or through signaling intermediates. Which step and which intermediates are utilized for crosstalk between the receptors is a critical question. For neurotrophins, their primary function in sustaining the viability of neurons is counterbalanced by a receptor mechanism to eliminate cells by an apoptotic mechanism. It is conceivable that this bidirectional system may be utilized selectively during development and in neurodegenerative diseases.

Acknowledgements This work was supported by grants from the NIH (MVC) and the American Heart Association (HY).

References Angeletti, R.H., Bradshaw, R.A., 1971. The amino acid sequence of 2.5S mouse submaxillarygland nerve growth factor. Proc. Natl. Acad. Sci. 68, 2417-2420. Arakawa, T., Haniu, M., Narhi, L.O., Miller, J.A., Talvenheimo,J., Philo,


J.S., Chute, H.T., Matheson, C., Carnahan, J., Louis, J.-C., Yan, Q., Welcher, A.A., Rosenfeld, R., 1994. Formation of heterodimers from three neurotrophins, nerve growth factor, neurotrophin-3 and brainderived neurotrophic factor. J. Biol. Chem. 269, 27833-27839. Bamji, S., Majdan, M., Pozniak, C.D., Belliveau, D.J., Aloyz R, J.K., Causing, C.G., Miller, F.D., 1998. The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death. J. Cell Biol. 140, 911-923. Barbacid, M., 1994. The trk family of neurotrophin receptors. J. Neurobiol. 25, 1386-1403. Barde, Y.A., 1989. Trophic factors and neuronal survival. Neuron 2, 1525-1534. Barker, P.A., Shooter, E.M., 1994. Disruption of NGF binding to the low-affinity neurotrophin receptor p75 reduces NGF binding to trkA on PC 12 cells. Neuron 13, 203-215. Benedetti, M., Levi, A., Chao, M.V., 1993. Differential expression of nerve growth factor receptors leads to altered binding affinity and neurotrophin responsiveness. Proc. Natl. Acad. Sci. U.S.A. 90, 78597863. Berg, M.M., Sternberg, D.W., Hempstead, B.L., Chao, M.V., 1991. The low affinity nerve growth factor (NGF) receptor mediates NGF-induced tyrosine phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 88, 857-866. Berg, M.M., Sternberg, D., Parada, L.F., Chao, M.V., 1992. K-252a inhibits NGF-induced trk proto-oncogene tyrosine phosphorylation and kinase activity. J. Biol. Chem. 267, 13-16. Bibel, M., Hoppe, E., Barde, Y.-A., 1999. Biochemical and functional interactions between the neurotrophin receptors trk and p75 NTR. The EMBO J. 18, 616-622. Bilderback, T.R., Gazula, V.-R., Lisanti, M.P., Dobrowsky, R.T., 1999. Caveolin interacts with TrkA and p75 NvR and regulates neurotrophin signaling pathways. J. Biol. Chem. 274, 257-263. Brojatsch, J., Naughton, J., Rolls, M.M., Zingler, K., Young, J.A.T., 1996. CAR1, a TNFR-related protein is a cellular receptor for cytopathic avian leukosis-sarcoma viruses and mediates apoptosis. Cell 87, 845-855. Carter, B.D., Kaltschmidt, C., Kaltschmidt, B., Offenhauser, N., BohmMatthaei, R., Baeuerle, P.A., Barde, Y.-A., 1996. Selective activation of NK-KB by nerve growth factor through the neurotrophin receptor p75. Science 272, 542-545. Casaccia-Bonnefil, P., Carter, B.D., Dobrowsky, R.T., Chao, M.V., 1996. Death of oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature 383, 716-719. Casaccia-Bonnefil, P., Kong, H., Chao, M.V., 1998. Neurotrophins: the biological paradox of survival factors eliciting apoptosis. Cell Death and Differentiation 5, 357-364. Chao, M.V., 1992a. Neurotrophin receptors: a window into neuronal differentiation. Neuron 9, 583-593. Chao, M.V., 1992b. Growth factor signaling: where is the specificity? Cell 68, 995-997. Chao, M.V., 1994. The p75 neurotrophin receptor. J. Neurobiol. 25, 1373-1385. Chinnaiyan, A.M., O'Rourke, K., Yu, G.-L., Lyons, R.H., Garg, M., Duan, D.R., Xing, L., Gentz, R., Ni, J., Dixit, V.M., 1996. Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Science 274, 990-992. Cleveland, J.L., Ihle, J.N., 1995. Contenders in FasL/TNF death signaling. Cell 81,479-482. Davies, A.M., 1994. Neurotrophic factors switching neurotrophin dependence. Curr. Biol. 4, 273-276. Dechant, G., Barde, Y.-A., 1997. The neurotrophin receptor p75 binds neurotrophin-3 on sympathetic neurons with high affinity and specificity. J. Neurosci. 17, 5281-5287. Dechant, G., Biffo, S., Okazawa, H., Kolbeck, R., Pottgiesser, J., Barde, Y.-A., 1993. Expression and binding characteristics of the BDNF receptor chick trkB. Development 119, 545-558. Distefano, P.S., Friedman, B., Radziejewski, C., Alexander, C., Boland,

259

P., Schick, C.M., Lindsay, R.M., Wiegand, S.J., 1992. The neurotrophins BDNF, NT-3 and NGF display distinct patterns of retrograde axonal-transport in peripheral and central neurons. Neuron 8, 983-993. Dobrowsky, R.T., Werner, M.H., Castellino, A.M., Chao, M.V., Hannun, Y.A., 1994. Activation of the sphingomyelin cycle through the lowaffinity neurotrophin receptor. Science 265, 1596-1599. Dobrowsky, R.T., Jenkins, G.M., Hannun, Y.A., 1995. Neurotrophins induce sphingomyelin hydrolysis. J. Biol. Chem. 270, 22135-22142. Dudek, H., Datta, S.R., Franke, T.F., Birnbaum, M.J., Yao, R., Cooper, G.M., Segal, R.A., Kaplan, D.R., Greenberg, M.E., 1997. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275, 661-665. Feinstein, E., Kimchi, A., Wallach, D., Boldin, M., Varfolomeev, E., 1995. The death domain: a module shared by proteins with diverse cellular functions. TIBS 20, 342-344. Frade, J.M., Rodriguez-Tebar, A., Barde, Y.-A., 1996. Induction of cell death by endogenous nerve growth factor through its p75 receptor. Nature 383, 166-168. Gargano, N., Levi, A., Alema, S., 1997. Modulation of nerve growth factor internalization by direct interaction between p75 and TrkA receptors. J. Neurosci. Res. 50, 1-12. Gedrich, R.W., Gilfillan, M.C., Duckett, C.S., Van Dongen, J.L., Thompson, C.B., 1996. CD30 contains two binding sites with different specificities for members of the tumor necrosis factor receptor-associated factor family of signal transducing proteins. J. Biol. Chem. 271, 12852-12858. Grob, P.M., Ross, A.H., Koprowski, H., Bothwell, M., 1985. Characterization of the human melanoma nerve growth factor receptor. J. Biol. Chem. 260, 8044-8049. Gu, C., Casaccia-Bonnefil, P., Srinivasan, A., Chao, M.V., 1999. Oligodendrocyte apoptosis mediated by caspase activation. J. Neurosci. 19, 3043-3049. Hantzopoulos, P.A., Suri, C., Glass, D.J., Goldfarb, M.P., Yancopoulos, G.D., 1994. The low affinity NGF receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins. Neuron 13, 187-207. Hempstead, B.L., Martin-Zanca, D., Kaplan, D.R., Parada, L.F., Chao, M.V., 1991. High-affinity NGF binding requires co-expression of the trk proto-oncogene and the low-affinity NGF receptor. Nature 350, 678-683. Hempstead, B.L., Rabin, S.J., Kaplan, L., Reid, S., Parada, L.F., Kaplan, D.R., 1992. Overexpression of the trk tyrosine kinase rapidly accelerates nerve growth factor-induced differentiation. Neurons 9, 883-896. Hsu, H., Shu, H.-B., Pan, M.-P., Goeddel, D.V., 1996. TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor-1 signal transduction pathways. Cell 84, 299-308. Huber, L.J., Chao, M.V., 1995. A potential interaction of p75 and trkA NGF receptors revealed by affinity crosslinking and immunoprecipitation. J. Neurosci. Res. 40, 557-563. Jing, S.Q., Tapley, P., Barbacid, M., 1992. Nerve growth-factor mediates signal transduction through trk homodimer receptors. Neuron 9, 1067-1079. Jungbluth, S., Bailey, K., Barde, Y.A., 1994. Purification and characterization of a brain-derived neurotrophic factor neurotrophin-3 (bdnf/nt3) heterodimer. Eur. J. Biochem. 221,677-685. Kaplan, D.R., Stephens, R.M., 1994. Neurotrophin signal transduction by the trk receptor. J. Neurobiol. 25, 1404-1417. Khursigara, G., Orlinick, J.R., Chao, M.V., 1999. Association of TRAF6 with the p75 neurotrophin receptor. J. Biol. Chem. 274, 2597-2600. Kouhara, H., Hadari, Y.R., Spivak-Kroizman, T., Schilling, J., Bar-Sagi, D., Lax, I., Schlessinger, J., 1997. A lipid-anchored Grb2-binding protein that links FGF-receptor activation to the Ras/MAPK signaling pathway. Cell 89, 693-702. Leibrock, J., Lottspeich, F.H., Holn, A., Hofer, M., Hengerer, B., Masiakowski, P., Thoenen, H., Barde, Y.A., 1989. Molecular cloning and expression of brain-derived neurotrophic factor. Nature 341, 149-152.

260

H. Yano, M.V. Chao / Pharmaceutica Acta Helvetiae 74 (2000) 253-260

Levi-Montalcini, R., 1987. The nerve growth factor: thirty-five years later. Science 237, 1154-1164. Liepinsh, E., Ilag, L.L., Otting, G., Ibanez, C.F., 1997. NMR structure of the death domain of the p75 neurotrophin receptor. EMBO J. 16, 4999-5005. Lindsay, R.M., Wiegand, S.J., Altar, C.A., Distefano, P.S., 1994. Neurotrophic factors - - from molecule to man. Trends Neurosci. 17, 182-190. Mahadeo, D., Kaplan, L., Chao, M.V., Hempstead, B.L., 1994. High affinity nerve growth factor binding displays a faster rate of association than p l40(trk) binding-implications for multisubunit polypeptide receptors. J. Biol. Chem. 269, 6884-6891. Maisonpierre, P.C., Belluscio, L., Squinto, S., Ip, N.Y., Furth, M.E., Lindsay, R.M., Yancopoulos, G.D., 1990. Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Science 247, 1446-1451. McDonald, N.Q., Hendrickson, W.A., 1993. A new structural superfamily of growth factors defined by a cystine knot motif. Cell 73, 421-424. McDonald, J.I., Meakin, S.O., 1996. Deletions in the extracellular domain of rat trkA lead to an altered differentiative phenotype in neurotrophin responsive cells. Mol. Cell. Neurosci. 7, 371-390. McDonald, N.Q., Lapatto, R., Murray-Rust, J., Gunning, J., Wlodawer, A., Blundell, T.L., 1991. A new protein fold revealed by a 2.3 A resolution crystal structure of nerve growth factor. Nature 354, 411414. Montgomery, R.I., Warner, M.S., Lum, B.J., Spear, P.G., 1996. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87, 427-436. Nakano, H., Oshima, H., Chung, W., Williams-Abbott, L., Ware, C.F., Yagita, H., Okumura, K., 1996. TRAF5, an activator of NF-KB and putative signal transducer for the lymphotoxin-[3 receptor. J. Biol. Chem. 271, 14661-14664. Nicola, N.A., Metcalf, D., 1992. Subunit promiscuity among hemopoietic growth factor receptors. Cell 67, 1-4. Nishi, R., 1994. Neurotrophic factors: two are better than one. Science 265, 1052-1053. Pan, G., O'Rourke, K., Chinnaiyan, A.M., Gentz, R., Ebner, R., Ni, J., Dixit, V.M., 1997. The receptor for the cytotoxic ligand TRAIL. Science 276, 111-113. Perez, P., Coil, P.M., Hempstead, B.L., Martin-Zanca, D., Chao, M.V., 1995. NGF binding to the trk tyrosine kinase receptor requires the extracellular immunoglobulin-like domains. Mol. Cell. Neurosci. 6, 97-105. Qian, X., Riccio, A., Zhang, Y., Ginty, D.D., 1998. Identification and characterization of novel substrates of Trk receptors in developing neurons. Neuron 21, 1017-1029. Qui, M.-S., Green, S.H., 1991. NGF and EGF rapidly activate p21ras in PC12 cells by distinct, convergent pathways involving tyrosine phosphorylation. Neuron 7, 937-946. Qui, M.-S., Green, S.H., 1992. PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity. Neuron 9, 705-717.

Rabizadeh, S., Oh, J., Zhong, L.T., Yang, J., Bitler, C.M., Butcher, L.L., Bredesen, D.E., 1993. Induction of apoptosis by the low-affinity NGF receptor. Science 261,345-348. Radziewjewski, C., Robinson, R.C., 1993. Heterodimers of the neurotrophic factors-formation, isolation and differential stability. Biochemistry 32, 13350-13356. Rodriguez-Tebar, A., Dechant, G., Gotz, R., Barde, Y.A., 1992. Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth-factor and brain-derived neurotrophic factor. EMJO J. 11, 917-922. Rothe, M., Wong, S.C., Henzel, W.J., Goeddel, D.V., 1994. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681-692. Rothe, M., Sarma, V., Dixit, V.M., Goeddel, D.V., 1995. TRAF2-mediated activation of NF-kB by TNF receptor 2 and CD40. Science 269, 1424-1427. Smith, C.A., Farrah, T., Goodwin, R.G., 1994. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation and death. Cell 76, 959-962. Sutter, A., Riopelle, R.J., Harris-Warrick, R.M., Shooter, E.M., 1979. NGF receptors. Characterization of two distinct classes of binding sites on thick embryo sensory ganglia ceils. J. Biol. Chem. 254, 5972-5982. Thoenen, H., 1995. Neurotrophins and neuronal plasticity. Science 270, 593-598. Urfer, R., Tsoulfas, P., O'Connell, L., Shelton, D.L., Parada, L.F., Presta, L.G., 1995. An immunoglobulin-like domain determines the specificity of neurotrophin receptors. EMBO J. 14, 2795-2805. Verdi, J.M., Birren, S.J., Ibanez, C.F., Persson, H., Kaplan, D.R., Benedetti, M., Chao, M.V., Anderson, D.J., 1994. p75(LNGFR) regulates trk signal transduction and NGF-induced neuronal differentiation in MAH cells. Neuron 12, 733-745. von Bartheld, C.S., Williams, R., Lefcort, F., Clary, D.O., Reichardt, L.F., Bothwell, M., 1995. Retrograde transport of neurotrophins from the eye to the brain in chick embryos: roles of the p75 NTR and trkB receptors. J. Neurosci. 16, 2995-3008. Wolf, D.E., McKinnon, C.A., Daou, M.-C., Stephens, R.M., Kaplan, D.R., Ross, A.H., 1995. Interaction with trkA immobilizes gp75 in the high affinity nerve growth factor receptor complex. J. Biol. Chem. 270, 2133-2138. Wyatt, S., Davies, A.M., 1993. Regulation of expression of mRNAs encoding the nerve growth factor receptors p75 and trkA mRNA in developing sensory neurons. Development 119, 635-647. Yao, R., Cooper, G.M., 1995. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267, 2003-2006. Yoon, S.O., Casaccia-Bonnefil, P., Carter, B., Chao, M.V., 1998. Competitive signaling between TrkA and p75 nerve growth factor receptors determines cell survival. J. Neurosci. 18, 3273-3281.



The RET receptor tyrosine kinase" activation, signalling and significance in neural development and disease Ivor Mason

*

MRC Brain Development Programme, Department of Developmental Neurobiology, King's College London, New Hunt's House, Guy's Campus, London SE1 9RT, UK

Abstract The RET receptor tyrosine kinase was first identified in a screen for human oncogenes and has subsequently been linked to several human syndromes: Hirschprung's disease, multiple endocrine neoplasia types 2A and 2B and familial thyroid carcinoma. Interestingly, all of the tissues affected by mutations in RET are derived from the neural crest during development. RET transduces a signal following activation by ligands of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophins which currently comprises GDNF, neuturin (NTN), artemin (ART) and persephin (PSP). To activate RET they form a tripartite complex with RET and a member of a family of four extracellular, GPI-linked alpha receptors (GFRctl-4). Specificity is achieved by each GFRet binding only one member of the GDNF family with high affinity. Current evidence indicates that signal transduction by RET activates several second messenger systems including the PLC ~/, Ras, JNK and inositol phosphate pathways. Targeted mutagenesis in transgenic mice has shown that Ret, GFRoL1 and GDNF are required for multiple developmental events including development of the enteric nervous system (ENS) affected in Hirschsprung's disease. We describe experiments in chick neural crest cells which provide evidence for the normal function of RET and the basis of the defect in Hirschsprung's disease. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: RET; Receptor tyrosine kinase; GDNF

1. Beginnings: identification of the RET proto-oncogene

2. GDNF: a ligand for RET

RET was first identified as one part of an oncogenic fusion protein generated as a result of a chromosomal translocation. Cloning of the normal R E T gene showed that it encoded a transmembrane receptor tyrosine kinase (RTK) having an extracellular domain whose structure was unique among the RTK superfamily. This comprised a cysteine-rich region and a domain with homology to the intermolecular binding region of the cadherin family of cell adhesion molecules. However, to date, no adhesive properties have been demonstrated for RET. Furthermore, amino acid residues, required to form the homophilic binding interface of other cadherins, are absent from the RET cadherin domain. Consequently, homophilic adhesive properties or interactions with other cadherins are unlikely and RET remained an orphan receptor until 1996.

Glial cell line-derived neurotrophic factor (GDNF) was purified from conditioned media as a protein dimer of about 32 kDa during a search for factors capable of promoting the survival of midbrain dopaminergic neurons in vitro and, therefore, a possible therapeutic agent in the treatment of Parkinson's disease. It is a potent trophic factor for these cells in excitotoxic-lesioned tissue in vivo and is also a survival factor for sensory, sympathetic or ciliary neurons and axotomised or cultured motor neurons. A direct link between RET and GDNF was established following chemical cross-linking of GDNF to a motor neuron cell hybrid line which identified a high molecular weight complex that could be immunoprecipitated using either anti-phosphotyrosine antibodies or RET antisera.

3. Formation of an active R E T / G D N F signalling complex m a m6nage h trois * Tel.: +44-171-955-5000; fax: +44-171-955-4886; e-mail: [email protected]

At the same time, other groups were using assays based upon ligand-binding to identify GDNF receptors; two can-

0031-6865/00/$ - see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S003 1-6865(99)00048-5

262

I. Mason/Pharmaceutica Acta Helvetiae 74 (2000) 261-264

didate proteins were identified in these studies. One was RET but the other was a novel glycophosphatidyl (GPI)anchored protein, now called GFR-oL1. Transfection of GFR-oLl-expressing constructs into cell lines lacking detectable GDNF-binding activity promoted high affinity binding of the ligand. Moreover, radiolabelled GDNF could be immunoprecipitated with anti-RET antibodies as a complex also containing GFR-oL1. Thus, it seemed likely that GDNF signalled through a complex including both RET and GFR-c~ 1. Evidence that RET transduces a signal from GDNF is strong. Treatment of RET-transfected cell lines or primary cultures of motor neurons with GDNF followed by immunoprecipitation with RET antisera showed phosphorylation of RET in a dose-dependent manner. This was independent of whether GDNF was associated with GPIanchored or soluble GFR-oL1. More importantly, it was inhibited by soluble RET-Fc chimeric protein providing support for a direct interaction between a GDNF-GFR-oL 1 complex and RET. Current evidence suggests that GDNF binds directly to GFR-oL 1 and indirectly with RET through this association. Notably, expression cloning approaches using GDNF as "bait" resulted only in the isolation of GFR-oL1. Transfection of cells, which expressed no detectable RET and did not appreciably bind GDNF, with a construct expressing GFR-a 1 was sufficient to promote high affinity binding of GDNF. Moreover, transfection of these cells with vectors expressing either RET alone or both RET and GFR-oL1 followed by exposure to GDNF and immunoprecipitation with anti-GDNF antisera and Western blotting with antiRET antisera, showed no interaction between RET and GDNF in the absence of GFR-oL1. Furthermore, Neuro2a neuroblastoma cells which expressed high levels of RET did not bind GDNF unless transfected with a construct expressing GFR-o~ 1. It should be noted, however, that one group showed that transfection with a RET-expressing construct alone was sufficient to confer high-affinity GDNF binding to fibroblasts and the lack of significant binding to untransfected cells suggested the absence of GFR-a 1 from the surface of these cells. Thus there still remains uncertainties about the topology and the stoichiometry of the interactions between these three proteins.

4. More ligands, more alpha receptors GDNF is now known to be the founder-member of a multigene family, comprising GDNF, neuturin (NTN), artemin (ART) and persephin (PSP). This followed the purification and cloning of NTN, a trophic factor for sympathetic neurons. The similarities between the GDNF and NTN sequences allowed two further members to be isolated by standard approaches used for multigene families. Currently, the GDNF family of proteins form a unique

subdivision of the TGF[3 superfamily, being the only members of that family to transduce signals via an RTK. Likewise, three further GFR-oL proteins have been identified (GFR-oL1-3), but by contrast, the RET receptor has remained unique with only a single gene known from all vertebrate classes and also from Drosophila (but see later). Ligand specificity appears to be achieved by each GDNF family member only binding with high affinity to one GFR-c~ protein in order to activate RET. Thus GDNF binds preferentially to GFR-oL1, NTN to GFR-a 2, ART to GFR-a3 and PSP to GFR-oL4. However, studies of responses of cells to the various ligands in vitro, suggest additional complexity and that lower affinity interactions (e.g., between NTN and GFR-oL1) may be sufficient for formation of a functional signalling complex.

5. Second messenger pathways Alternative mRNA splicing generates cytoplasmic domains of RET having either 12 or 14 tyrosine residues which are potential targets for trans-phosphorylation by the tyrosine kinase enzyme functions of activated RET signalling complexes. Four of these residues have been identified as the docking sites for various cytoplasmic adaptor proteins G r b 7 / 1 0 (Tyr9~ PLC~(Tyrl~ Shc/ ENIGMA (Tyr 1~ and Grb2 (Tyrl~ This is consistent with data indicating that several second messenger pathways are activated by RET including PLC ~/, Jun-N-terminal kinase (JNK), phosphatidylinositol-3-kinase (PI3K) and Ras-MAP kinase pathways. Interestingly, different second messenger pathways appear to be associated with tumorigenic activity of RET in different forms of human multiple endocrine neoplasia (see below).

6. Can GDNF family proteins signal in the absence of RET? Unexpectedly, GDNF is able to stimulate the survival of certain neuronal populations (e.g., postnatal cochlear neurons) which express GFR-oL1 but lack any detectable RET and also of sensory neurons isolated from transgenic mice lacking functional RET protein. In the latter case, stimulation by GDNF is accompanied by activation of PLC~/ and Src pathways and Src has been shown to coprecipitate with GFR-oL1. However, the identity of the protein(s) or other molecules which mediate the interaction of GPI-linked GFR-oL1 with Src remains elusive.

7. RET, human Hirschsprung's disease and familial cancer syndromes

RET is implicated in several inherited human diseases. These include familial Hirschsprung's disease, which is


characterised by varying degrees of loss of the enteric nervous system (ENS), and several inherited cancer syndromes: multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma (FMTC). Certain forms of Hirschsprung's disease are the result of loss-of-function mutations in RET while the cancer syndromes seem to be gain-of-function mutations. Remarkably, all of these lesions affect neural crest derivatives, although the significance of this is, as yet, unclear. In addition to the RET locus, a number of other chromosomal loci are associated with aganglionosis of the bowel in humans and are also classified as Hirschsprung's disease. Some of these affect the endothelin-3 gene or that of its receptor ENDR-B, but others remain to be identified and may include the GDNF and GFR-al loci.

8. Insights from transgenic mice The requirement for RET in the formation of the ENS was confirmed by a transgenic mouse mutant in which targeted insertional mutagenesis was used to disrupt the RET kinase domain. Mice homozygous for the mutant allele lacked enteric neurons posterior to the stomach, and they also lacked kidneys and the superior cervical ganglion. Three groups "knocked out" the Gdnf gene in transgenic mice and the similarity of the resulting phenotype with that of the RET kinase deficient animals was striking. Homozygous null animals died soon after birth and were found to lack kidneys, enteric neurons and to have depletions in a number of other neuronal populations. Moreover, Gfr-al-null mice show similar defects. The similarity between some of the defects seen in the Ret, Gfr-al and Gdnf mutant mice is clearly attributable to their functional relationship and correlates well with expression data obtained from in situ hybridisation analyses. However, it is noteworthy that in some tissues (e.g., gut) Ret and Gfr-al are expressed in adjacent cell populations rather than the same cell, raising the possibility that the latter protein can also present GDNF ligand either in trans or as a soluble form following cleavage from the membrane. Indeed, there are some data from studies in vitro that support this contention. Defects in the ENS are significant in view of the association of RET with Hirshsprung's disease. The ENS derives from neural crest cells which express Ret when in the developing gut and both Gdnf and Gfr-al are expressed in the gut mesenchyme. Lack of kidneys in mutant mouse embryos is due to the failure of the ureteric bud (which expresses Ret) to develop from the Wolffian duct and expand into the metanephric blastema (which expresses Gdnf). This would normally initiate the formation of the metanephric (adult) kidney, but in the absence of ligand or receptor the blastemal cells eventually die.

263

By contrast, mice lacking either NTN or Gfr-e~l are both viable and fertile. However, they do exhibit similar optic, enteric and parasympathetic nervous defects and this provides further evidence in support of a functional partnership in vivo. Yet, the GFR-a/-null mouse shows growth defects that are not observed in the NTN mutant. Mutant animals for the remaining ligands and alpha receptors are being generated by several groups and can be expected to be published shortly.

9. Can RET be the only receptor for GDNF family ligands? Interestingly, the reported phenotypes of the RET and GDNF null animals do not match completely. Notably, the Gdnf mutant mouse showed partial loss of a number of neuronal populations that was not reported in the Ret mutant. A number of explanations are possible for this. The differences between the various mutant phenotypes might simply be due to differences in either the genetic background of the mice into which the mutant genes have been introduced or to differences in the genetic material inherited from the ES cells. The GDNF/GFR-e~ 1 complex may sometimes function independently of RET (see above). Alternatively, the incomplete overlap may point to the existence of other receptors, closely related RET. However, attempts to identify other members of a possible RET multigene family have not met with success to date.

10. Studies of avian neural crest cells in vitro suggest a function for RET during ENS development Whether the loss of enteric neurons in Hirschsprung's disease and in mutant mice is due to abnormal proliferation, survival or differentiation is unclear, although migration of their neural crest precursors appears normal. Our greatest understanding of development of the ENS has derived from studies of chick embryos and from avian neural crest cells in vitro. These studies established that the ENS derives from neural crest cells which migrate and differentiate within the mesenchyme of the developing gut to form the submucous and myenteric plexi. Most of these cells derive from the posterior hindbrain and anterior spinal cord (vagal region) but a second population of neural crest cells, originating in the most posterior spinal cord (sacral region), also contributes to the posterior gut nervous system. Ret is not expressed in chick neural crest cells as they leave the neural tube but transcripts are detected in cells within the gut mesenchyme with an anterior-to-posterior temporal progression and, subsequently, are maintained in the developing plexi. Gdnf and GFR-al are expressed in the mesenchyme of the gut. We have shown that consistent

264


with an undifferentiated stem cell phenotype, neural crest cells originating from vagal neural tube explants do not express Ret in vitro. These cells remain largely undifferentiated in culture for seven days, thereafter an increasing population of melanocytes differentiate from them. Introduction of an activated RET protein isoform to these cells using a retroviral vector has no effect on either division or survival, however it promotes the differentiation of neurons. In control cultures fewer than 1% of the cells differentiate into neurons, whereas one third of the retroviral-infected cells do so. These data suggest that the lack of enteric neurons in RET-null mice or Hirschsprung's patients is due to a failure of differentiation rather than survival or division.

Edery, P., Eng, C., Munnich, A., Lyonnet, S., 1997. RET in human development and oncogenesis. BioEssays 19, 389-395. Lindsay, R.M., Yancopoulos, G.D., 1996. GDNF in a bind with known orphan: accessory implicated in new twist. Neuron 17, 571-574. Mason, I., 1996. The GDNF receptor: recent progress and unanswered questions. Mol. Cell. Neurosci. 8, 112119. Passini, B., Ceccherini, I., Romeo, G., 1996. RET mutations in human disease. Trends Genet. 12, 138-144. Robertson, K., Mason, I., 1997. The GDNF-RET signalling partnership. Trends Genet. 13, 1-3. Robertson, K., Mason, I., Hall, S., 1997. Hirschsprung's disease: genetic mutations in mice and men. Gut 41, 436-441.

11. Further reading and reviews Airaksinen, M.S., Tiievsky, A., Saarma, M., 1999. GDNF family neurotrophic factor signalling: four masters, one servant? Mol. Cell. Neurosci. 13, 313-325.

Acknowledgements Studies of Ret in the author's laboratory have been supported by the MRC.

PHA1NACEUTICA ACTAHELVETIAE ELSEVIER

Pharmaceutica Acta Helvetiae 74 (2000) 265-272

ii

www.elsevier.com/locate/pharmactahelv

The ciliary neurotrophic factor and its receptor, CNTFRoL Mark W. Sleeman *, Keith D. Anderson, Philip D. Lambert, George D. Yancopoulos, Stanley J. Wiegand Regeneron Pharmaceuticals, 777 Old Saw Mill Riuer Road, Tarrytown, NY 10591-6707, USA

Abstract Ciliary neurotrophic factor (CNTF) is expressed in glial cells within the central and peripheral nervous systems. CNTF stimulates gene expression, cell survival or differentiation in a variety of neuronal cell types such as sensory, sympathetic, ciliary and motor neurons. In addition, effects of CNTF on oligodendrocytes as well as denervated and intact skeletal muscle have been documented. CNTF itself lacks a classical signal peptide sequence of a secreted protein, but is thought to convey its cytoprotective effects after release from adult glial cells by some mechanism induced by injury. Interestingly, mice that are homozygous for an inactivated CNTF gene develop normally and initially thrive. Only later in adulthood do they exhibit a mild loss of motor neurons with resulting muscle weakness, leading to the suggestion that CNTF is not essential for neural development, but instead acts in response to injury or other stresses. The CNTF receptor complex is most closely related to, and shares subunits with the receptor complexes for interleukin-6 and leukemia inhibitory factor. The specificity conferring ot subunit of the CNTF complex (CNTFRoL), is extremely well conserved across species, and has a distribution localized predominantly to the nervous system and skeletal muscle. CNTFRoL lacks a conventional transmembrane domain and is thought to be anchored to the cell membrane by a glycosyl-phosphatidylinositol linkage. Mice lacking CNTFRot die perinatally, perhaps indicating the existence of a second developmentally important CNTF-like ligand. Signal transduction by CNTF requires that it bind first to CNTFRoL, permitting the recruitment of gpl30 and LIFR[3, forming a tripartite receptor complex. CNTF-induced heterodimerization of the [3 receptor subunits leads to tyrosine phosphorylation (through constitutively associated JAKs), and the activated receptor provides docking sites for SH2-containing signaling molecules, such as STAT proteins. Activated STATs dimerize and translocate to the nucleus to bind specific DNA sequences, resulting in enhanced transcription of responsive genes. The neuroprotective effects of CNTF have been demonstrated in a number of in vitro cell models as well as in vivo in mutant mouse strains which exhibit motor neuron degeneration. Intracerebral administration of CNTF and CNTF analogs has also been shown to protect striatal output neurons in rodent and primate models of Huntington's disease. Treatment of humans and animals with CNTF is also known to induce weight loss characterized by a preferential loss of body fat. When administered systemically, CNTF activates downstream signaling molecules such as STAT-3 in areas of the hypothalamus which regulate food intake. In addition to its neuronal actions, CNTF and analogs have been shown to act on non-neuronal cells such as glia, hepatocytes, skeletal muscle, embryonic stem cells and bone marrow stromal cells. 9 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Ciliary neurotrophic factor; Body weight; Obesity; STAT

1. Introduction Ciliary neurotrophic factor (CNTF) is a naturally occurring protein with a molecular mass of approximately 22 kDa. It was initially identified by its ability to support the survival of parasympathetic neurons of the chick ciliary ganglion in vitro (Adler et al., 1979) and subsequently purified to homogeneity from sciatic nerves (Lin et al., 1989; StiSckli et al., 1989). C N T F enhances the survival of


sensory neurons, motor neurons, cerebral neurons and hippocampal neurons (Skaper et al., 1986; Ip et al., 1991; Sendtner et al., 1992; W o n g et al., 1993; Larkfors et al., 1994). It promotes cholinergic differentiation of sympathetic neurons (Ernsberger et al., 1989; Saadat et al., 1989), the differentiation of glial progenitors into astrocytes (Hughes et al., 1988; Lillien et al., 1988) and the maturation and survival of oligodendrocytes (Louis et al., 1993). Some peripheral tissues, notably skeletal muscle, are also known to respond to C N T F (Conover et al., 1993; Helgren et al., 1994). C N T F is structurally and functionally related to members of a family of cytokines that includes leukemia in-

0031-6865/00/$ - see front matter 9 2000 Published by Elsevier Science B.V. All rights reserved. PII: S0031-6865(99)00050-3

266

M. W. Sleeman et al. / Pharmaceutica Acta Helvetiae 74 (2000) 265-272

hibitory factor (LIF), interleukin-6 (IL-6), interleukin-11 and oncostatin M (OSM), and as a result was predicted to have a four-helix bundle structure characteristic of most cytokines. Crystallographic (McDonald et al., 1995) and NMR (Panayotatos et al., 1995) studies have confirmed the structure of CNTF as consisting of four anti-parallel oLhelices (A,B,C,D) connected by two cross-over loops (between A - B and C-D) and one short loop between helices B and C (Kallen et al., 1999). The amino acid sequence of CNTF shows the features of a cytosolic protein with no signal peptide, no consensus sequences for glycosylation and only one free cysteine residue at position 17. Amino acid substitution within specific sites has produced variants with enhanced receptor specificity and hence biological activity (Saggio et al., 1995) and other variants withtpartial activities (Di Marco et al., 1996). Axokine | is our designation for a recombinant human CNTF variant that has three modifications to the parent molecule. Glutamine is replaced by arginine at position 63, a mutation that increases the potency of CNTF, both in vitro and in vivo by 4-5-fold. Additionally, the free cysteine at position 17 is replaced by alanine resulting in greater physical and chemical stability, and the last 15 amino acids are removed, improving solubility (Panayotatos et al., 1993). Mutagenesis studies have made it possible to map the receptor recognition sites of CNTF, IL-6 and LIF cytokines. This has allowed "modules" to be swapped creating chimeric cytokines with known sites of interaction with the [3-receptor components and thus modulating the activity of the chimeric protein on cells expressing receptor components (Kallen et al., 1999, and reference therein).

2. Receptor distribution and interaction with ligand Cloning of the CNTF receptor subunit (CNTFRo~) revealed that it was related to one of the two components of the IL-6 receptor, the IL-6Ra, and its initial distribution was described as being predominantly within neural tissue (Davis et al., 1991). Outside the central nervous system (CNS), relatively high expression was also detected in skeletal muscle, with lower expression in the adrenal gland, sciatic nerve, skin, liver, kidney and testis (Davis et al., 1991; Ip et al., 1992; and Fig. 1). In contrast to other known cytokine receptors, CNTFRoL does not have a transmembrane or cytoplasmic region, but is anchored to the cell surface membrane by a glycosyl-phosphatidylinositol linkage. The CNTFRoL can function in either the membrane-bound form or soluble form, the latter being produced by phospholipase C-mediated cleavage of the membrane-bound receptor. The soluble CNTFRe~ has been detected in cerebrospinal fluid and serum, and has been shown to provide signaling capabilities to cells that are not highly responsive to CNTF alone (Davis et al., 1993; Helgren et al., 1994). Other examples of soluble receptor forms that have been found in body fluids include IL-6RoL,

gpl30 and the LIFRc~ (see Zhang et al., 1998). The high-affinity biological actions of CNTF require binding first to CNTFRoL, in a 1:1 stoichiometry, permitting the recruitment of gpl30 and LIFR[3 membrane spanning signal transducing units (Stahl and Yancopoulos, 1994). Recent immunoprecipitation experiments have suggested that CNTF receptor complexes are hexamers, consisting of CNTF, CNTFRc~, gpl30 and LIFR in a 2:2:1:1 ratio (De Serio et al., 1995). CNTF-induced heterodimerization of the [3 receptor subunits leads to tyrosine phosphorylation, through constitutively associated JAKs, and the activated receptor provides docking sites for SH2 containing signaling molecules, such as STAT-3, PTP1D and perhaps other undetermined substrates (Stahl et al., 1995). Activated STAT-3 dimerizes with itself or other STAT proteins (e.g. STAT-1), and translocates to the nucleus where it is able to bind specific DNA sequences. This sequence of events has been correlated with enhanced transcriptional activity of responsive genes including acute phase plasma proteins. Celiac ganglion neurons, other peripheral neurons, neuron-like cells (sensory, sympathetic adrenal chromaffin), central neurons (septal, hippocampal, spinal cord) as well as non-neuronal cells, including skeletal muscle and adrenal (Helgren et al., 1994) have shown activation of tis-ll, an early immediate gene, in response to exogenous CNTF. Recently, a new class of genes encoding suppressors of cytokine signaling (SOCS) have been shown to be induced by cytokines (Yoshimura, 1998) and specific members are induced in response to peripheral CNTF administration (BjCrb~ek et al., 1999). In addition to the JAK/STAT pathway, CNTF and other cytokines have been shown to induce tyrosine phosphorylation of a phospholipase C~/, PTP1D, ppl20, Shc, Grb2, Raf-1 and ERK1 and ERK2 (Boulton et al., 1994). Recent reviews extensively cover the signaling mechanisms of this class of cytokines (Hirano et al., 1997). In contrast to mice which lack CNTF, CNTFRe~ knockout mice die perinatally, and display severe motor neuron deficits (DeChiara et al., 1995). Similarly, mice that lack the common signal transducer gpl30 display embryonic lethality and have diverse embryonic defects (Yoshida et al., 1993), while mice that lack the shared LIF receptor die in the perinatal development period and exhibit placental, skeletal, neural and metabolic defects (Li et al., 1995; Ware et al., 1995).

3. Biological properties of CNTF and analogs The family of cytokines to which CNTF belongs has pleiotropic effects on multiple tissues, and the use of genetic ablations studies to determine primary or secondary requirements for individual members is often difficult. Mice that are homozygous for an inactivated CNTF gene develop normally, although in late adulthood, they may exhibit a mild loss of motor neurons with resulting


267

A

Arcuate

,,~

~

Nucleus

B

Median e m i n e n c e

Distribution of CNTFRa in Postnatal Rodent STRUCTURE

TECHNIQUE

Hvoothalamus Nuclei oArcuate .Paraventricular oSupraoptic oLat. Hypothal. Area oTuberomammilary Autonomic Nuclei/Ganglie, lntermediolateral 9 N. (Pregang. Symp.) oSCG ,,Adrenal ,,Ciliary G. .DRG ~

G.

Perioheral Oraans =Skeletal Mus~ .Skin ,,Liver .Kidney -Testes =Lung Bone 9 Marrow

Reference

ISH ISH IHC

Gloaguen et ai 1997 Gloaguen et al 1997; Lee et al 1997 Kordower et al 1997; MacLennan et al 1996

ISH IHC ISH

Lee et al 1997 Kordower et al 1997 Lee et al 1997

IHC ISH NRT 1~ NRT ISH ISH IHC NRT

Kordower et al 1997; MacLennan et al 1996 Ip et al 1993 Kotzbauer et al 1994 Kotzbauer et al 1994; Wong et al 1995 Davis et al 1991 Ip et al 1993 Ip et al 1993 MacLennan et al 1996 Thaler et al 1994

NRT NRT NRT WB 1~ NRT ISH NRT 1o

Davis et al 1991; Ip et al 1993 ip et al 1993; MacLennan et al 1994 Ip et al 1993 Kirsh & Hofmann 1994 Nesbit et al 1996; Schooltink et al 1992 Ip et al 1993 Ip et ai 1993 MacLennan et al 1994 Bellido et al 1996

Fig. 1. (A) In situ localization of CNTFRe~ mRNA in coronal section of adult mouse brain. Shown is a low-power dark-field photomicrograph of 35 emulsion-dipped slides of mouse sections hybridized with CNTFRoL antisense S-labeled RNA probe. (B) List of reported distribution of the CNTFRe~ and method of assessment. Detection of CNTFRc~ mRNA by Northern Blot, RNase Protection assay or RT-PCR (NRT) or in situ hybridization (ISH). Detection of CNTFRe~ protein by Western Blot (WB) or immunohistochemistry (IHC). Detection in in vitro primary cell culture (1~

muscle weakness (Masu et al., 1993). This would suggest that endogenous CNTF does not play a critical role in development of motor neurons and other neuronal populations. Consistent with this conclusion, individuals with an inactivated CNTF gene show little adverse effects and no sign of neurological abnormalities (Takahashi et al., 1994). This may suggest some degree of degeneracy within this

class of cytokine and a second ligand for the CNTFRe~ has been proposed on the basis of these results (Ip et al., 1993). The exact biological roles of CNTF in the mature CNS remain a matter of speculation, but application of exogenous CNTF has profound effects; it is capable of protecting diverse neuronal and glial cells populations following

268


nervous system injury. Thus, administration of CNTF has been shown to rescue sensory, motor neurons, thalamic, septal and striatal neurons as well as photoreceptors and oligodendrocytes. These results, together with the finding that the expression of CNTF mRNA and/or distribution of CNTF protein is profoundly altered by injury to the CNS or PNS (Friedman et al., 1992; Ip et al., 1993) led to the proposal that CNTF acts as a neurotrophic factor in response to nervous system damage. In the CNS, the increased expression of CNTF is restricted to astrocytes adjacent to the site of damage, supporting the idea that astrocytes play an important role in maintaining or restoring neuronal function following brain injury (Rudge et al., 1995). That treatment with exogenous CNTF can provide protection following CNS injury has been shown in several animal models of neuronal injury or disease including the ability to preserve striatal output neurons in a pharmacological model of Huntington's disease (Anderson et al., 1996; Emerich et al., 1996, 1997). This neuroprotective effect may occur through direct actions on striatal neurons, as there is clear expression of all components of the CNTFR complex in the striatum. A potential mechanism might be to modify the expression or function of glutamate receptors and hence modify the sensitivity to excitotoxic stimulation. In the intact adult CNS, injection of recombinant CNTF or adenovirally delivered CNTF, induces hypertrophy of astrocytes and significantly affects astrocyte phenotype (Winter et al., 1995; Clatterbuck et al., 1996). In vivo administration of CNTF prevents degeneration of chick spinal motoneurons during development (Oppenheim et al., 1991) and motor neurons are known to

.,,,

STAT 30min i~!!ii ii ili

express the CNTF receptor (Ip et al., 1993) and to retrogradely transport CNTF (Curtis et al., 1993). Sendtner et al. (1994) showed that exogenous administration of CNTF effectively blunts the progression of motorneuropathy, providing insight into the potential pharmacological potential of this factor and raising the hope for its possible application in human neurodegenerative diseases (Lindsay, 1994). Double-blind placebo controlled trials of rhCNTF in amyotrophic lateral sclerosis (ALS) patients showed no significant improvement in the end points of muscle strength tests, but did highlight significant effects of notably decreased food intake and weight loss (ALS CNTF Treatment Study Group, 1996; Miller et al., 1996). Administration of CNTF and CNTF analogs in various doses to normal and obese mice induces a dose- and time-dependent decrease in food intake with a subsequent reduction in body weight (Gloaguen et al., 1997; Anderson et al., 1998). CNTF and Axokine | are also highly effective at reducing weight in non-obese rats and humans (Fantuzzi et al., 1995; ALS CNTF Treatment Study Group, 1996; Xu et al., 1998). Interestingly, the effects of Axokine | on lean animals are transient, subsiding after 5-10 days, with a greater magnitude of effect in obese animals than in lean mice, with up to a 60-70% reduction in total body fat. We have used Axokine | to produce substantial reductions in adipose tissue in ob / ob, d b / db mice, as well as in leptin-resistant dietary obesity, with little impact on lean muscle mass except at high doses. Whilst fever and cachexia have been reported after systemic administration of CNTF (Shapiro et al., 1993; Espat et al., 1996; Martin et al., 1996; Matthys and Billiau,

pS" 'AT 30min

=~i :~,~I~:.:~,.":i,:i',~:'~,,~i: ~i i i ~,~,"

PB i~i~i!,~i!i!iii~ii?iU?;

Ax15

Fig. 2. Immunohistochemicaldetection of STAT-3 and pSTAT-3 in the basal hypothalamus of the mouse. Photomicrographs of 40-1xm coronal slices through the ob/ob mouse brain at the level of the arcuate nucleus and median eminence. Brains were collected 30 min after i.v. injection of phosphate buffer (PB) or Axokine| (0.1 mg/kg) and adjacent brain slices were processed for detection of STAT-3 (A,C) or phosphorylatedSTAT-3 (B,D).

269


A Veh

AXOKINE 0.1 0.3 1.0

IL-6 1.0 rr'~j/kg

D

30 min Veh

O.1

.....

pSTAT-3

STAT-3

..... .....

i p97

....

I f

..... ......... ~ p97 ~ ~ ...... 1"

~:~!'~

B

Veh

0.1

AXOKINE 0.3

1.0

IL-6

1.0 mg/kg

pSTAT-3 t

STAT-3

]:

C Veh

0.1

t

......~,~-

~:~~:~:.i.~/

AXOKINE 0.3

1.0

STAT-3 t

1,0

AXOKINE 0,1

0.3

IL-6

1.0

1.0

288

E

18S

AXOKINE

Veh

0.1

SOCS-3 mRNA

p97

Stain

120 min

30 min

.

0.3

IL-6 1,0

........ ~ ~.~ : ~

AXOKtNE

Veh

1D

0.1

0.3

1.0

IL-6 1.0

t"

~ W

Eth. Br.

m 1N

F 30 min

120 min

AXOKINE 0.1

~9 ~

1.(3

Veh

Eth. Br. Stain

Veh

pSTAT-3

0.3

IL-6

SOCS-3 mRNA

p97

IL-6 1.0 mg/kg

120 min

AXOKINE

...............................

i p97

SOCS-3 :~,[~i~ii

b ~ ~ ~ ~ w ,~

t " p97

Eth. Br. Stain

0.3

1.0

IL-6 1.0

Veh

AXOKINE 0.!

mRNA

03

1.0

IL-6 1.0

1==

Fig. 3. Assessment of signaling in peripheral tissue after systemic administration of Axokine | The left hand group of panels show Western Blot analysis for phosphorylated STAT-3 (upper panel) and STAT-3 (lower panel) in muscle (A), liver (B) and adipose (C) tissues from normal mice 30 min after i.p. injection of Axokine | (0.1-1.0 mg/kg), IL-6 (1 mg/kg) or Vehicle. The right hand group of panels shows Northern Blot analysis of mRNA for SOCS-3 (upper panel) in muscle (D), liver (E) and adipose (F) tissues from normal mice 30 or 120 min after i.p. injection of Axokine | (0.1-1.0 mg/kg), IL-6 (1 mg/kg) or vehicle.

1997), we and others have failed to find consistent effects on locomotor activity, body temperature, grooming behaviour, or conditional taste aversion after long-term administration of doses that normalize body weight in obese animals (Gloaguen et al., 1997; Anderson et al., 1998). In addition to the effects observed on body weight, there is a specific and perhaps independent effect to reduce both the fed and fasting glucose levels as well as insulin levels in both rodent models of obesity/diabetes 1 (Gloaguen et al., 1997).

Sleeman, M.W., Lambert, P.D., Wiegand, S.J., unpublished observation.

4. Mechanism of action in obesity

4.1. Central effects Treatment of animals with CNTF is known to suppress appetite and induces a preferential loss of fat stores in the o b / o b mouse (Gloaguen et al., 1997; Anderson et al., 1998). These effects may be mediated by specific activation of hypothalamic areas known to regulate food intake and body weight and which express the CNTFRoL (Fig. 1A). Several of these brain areas, such as the arcuate nucleus, are accessible from the periphery due to their proximity to circumventricular organs, whose capillaries

270


do not exhibit barrier properties. Peripheral administration of CNTF is able to elicit a specific activation of downstream signaling genes (e.g., SOCS-3; BjCrba~k et al., 1999) as well as suppress the expression of neuropeptide Y (NPY) in the hypothalamus of fasted animals (Xu et al., 1998). In our evaluation of the effects of exogenous CNTF's central actions, we have observed that systemic administration of Axokine | is able to specifically activate STAT-3 (Fig. 2) in the median eminence and arcuate nucleus, a key brain area involved in the regulation of feeding. Interestingly, Axokine | also induced upregulation of Fos and Egr-1 in a number of circumventricular organs including the subfornical organ, the preoptic area adjacent to the vascular organ of the lamina terminalis, the median eminence and tanycytes in the ventral part of the third ventricle. 2 Axokine | is more potent at inducing weight loss when delivered intracerebroventricularly than when given systemically (Anderson et al., 1998). The actions of CNTF have been compared to those of leptin on the basis of homology at the level of the receptor and signaling events. CNTF or Axokine | is more effective than leptin at reducing weight in mice with diet-induced obesity and has been shown to reduce body weight and normalize serum mice insulin levels in leptin receptor-deficient d b / d b (Gloaguen et al., 1997; Anderson et al., 1998; Marsh et al., 1999). Further differences in the way in which leptin and Axokine | reduce body weight are discussed elsewhere (Anderson et al., 1998).

5. Effects on non-neural tissues In addition to its neuronal actions, CNTF and analogs have been shown to exert a number of biological effects on non-neuronal cells such as glia, hepatocytes, skeletal muscle, embryonic stem cells and bone marrow stromal cells. Previous studies have demonstrated the induction of t i s - l l in peripheral tissues such as soleus muscle, and also that repeated administration in vivo resulted in a decreased level of CNTF receptor protein and tyrosine phosphorylation of receptor components and reduced induction of t i s - l l (DiStefano et al., 1996). Interestingly, no functional desensitization was observed in the face of prolonged administration of the factor. This correlates with the prolonged effects of repeated daily Axokine | administration on body weight and food intake in the different models of obesity tested to date. We have investigated peripheral signaling with in vivo administration of Axokine | and have shown a rapid and dose-dependent activation of STAT-3 protein and subsequent transcriptional activation of the downstream targets, such as SOCS-3, in a number of peripheral tissues (Fig. 3). An effect at the level of the

2 Lambert, P.D., Wiegand, S.J., unpublished observation.

liver is not surprising, given other reports that CNTF upregulates fibrinogen gene expression in primary hepatic cells (Nesbitt et al., 1993), and stimulates hepatic lipid metabolism (Nonogaki et al., 1996). Axokine | also activates signaling in tissues not previously known to express C N T F R a (Davis et al., 1991) and may further support the notion of a soluble form of the C N T F R a receptor (Davis et al., 1993). A careful and extensive analysis of the expression of receptor components and binding studies in peripheral tissues is currently being conducted to assess this question.

Acknowledgements We would like to thank the contribution of Karen Garcia, Cherrie Liu, Jennifer Marden, Hiliary Cox and Thomas Corcoran for assistance in animal treatment, tissue collection and subsequent analysis. Axokine | is a registered trademark of Regeneron Pharmaceuticals.

References Adler, R., Landa, K.B., Manthorpe, M., Varon, S., 1979. Cholinergic neuronotrophic factors. Intraocular distribution of soluble trophic activity for ciliary neurons. Science 204, 143-1436. ALS CNTF Treatment Study Group, 1996. Neurology 46, 1244-1249. Anderson, K.D., Panayotatos, N., Corcoran, T.L., Lindsay, R.M., Wiegand, S.J., 1996. Ciliary neurotrophic factor protects striatal output neurons in an animal model of Huntington disease. Proc. Natl. Acad. Sci. U. S. A. 93, 7346-7351. Anderson, K.D., Wong, V., Corcoran, T.L., Murray, J.D., Wang, Q., Nelson, G. et al., 1998. Weight-loss effects of central and peripheral administration of Axokine (an analogue of ciliary neurotrophic factor) and leptin. Soc. Neurosci. Abs. 24, 621. Bjcrb~ek, C., Elmquist, J.K., E1-Haschimi, K., Kelly, J., Ahima, R.S., Hileman, S., Flier, J.S., 1999. Activation of SOCS-3 messenger ribonucleic acid in the hypothalamus by ciliary neurotrophic factor. Endocrinology 140 (5), 2035-2043. Boulton, T.G., Stahl, N., Yancopoulos, G.D., 1994. Ciliary neurotrophic factor/leukemia inhibitory factor/interleukin 6/oncostatin M family of cytokines induces tyrosine phosphorylation of a common set of proteins overlapping those induced by other cytokines and growth factors. J. Biol. Chem. 269 (15), 11648-11655. Clatterbuck, R.E., Price, D.L., Koliatsos, V.E., 1996. Ciliary neurotrophic factor stimulates the expression of glial fibrillary acidic protein by brain astrocytes in vivo. J. Comp. Neurol. 369 (4), 543-551. Conover, J.C., Ip, N.Y., Poueymirou, W.T., Bates, B., Goldfarb, M.P., DeChiara, T.M., Yancopoulos, G.D., 1993. Ciliary neurotrophic factor maintains the pluripotentiality of embryonic stem cells. Development 119 (3), 559-565. Curtis, R., Adryan, K.M., Zhu, Y., Harkness, P.J., Lindsay, R.M., DiStefano, P.S., 1993. Retrograde axonal transport of ciliary neurotrophic factor is increased by peripheral nerve injury. Nature 365 (6443), 253-255. Davis, S., Aldrich, T.H., Valenzuela, D.M., Wong, V., Furth, M.E., Squinto, S.P., Yancopoulos, G.D., 1991. The receptor for ciliary neurotrophic factor. Science 253, 59-63. Davis, S., Aldrich, T.H., Ip, N.Y., Stahl, N., Scherer, S., Farruggella, T.,

M. W. Sleeman et al. / Pharmaceutica Acta Heluetiae 74 (2000) 265-272

DiStefan, P.S., Curtis, R., Panayotatos, N., Gascan, H., Chevalier, S., Yancopoulos, G.D., 1993. Released form of CNTF receptor alpha component as a soluble mediator of CNTF responses. Science 259 (5102), 1736-1739. DeChiara, T.M., Vejsada, R., Poueymirou, W.T., Acheson, A., Suri, C., Conover, J.C., Friedman, B., McClain, J., Pan, L., Stahl, N., Ip, N.Y., Kato, A., Yancopoulos, G.D., 1995. Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth. Cell 83 (2), 313-322. De Serio, A., Graziani, R., Laufer, R., Ciliberto, G., Paonessa, G., 1995. In vitro binding of ciliary neurotrophic factor to its receptors: evidence for the formation of an IL-6-type hexameric complex. J. Mol. Biol. 254 (5), 795-800. Di Marco, A., Gloaguen, I., Graziani, R., Paonessa, G., Saggio, I., Hudson, K.R., Laufer, R., 1996. Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists. Proc. Natl. Acad. Sci. U. S. A. 93 (17), 9247-9252. DiStefano, P.S., Boulton, T.G., Stark, J.L., Zhu, Y., Adryan, K.M., Ryan, T.E., Lindsay, R.M., 1996. Ciliary neurotrophic factor induces downregulation of its receptor and desensitization of signal transduction pathways in vivo: non-equivalence with pharmacological activity. J. Biol. Chem. 271 (37), 22839-22846. Emerich, D.F., Lindner, M.D., Winn, S.R., Chen, E.-Y., Frydel, B.R., Kordower, J.H., 1996. Implants of encapsulated human CNTF-producing fibroblasts prevent behavioral deficits and striatal degeneration in a rodent model of Huntington's disease. J. Neurosci. 16, 5168-5181. Emerich, D.F., Winn, S.R., Hantraye, P.M., Peschanski, M., Chen, E.Y., Chu, Y., McDermott, P., Baetge, E.E., Kordower, J.H., 1997. Protective effect of encapsulated cells producing neurotrophic factor CNTF in a monkey model of Huntington's disease. Nature 386, 395-399. Ernsberger, U., Sendtner, M., Rohrer, H., 1989. Proliferation and differentiation of embryonic chick sympathetic neurons: effects of ciliary neurotrophic factor. Neuron 2 (3), 1275-1284. Espat, N.J., Auffenberg, T., Rosenberg, J.J., Rogy, M., Martin, C.H., Hasselgren, P.O., Copeland, E.M., Moldawer, L.L., 1996. Ciliary neurotrophic factor is catabolic and shares with IL-6 the capacity to induce an acute phase response. Am. J. Physiol. 271, R185-R190. Fantuzzi, G., Benigni, F., Sironi, M., Conni, M., Carelli, M., Cantoni, L., Shapiro, L., Dinarello, C.A., Sipe, J.D., Ghezzi, P., 1995. Ciliary neurotrophic factor (CNTF) induces serum amyloid A, hypoglycaemia and anorexia, and potentiates IL-1 induced corticosterone and IL-6 production in mice. Cytokine 7 (2), 150-156. Friedman, B., Scherer, S.S., Rudge, J.S., Helgren, M., Morrisey, D., McClain, J., Wang, D.Y., Wiegand, S.J., Furth, M.E., Lindsay, R.M., Ip, N.Y., 1992. Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo. Neuron 9, 295-305. Gloaguen, I., Costa, P., Demartis, A., Lazzaro, D., Marco, A.D., Graziani, R., Paonessa, G., Chert, F., Rosenblum, C.I., Ploeg, L.H.T.v.d., Cortese, R., Ciliberto, G., Laufer, R., 1997. Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance. Proc. Natl. Acad. Sci. U. S. A. 94, 6456-6461. Helgren, M.E., Squinto, S.P., Davis, H.L., Parry, D.J., Boulton, T.G., Heck, C.S., Zhu, Y., Yancopoulos, G.D., Lindsay, R.M., DiStefano, P.S., 1994. Trophic effect of ciliary neurotrophic factor on denervated skeletal muscle. Cell 76 (3), 493-504. Hirano, T., Nakajima, K., Hibi, M., 1997. Signaling mechanisms through gp130: a model of the cytokine system. Cytokine Growth Factor Rev. 8 (4), 241-252. Hughes, S.M., Lillien, L.E., Raft, M.C., Rohrer, H., Sendtner, M., 1988. Ciliary neurotrophic factor induces type-2 astrocyte differentiation in culture. Nature 335 (6185), 70-73. Ip, N.Y., Li, Y.P., van de Stadt, I., Panayotatos, N., Alderson, R.F., Lindsay, R.M., 1991. Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures. J. Neurosci. 11 (10), 3124-3134.

271

Ip, N.Y., Nye, S.H., Boulton, T.G., Davis, S., Taga, T., Li, Y., Birren, S.J., Yasukawa, K., Kishimoto, T., Anderson, D.J., Stahl, N., Yancopoulos, G.D., 1992. CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gpI30. Cell 69 (7), 112I-1132. Ip, N.Y., McClain, J., Barrezueta, N.X., Aldrich, T.H., Pan, L., Li, Y., Wiegand, S.J., Friedman, B., Davis, S., Yancopoulos, G.D., 1993. The oL component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development. Neuron 10, 89-102. Kallen, K.J., Grotzinger, J., Lelievre, E., Vollmer, P., Aasland, D., Renne, C., Mullberg, J., Myer zum Buschenfelde, K.H., Gascan, H., Rose-John, S., 1999. Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6. J. Biol. Chem. 274 (17), 11859-11867. Larkfors, L., Lindsay, R.M., Alderson, R.F., 1994. Ciliary neurotrophic factor enhances the survival of Purkinje cells in vitro. Eur. J. Neurosci. 6 (6), 1015-1025. Li, M., Sendtner, M., Smith, A., 1995. Essential function of LIF receptor in motor neurons. Nature 378 (6558), 724-727. Lillien, L.E., Sendtner, M., Rohrer, H., Hughes, S.M., Raft, M.C., 1988. Type-2 astrocyte development in rat brain cultures is initiated by a CNTF-like protein produced by type-1 astrocytes. Neuron 1 (6), 485-494. Lin, F.-L.H., Mismer, D., Lile, J.D., Armes, L.G., Butler, E.T., Vannice, J.L., Collins, F., 1989. Purification, cloning and expression of ciliary neurotrophic factor (CNTF). Science 246, 1023-1025. Lindsay, R.M., 1994. Trophic protection of motor neurons: clinical potential in motor neuron diseases. J. Neurol. 242 (1), $8-S 11, Suppl. 1. Louis, J.C., Magal, E., Takayama, S., Varon, S., 1993. CNTF protection of oligodendrocytes against natural and tumor necrosis factor-induced death. Science 259 (5095), 689-692. Marsh, D.J., Hollopeter, G., Huszar, D., Laufer, R., Yagaloff, K.A., Fisher, S.L., Burn, P., Palmiter, R.D., 1999. Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides. Nat. Genet. 21 (1), 119-122. Masu, Y., Wolf, E., Holtmann, B., Sendtner, M., Brem, G., Thoenen, H., 1993. Disruption of the CNTF gene results in motor neuron degeneration. Nature 365 (6441), 27-32. Matthys, P., Billiau, A., 1997. Cytokines and cachexia. Nutrition 13, 763-770. McDonald, N.Q., Panayotatos, N., Hendrickson, W.A., 1995. Crystal structure of dimeric human ciliary neurotrophic factor determined by MAD phasing. EMBO J. 14 (12), 2689-2699. Miller, R.G., Petajan, J.H., Bryan, W.W., Armon, C., Barohn, R.J., Goodpasture, J.C., Hoagland, R.J., Parry, G.J., Ross, M.A., Stromatt, S.C., rhCNTF ALS Study Group, 1996. A placebo-controlled trial of recombinant human ciliary neurotrophic (rhCNTF) factor in Amyotrophic Lateral Sclerosis. Ann. Neurol. 39, 256-260. Nesbitt, J.E., Fuentes, N.L., Fuller, G.M., 1993. Ciliary neurotrophic factor regulates fibrinogen gene expression in hepatocytes by binding to the interleukin-6 receptor. Biochem. Biophys. Res. Commun. 190, 544-550. Nonogaki, K., Pan, X.M., Moser, A.H., Shigenaga, J., Staprans, I., Sakamoto, N., Grunfeld, C., Feingold, K.R., 1996. LIF and CNTF, which share the gpl30 transduction system, stimulate hepatic lipid metabolism in rats. Am. J. Physiol. 271 (3), E521-E528, Pt. 1. Oppenheim, R.W., Prevette, D., Yin, Q.W., Collins, F., MacDonald, J., 1991. Control of embryonic motoneuron survival in vivo by ciliary neurotrophic factor. Science 251 (5001), 1616-1618. Panayotatos, N., Radziejewska, E., Acheson, A., Pearsall, D., Thadani, A., Wong, V., 1993. Exchange of a single amino acid interconverts the specific activity and gel mobility of human and rat ciliary neurotrophic factors. J. Biol. Chem. 268, 19000-19003.

272


Panayotatos, N., Radziejewska, E., Acheson, A., Somogyi, R., Thadani, A., Hendrickson, W.A., McDonald, N.Q., 1995. Localization of functional receptor epitopes on the structure of ciliary neurotrophic factor indicates a conserved, function-related epitope topography among helical cytokines. J. Biol. Chem. 270 (23), 14007-14014. Rudge, J.S., Pasnikowski, E.M., Hoist, P., Lindsay, R.M., 1995. Changes in neurotrophic factor expression and receptor activation following exposure of hippocampal neuron/astrocyte cocultures to kainic acid. J. Neurosci. 15 (10), 6856-6867. Saadat, S., Sendtner, M., Rohrer, H., 1989. Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture. J. Cell Biol. 108 (5), 1807-1816. Saggio, I., Gloaguen, I., Poiana, G., Laufer, R., 1995. CNTF variants with increased biological potency and receptor selectivity define a functional site of receptor interaction. EMBO J. 14 (13), 3045-3054. Sendtner, M., Schmalbruch, H., StSckli, K.A., Carroll, P., Kreutzberg, G.W., Thoenen, H., 1992. Ciliary neurotrophic factor prevents degeneration of motor neurons in mouse mutant progressive motor neuronopathy. Nature 358, 502-504. Shapiro, L., Zhang, X., Rupp, R.G., Wolff, S.M., Dinarello, C.A., 1993. Ciliary neurotrophic factor is an endogenous pyrogen. Proc. Natl. Acad. Sci. U. S. A. 90, 8614-8618. Skaper, S.D., Montz, H.P., Varon, S., 1986. Control of Na +, K +-pump activity in dorsal root ganglionic neurons by different neuronotrophic agents. Brain Res. 386 (1-2), 130-135. Stahl, N., Yancopoulos, G.D., 1994. The tripartite CNTF receptor complex: activation and signaling involves components shared with other cytokines. J. Neurobiol. 25, 1454-1466. Stahl, N., Farruggella, T.J., Boulton, T.G., Zhong, Z., Darnell, J.E. Jr., Yancopoulos, G.D., 1995. Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors. Science 267 (5202), 1349-1353. StiSckli, K.A., Lottspeich, F., Sendtner, M., Masiakowski, P., Carroll, P., GiStz, R., Lindholm, D., Thoenen, H., 1989. Molecular cloning,

expression, regional distribution of rat ciliary neurotrophic factor. Nature 342, 920-923. Takahashi, R., Yokoji, H., Misawa, H., Hayashi, M., Hu, J., Deguchi, T., 1994. A null mutation in the human CNTF gene is not causally related to neurological diseases. Nat. Genet. 1, 79-84. Ware, C.B., Horowitz, M.C., Renshaw, B.R., Hunt, J.S., Liggitt, D., Koblar, S.A., Gliniak, B.C., McKenna, H.J., Papayannopoulou, T., 1995. Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death. Development 121 (5), 12831299. Winter, C.G., Saotome, Y., Levison, S.W., Hirsh, D., 1995. A role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury. Proc. Natl. Acad. Sci. U. S. A. 92 (13), 5865-5869. Wong, V., Arriaga, R., Ip, N.Y., Lindsay, R.M., 1993. The neurotrophins BDNF, NT-3 and NT-4/5, but not NGF, up-regulate the cholinergic phenotype of developing motor neurons. Eur. J. Neurosci. 5 (5), 466-474. Xu, B., Kalra, P.S., Moldawer, L.L., Kalra, S.P., 1998. Increased appetite augments hypothalamic NPY Y1 receptor gene expression: effects of anorexigenic ciliary neurotropic factor. Regul. Pept. 75-76, 391-395. Yoshida, T., Satoh, M., Nakagaito, Y., Kuno, H., Takeuchi, M., 1993. Cytokines affecting survival and differentiation of an astrocyte progenitor cell line. Brain. Res. Dev. Brain Res. 76 (1), 147-150. Yoshimura, A., 1998. The CIS family: negative regulators of JAK-STAT signaling. Cytokine Growth Factor Rev. 9 (3-4), 197-204. Zhang, J.G., Zhang, Y., Owczarek, C.M., Ward, L.D., Moritz, R.L., Simpson, R.J., Yasukawa, K., Nicola, N.A., 1998. Identification and characterization of two distinct truncated forms of gpl30 and a soluble form of leukemia inhibitory factor receptor alpha-chain in normal human urine and plasma. J. Biol. Chem. 273 (17), 1079810805.



Rediscovering good old friend IGF-I in the new millenium: possible usefulness in Alzheimer's disease and stroke Sylvain Dor~, Satyabrata Kar, When-Hua Zheng, R6mi Quirion

*

Douglas Hospital Research Centre, Department of Psychiatry, McGill University, Montreal, Quebec, Canada H4H 1R3

Abstract

Much research has been done over the past two decades on the role of insulin-like growth factors I and II (IGF) in the maintenance of normal body homeostasis, especially in regard to various endocrine functions, growth and aging. For example, IGF-I is a well established promoter of tissue growth and has been used in the clinics for the treatment of growth related disorders, even being abused by athletes to enhance performance in competitions. In contrast, comparatively limited attention has been given to the potential significance of the IGFs in the central nervous system. Over the past few years, we have studied the trophic as well as neuromodulatory roles of the IGFs in the brain. IGF-I and IGF-II are potent modulators of acetylcholine release; IGF-I inhibiting release while IGF-II is a potent stimulant. Moreover, only the internalization of the IGF-I receptor complex was blocked by an inhibitor of phosphotyrosylation. This is in accordance with the differential nature of the IGF-I and IGF-II receptors, the former being a tyrosine kinase receptor while the later is a single transmembrane domain protein bearing binding sites for 6-mannose phosphate containing residues. The activation of IGF-I receptors protected neurons against cell death induced by amyloidogenic derivatives likely by an intracellular mechanism distinct from those involved in the regulation of acetylcholine release and neuronal growth. The stimulation of IGF-I receptors can activate intracellular pathways implicating a PI3/Akt kinase and CREB phosphorylation or modulate the production of free radicals. The effects, particularly those of IGF-I on key markers of the Alzheimer's (AD) brains namely cholinergic dysfunction, neuronal amyloid toxicity, tau phosphorylation and glucose metabolism suggest the potential usefulness of this growth factor in the treatment of neurodegenerative diseases. However, the poor bioavailability, enzymatic stability and brain penetration of IGF-I hamper progress in this regard. The recent development of a small, non-peptidyl mimetic of insulin able to directly activate the insulin receptor [Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M.T., Pelaez, F., Ruby, C., Kendall, R.L., Mao, X., Griffin, P., Calaycay, J., Zierath, J.R., Heck, J. V., Smith, R.G., Moller, D.E., 1999. Science, 284, 974-977] suggests that a similar strategy could be used for IGF-I and the IGF-I receptor leading to the characterization of IGF-I mimics of potential clinical usefulness. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: Insulin-like growth factor; Mimetic; Analogue; Neurodegenerative disease; Amyloid; Ischemia; Alzheimer's disease; Stroke; Receptor; Tyrosine kinase; Aging; Amyotrophic lateral sclerosis

Abbreviations: A[3, [3-amyloid; AD, Alzheimer's disease; BP, Binding protein; Ca, calcium; CNS, central nervous system; IGF, insulin-like growth factor; M6P, mannose-6-phosphat * Corresponding author. Tel.: +1-514-762-3048; fax: +1-514-762-3034; e-mail: [email protected]


274

S. Dor~ et al. / Pharmaceutica Acta Helvetiae 74 (2000) 273-280

1. Introduction

Activity of IGF-I like proteins has been discovered in 1957. It was first named sulfatation factor, later the term somatomedin C has been used. The precise action of IGF-I was poorly understood until the production of recombinant human IGF-I in the 1980s. It has been well characterized first as a factor mediating the action of growth hormone (Isaksson et al., 1991). It has been extensively studied in the development of bone and cartilage (Johansson et al., 1993) and proposed that it could play a significant role in the repair process in patients suffering from arthritis (Dor~ et al., 1994). IGF-I is found in high levels in the blood and believed to originate mainly from the liver (Pankov, 1999). Subsequent gene expression studies revealed its detection in several organs including the brain (Rotwein et al., 1988). The structure of IGF-I, a peptide of 70 amino acids, is very much similar to that of insulin (Isaksson et al., 1991) (Fig. 1). Interestingly, given the evidence that insulin level in the brain is quite low, researchers have suggested that IGF-I could exert insulin-like activity in the central nervous system. In fact, IGF-I has the capacity to bind to the family of IGF-like receptors including the insulin receptor (Fig. 2). Additionally, IGF-I binds to IGF binding proteins (IGFBPs) (Clemmons, 1998). The level of expression of these IGFBPs in the brain seems to be much lower than in periphery, and their role in the nervous system is still puzzling although researchers consider that they would be mostly inhibitory proteins limiting the availability of IGF-I.

IGF-I is a well established stimulator of cell growth, proliferation and differentiation, in addition to activate glucose transport and to prevent cell death (Dor~ et al., 1997c). The latter effects are critical for the post mitotic neurons. It is hence not surprising that the IGF receptors are tightly regulated and subject to rapid changes following various surgical and pharmacological manipulations (for examples, see Kar et al., 1993; Dor~ et al., 1996; Dor~ et al., 1999).

2. Mechanisms of action of IGF-I

A plethora of experimental approaches have shown that the IGFs participate in the development of the nervous system by promoting neural growth, survival, and the differentiation of neurons and glia (Lenoir and Honegger, 1983; Aizenman and de Vellis, 1987; Shemer et al., 1987; Baker et al., 1993; Carson et al., 1993; de Pablo and de la Rosa, 1995). In the adult nervous system, these growth factors are considered to play a role in trophism and repair as well as in the activity-dependent functioning of the brain (Berelowitz et al., 1981; Nilsson et al., 1988; Araujo et al., 1989; Lee et al., 1992; Kar et al., 1993; Beilharz et al., 1995; Guthrie et al., 1995; Ballard et al., 1996; Castro-Alamancos et al., 1996). IGF-I like immunoreactivity has been shown to be selectively localized to various regions of the brain and its physiological responses are presumed to be mediated by specific interactions with cell

CI v~..,_e3o AI

92

Fig. 1. Amino acid sequence of IGF-I. Key features are S-S bridges between A and B chains as observed for insulin (Adapted from Humbel and Rinderknecht, 1979).

S. Dord et al. / Pharmaceutica Acta Helvetiae 74 (2000) 273-280

Insulin,. , "" ". . . . . . . . . . . . . . . . illl

N

l"

275

. .,,. IGF-II '"j IGF-I

II Illl t ii i

N

am

m

Ligand binding site

Juxtamembrane (61%) Tyrosine

kinase(84%)

C-terminal (44%)

1316Y '-" 1322Y [nsu[ill

Receptor

;

~

" I~)~ ---1316Y

IGF-I ;Receptor

Activationtyrosinekinase hosphorylation of docking proteins

Recruitment of SH2/SH3domain containing proteins (Grb2/Sos, p85 (PI3-kinase), SH-PTP2, Syp, Nck, Fyn, SERCA...)~ T Activation of downstream signaling pathways (Ras/Raf/MAP Kinase/p90 $6 kinase pathways, Pl3-kinase/p70 $6 kinase pathways, Protein kindle B/Akt pathways .... )

Effect on proteinsynthesis, proliferation, differentiation, glucose transport, apoptosis... Fig. 2. Insulin and IGF-I receptors. Receptor domains are described on the left and the percentage of homology is indicated. Tyrosine residues undergoing phosphorylation upon receptor activation is also shown. Binding of the ligand to the extracellular oL-subunits leads to autophosphorylation of specific tyrosine residues on the intracellular part of the [3-subunits through a transphosphorylation mechanism that results in the activation of their tyrosine kinase domain. This enable the receptor kinase to phosphorylate intermediate docking proteins which subsequently recruit various intracellular proteins. This results in the activation of downstream signaling pathways (Adapted from Lamothe et al., 1998 and Butler et al., 1998).

surface receptors (LeRoith et al., 1995" Dor6 et al., 1997b). The actions of IGF-I are mediated by the IGF receptors and modulated by the presence of the IGFBPs. These binding proteins can bind IGF-I and IGF-II, but not insulin, with high affinities. IGF-I and insulin mediate their actions by binding to insulin a n d / o r IGF-I receptors which are structurally related disulfide-bonded heterotetrameric membrane glycoprotein proteins of about 330-350 kDa (Fig. 2). The IGF-I receptor is a 1367 residue protein having higher affinity for IGF-I than for IGF-II or insulin, and consists of an heterotetramer consisting of two oL- (115 kDa) and two [3- (94 kDa) subunits joined by disulfide bridges (LeRoith et al., 1995). The binding of the ligand to

the two oL extracellular subunits induces conformational changes which lead to the phosphorylation of certain residues (Tyr-ll31, 1135 and 1136) and the activation of intracellular effector molecules. The phosphorylation of Tyr-950 localized in the NPXY motif is required for IGF-I receptor internalization. The adaptor proteins SHC and IRS-I are also known to bind to this site. These events stimulate the activation of a cascade of intracellular signaling pathways. The IGF-II/mannose-6-phosphate receptor has a higher affinity for IGF-II than IGF-I and does not bind insulin. It comprises a single polypeptide chain with a large extracellular domain and a short cytoplasmic tail (LeRoith et al.,

276

S. Dord et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 273-280

1995; Clemmons, 1998). Furthermore, while the role of the IGF-II receptor in transporting lysosomal enzymes and internalizing various compounds appears to be well established, its function in signal transduction, unlike that of the IGF-I receptor, remains controversial (LeRoith et al., 1995; Dor6 et al., 1997b; Kar et al., 1997). The process of receptor internalization is also strikingly different between the IGF-I and IGF-II receptors. Since the IGF-I receptor is coupled to tyrosine kinase activity, tyrphostin 47, a specific tyrosine kinase inhibitor, was used in cultured hippocampal cells and shown to inhibit [~25I]IGF-I, but not [125I]IGF-II, receptor internalization (Dor6 et al., 1997b). Accordingly, IGF-I is apparently internalized mostly via the IGF-I tyrosine kinase receptor while IGF-II is not. The activation of the IGF-I tyrosine kinase receptor by its endogenous agonist (Traxler and Furet, 1999) leads to an association with the SH2 domain of proteins transducing the signal to a downstream cascade (for review see Daly, 1998). Such a protein for both the IGF-I and insulin receptors is the insulin receptor substrate-1 (IRS-1). Recently, several proteins with similar structures and different tissue distributions have been cloned (IRS-2, -3, -4) (Butler et al., 1998). IGF-I receptor/ IRS-1 association subsequently activates phosphoinositide 3-kinase, phosphotyrosine phosphatases, $6 kinase, Ras-MAP kinase and transcription factors leading to alterations in Ca 2+ mobilization, and mitochondrial respiration (Lienhard, 1994; Jones and Clemmons, 1995; LeRoith et al., 1995; Waters et al., 1996). It has also recently been shown that IGF-I can modulate glutamate-induced gamma aminobutyric acid release from the cerebellum by the simultaneous activation of protein kinase C and nitric oxide signaling pathways (Castro-Alamancos et al., 1996). Moreover, we have evidence suggesting that the modulation of ACh release by IGF-I implicates a distinct intracellular pathway compared to its neuroprotective action against the amyloids. In PC12 cells, we have recently demonstrated that the stimulation of the IGF-I receptor result in the activation of pathway implicating PI3/Akt kinase and CREB phosphorylation (Zheng et al., 1999). Treatment of cells with low nM concentrations of IGF-I caused a 3-5 fold increase in Akt serine/threonine kinase phosphorylation which is blocked by a PI3 kinase inhibitor. A similar treatment with IGF-I increased CREB phosphorylation by 2-3 folds; this effect of IGF-I was significantly blocked by MAP kinase inhibitors. Interestingly, treatment of PC12 cells with phorbol esters decreased IGF-I induced IRS-1 tyrosine phosphorylation and the activation of Akt (Zheng et al., 1999). It also appears that all 3 isoforms of Akt are expressed and activated by IGF-I in primary neurons. Moreover, the activation of Akt has been suggested to mediate anti-apoptotic functions due to the phosphorylation of caspase 9 (Cardone et al., 1998), bad (Brunet et al., 1999) and Forkhead transcription factors (Brunet et al., 1999). Additionally, the activation of Akt kinases has been shown to

phosphorylate and modulate nitric oxide synthase activity (Fulton et al., 1999) and to rapidly modulate L-type Ca 2+ channels (Blair et al., 1999). Taken together, these results indicate that IGF-I can act on different intracellular signaling pathways to induce its plethora of biological effects.

3. Design of active IGF-related analogues

3.1. Peptidyl analogues Scientists at Neurocrine B iosciences hypothesized that displacement in the brain of a large pool of endogenous IGF-I from the "reservoir" of inactive IGF-I bound to IGFBPs would elevate "free" IGF-I levels to elicit neuroprotective effects comparable to those produced by the administration of exogenous IGF-I. A human IGF-I analogue, [LeuZ4'59'6~ with high affinity for IGFBPs (K i = 0.3-3.9 nM) and no biological activity at the IGF-I receptors (K i = > 10,000 nM) was shown to increase the levels of "free", bioavailable IGF-I in the cerebrospinal fluid (Loddick et al., 1998). Intracerebroventricular administration of this analog up to 1 h after an ischemic insult to the rat brain had a potent neuroprotective action comparable to that of exogeneously applied IGF-I (Loddick et al., 1998) This novel strategy to increase bioavailable IGF levels in the brain may be useful for the treatment of stroke and other neurodegenerative diseases. The characterization and biological activity of IGF-I analogues with reduced affinity for the IGFBPs have been reported by scientists from Merck Research Laboratories (Bayne et al., 1990; Cascieri et al., 1991). These authors demonstrated that distinct domains (A-chain, B-chain) of IGF-I mediate binding to the IGF-I, IGF-II, and insulin receptors, as well as to IGFBPs and antibodies. They specifically reported that an h I G F - I analog, [LeuZ4'6~ had over a thousand fold loss in affinity for the IGF-I receptor and little measurable affinity for the IGF-II receptor (Bayne et al., 1990). To facilitate the synthesis and to promote the stability of this analog, the methionine residue at position 59 was replaced with a leucine. The resulting compound, [LeuZ4'59'6~ 31]hlGF-I, was tested for its capacity to displace [125I]hIGF-I binding from human IGFBP-1, -2, -3, -4, and -5, and for its biological activity in B A L B / c 3T3 fibroblast cells which are known to proliferate in response to the IGFs. Both hIGF-I and hIGF-II displayed higher affinities for IGFBP-1, -2, -3, -4, and -5 (K i values = 0.01-0.22 nM) than for the IGF-I and IGF-II receptors (K i values = 1.5 and 0.2 nM, respectively). In contrast to this relative lack of selectivity, [LeuZ4'59'6~ 31]hIGF-I had high affinity for IGFBP-1, -2, -3, -4, and -5 (K i values = 0.28-3.91 nM) while being inactive at the two IGF receptors (K i values > 10,000 nM). Furthermore, in contrast to hIGF-I, which concentration dependently stimulated DNA synthesis in 3T3 fibroblasts with an IC50 of 5-10 nM, [Leu24'59'6~

277

S. Dord et al. / Pharmaceutica Acta Helvetiae 74 (2000) 273-280

had no activity in this assay at concentrations of up to 8 ~M, indicating a lack of ability to directly stimulate the IGF-I receptor in this functional assay. Accordingly, these authors demonstrated that the IGFBP ligand inhibitor, [LeuZa'59'6~ 31]hIGF-I, is capable of interacting with the binding protein in a specific manner to displace IGF-I and hence release bioactive peptide. In additional experiments, adult male rats previously implanted with lateral cerebral ventricular guide cannula were subjected to ischemia with the permanent occlusion of the medial cerebral artery (MCAO), and the resulting brain lesion was visualized and quantified 24 h later. Animals that had received a single intracerebroventricular injection of the IGFBP ligand inhibitor (50 p~g) at the time of the MCAO had much smaller total lesion volumes than those injected with vehicle, primarily because of a reduction of the cerebral cortical infarct volume, although some protection was also evident in the striatum (Loddick et al., 1998). Moreover, the I G F B P ligand inhibitor [Leu24'59'6~ attenuated the loss of pyramidal neurons known to occur following an intrahippocampal administration of quinolinic acid (Loddick et al., 1998). The extent of protection was similar whether [LeuZ4'59'6~ 31]hIGF-I was administered concurrent with (0 h) or 1 h after the occlusion of the artery, providing a therapeutic window for the treatment. Another advantage in targeting IGFBPs is that it may be possible to identify non-peptide small molecules that act as selective competitors of IGF-I binding to these proteins with the potential for good blood-brain barrier penetration and oral activity.

3.2. Non-peptidyl analogues To identify small molecule insulin receptor (IR) activators, a group of scientists working at Merck (Zhang et al., 1999), designed a cell-based screening assay with Chinese hamster ovary cells overexpressing the human IR (CHO.IR). After incubation of intact cells with insulin or test compounds, IR-ligand complexes were immunopurifled and assayed for tyrosine kinase (IRTK) activity toward an exogenous substrate. Through extensive screening of over 50,000 mixtures of synthetic compounds and natural products, they identified a small molecule (L-783,281: Fig. 3) from a fungal extract (Pseudomassaria sp.) that was reproducibly active in the assay. At concentrations of 3 to 6 o~M, L-783,281 induced 50% of the maximal effect of insulin on IRTK activity. L-783,281 induced the phosphorylation of the IR subunit and IRS-1 in CHO.IR cells, as evidenced by anti-phosphotyrosine immunoblotting. In contrast, in CHO cells overexpressing the IGF-I receptor (CHO.IGFIR), L-783,281 (10 ~M) did not stimulate IGFIR or IRS-1 tyrosyl phosphorylation. No other L783,281-induced tyrosyl protein phosphorylation was evident, suggesting that the compound is selective for insulin receptor activation (Zhang et al., 1999). L-783,281 also

'%

H

I-I

-'~

L-783,281

Fig. 3. Chemical structure of the insulin agonist mimetic developed by Merck's scientists (Adapted from Zhang et al., 1999).

activated other components of the insulin receptor signal transduction pathway. It stimulated PI 3-kinase activity and the phosphorylation of Akt kinase in CHO.IR cells. L-783,281 also facilitated glucose uptake in rat primary adipocytes (263% over basal level at 10 p~M) and in isolated soleus muscle from lean mice (237% over basal level at 2 p~M). Several lines of evidence also suggested that L-783,281 directly activates the IR intracellular subunit (or tyrosine kinase domain) (Zhang et al., 1999). The discovery of L-783,281 demonstrates that a small, non-peptidyl molecule is capable of mimicking the in vitro and in vivo function of a protein hormone such as insulin by interacting with and activating its receptors. Vanadate is another orally active compound that can act in vivo as an insulin mimetic agent (Meyerovitch et al., 1987). However, unlike vanadate which augments tyrosyl phosphorylation of a wide variety of cellular proteins and functions in vitro as an inhibitor of protein tyrosine phosphatases (PTPases) (Denu et al., 1996), L-783,281 is selective for the insulin receptor and did not inhibit selected PTPases in vitro (Zhang et al., 1999). On the basis of the high degree of homology between the insulin and the IGF-I receptors, the discovery of L-783,281 as a non-peptidyl activator of the IR suggests that similar type of small molecules could be developed for the IGF-I receptor.

4. Potential usefulness of IGF-I in Alzheimer's disease

At the cellular level, IGF-I receptor sites are primarily enriched in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon's horn of the hippocampus whereas IGF-II binding sites are localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus (Dor~ et al., 1999). Interestingly, these regions are markedly affected in Alzheimer's disease (AD) (Selkoe, 1998). [3-amyloid (A[~I_40 , A ~ l _ 4 2 ) is believed to play a critical role in the neurodegenerative process occurring in AD (Roher et al., 1993; Harris et al., 1995). This protein is found deposited in extracellular neuritic plaques, one of the hallmarks of the AD brain (Price et al., 1995; Selkoe, 1998). The direct, toxic properties of A[3-related fragments

278

S. Dor~ et al. / Pharmaceutica Acta Helvetiae 74 (2000) 273-280

in cultured rat and human neurons and in in vivo are well established (Busciglio et al., 1993; Loo et al., 1993; Maurice et al., 1996), although the mechanism of action involved remains to be fully established (Selkoe, 1998). Using rat primary hippocampal neurons, we have shown that the activation of the IGF-I receptor has the capacity to protect and even to rescue neurons against cell death induced by [3-amyloid (Dor6 et al., 1997a). Accordingly, it may be that IGF-I and/or IGF-I mimetics could protect neurons against this type of toxicity believed to be an on-going feature in the progression of AD. Moreover, we reported that IGF-I was a potent modulator of acetylcholine release in rat hippocampal slices, in vitro (Kar et al., 1997). Hence, IGF-I can apparently act as a potent regulator of at least two key phenotypes of the AD brain, namely [3-amyloid toxicity (Selkoe, 1998) and neuronal cholinergic activities (Price et al., 1995; Auld et al., 1998; Selkoe, 1998). Interestingly, in support of a possible role for IGF-I in AD, it has been shown that IGF-I binding sites are increased in cortical areas of the AD brain (Crews et al., 1992) and immunoreactive IGF-I peptide and IGF-II receptors are localized to [3-amyloid containing neuritic plaques (Kar and Quirion, unpublished results). Moreover, it has recently been demonstrated that IGF-I can regulate tau phosphorylation in cultured human neurons (Hong and Lee, 1997). Tau is a neuronal microtubule-associated protein found predominantly in axons (Binder et al., 1985). The function of tau is to promote tubulin polymerization and to stabilize microtubules (Drechsel et al., 1992). The hyperphosphorylated form of tau is characteristic of the neurofibrillary tangles, another key phenotype of the AD brain (Greenberg and Davies, 1990; Lee et al., 1991; Goedert et al., 1992) which often correlates with the degree of dementia (Braak and Braak, 1991; Arriagada et al., 1992). Indeed, Hong and Lee (1997) demonstrated that IGF-I can affect tau phosphorylation through the inhibition of glycogen-synthase kinase-3. These effects of IGF-I are mediated through the inhibition of glycogen-synthase kinase-3 via the PI3-kinase/protein kinase B signaling pathway (Hong and Lee, 1997). Hence, IGF-I can apparently regulate tau phosphorylation, in addition to protect neurons against [3-amyloid toxicity. These two features alone certainly fully justify further investigations on the role and potential usefulness of IGF-I (or mimetics) in AD.

associated with increase in IGF protein levels (Gluckman et al., 1992; Beilharz et al., 1995; Breese et al., 1996; Lee et al., 1996; Sandberg Nordqvist et al., 1996) and IGF-1 receptors (Bergstedt and Wieloch, 1993). This raises the question as to whether the IGF-I presents in the bloodstream can get into and be used by the brain. There is one study showing that labeled IGF-I could be found in the brain following a peripheral injection of the iodinated peptide (Lee et al., 1992). It is however not clear whether the CNS-recovered labeled peptide is biologically active or if some inactive radioactive metabolites are mostly present. Interestingly, it has been suggested that the IGFBPs could act as specific transporters of the IGFs across the blood brain barrier (Pardridge, 1993; Reinhardt and Bondy, 1994), although this concept requires further investigation. One group has also proposed that a tripeptide (GPE) derived from IGF-I could pass the blood brain barrier and mimic some of the effects of the parent molecule in the brain (Sara et al., 1993). However, it is generally believed that peptides containing proline residues do not easily cross the blood brain barrier (Benrabh and Lefauconnier, 1996). Naturally, another aspect to consider in stroke is that the integrity of the blood brain barrier is most likely to be significantly altered possibly facilitating the penetration of large blood-borne peptides and trophic factors such as IGF-I. Additionally, many of animal studies on the possible role of IGF-I in brain ischemia have used infant animals, their blood brain barrier likely being much different and more permeable than that of an adult (Mitchell et al., 1982; Ziylan et al., 1992; Rosenstein, 1996). Given the evidence that brain injury is commonly associated with increases in IGFs and IGFBPs (Klempt et al., 1993; Breese et al., 1996; Lee et al., 1996; Tapanainen et al., 1997; Beilharz et al., 1998), it was hypothesized that displacement of the large "pool" of endogenous IGF-I from the IGFBPs would elevate free IGF-I levels to elicit neuroprotective effects comparable to those produced by the administration of exogenous IGF-I. We already discussed the potential usefulness of the human IGF-I analogue, [Leu 24' 59, 60, Ala31 ]hlGF-I, in that regard (section on peptidyl mimetics, Loddick et al., 1998). This type of intervention could certainly have potential clinical usefulness, at least for an acute, temporary treatment of the ischemic event.

6. Conclusion 5. Potential application of IGF-I in stroke It has been reported that intracerebroventricular and subcutaneous supplementations of IGF-I protect the immature and adult brain against hypoxic-ischemic injury (Cheng and Mattson, 1992; Gluckman et al., 1992; Zhu and Auer, 1994; Galli et al., 1995; Guan et al., 1996; Sortino and Canonico, 1996; Tagami et al., 1997; Loddick et al., 1998; Hoffmann et al., 1999). Interestingly, brain injury is often

Knowing the potential actions of IGFs on a variety of neurons and glia cells, it is possible to contemplate that IGF-I treatment could have therapeutic applications in a number of neurodegenerative disorders and in traumatic brain and spinal cord injuries. IGF-I has already been used in the clinics for a long time (Lewis et al., 1993; Yuen and Mobley, 1995). It has been extensively studied with respect to growth delay, diabetes and catabolic disorders. Its

s. Dor~ et al. ,/Pharmaceutica Acta Helvetiae 74 (2000) 273-280

safety has been quite well established, although some side-effects are important to consider, particularly in the context of a chronic treatment. One of the major counterindication is that IGF-I, due to its characteristic growth action, could p r o m o t e the progression of s o m e forms of cancer (Lewis et al., 1993; L o n n i n g and Lien, 1995). C o n c e r n i n g the nervous system, a few clinical trials have suggested some beneficial effects of I G F - I (with very little side effects) in the treatment of peripheral n e u r o d e g e n e r a t i v e disorders such as a m y o t r o p h i c lateral sclerosis although it did not reach satisfying level of significance (Lewis et al., 1993; D a m i a n o et al., 1999). The necessity to d e v e l o p n o n - p e p t i d y l I G F - I mimetics is evident in order to facilitate CNS tissue penetration and to improve stability. This is most certainly feasible now considering the recent d e v e l o p m e n t , as discussed earlier, of a non-peptidyl molecule, L-783,281, which acts as an insulin receptor activator (Zhang et al., 1999). The recent characterization of the t h r e e - d i m e n s i o n a l (3D) structure and I G F - I receptor (Garrett et al., 1998) and insulin receptor (Luo et al., 1999) will certainly be m o s t helpful toward the design of useful mimetic. Finally, a better understanding of the intracellular signaling events occurring following the activation of the I G F - I receptor could also prove useful in the d e v e l o p m e n t of selective mimetics. For example, it is only m o s t recently that s o m e differences have been reported b e t w e e n the intracellular cascades of events induced by the activation of the insulin vs. I G F - I receptors (Butler et al., 1998).

References Aizenman, Y., de Vellis, J., 1987. Brain Res. 406, 32-42. Araujo, D.M., Lapchak, P.A., Collier, B., Chabot, J.G., Quirion, R., 1989. Brain Res. 484, 130-138. Arriagada, P.V., Growdon, J.H., Hedley-Whyte, E.T., Hyman, B.T., 1992. Neurology 42, 631-639. Auld, D.S., Kar, S., Quirion, R., 1998. Trends Neurosci. 21, 43-49. Baker, J., Liu, J.P., Robertson, E.J., Efstratiadis, A., 1993. Cell 75, 73-82. Ballard, F.J., Wallace, J.C., Francis, G.L., Read, L.C., Tomas, F.M., 1996. Int. J. Biochem. Cell Biol. 28, 1085-1087. Bayne, M.L., Applebaum, J., Chicchi, G.G., Miller, R.E., Cascieri, M.A., 1990. J. Biol. Chem. 265, 15648-15652. Beilharz, E.J., Bassett, N.S., Sirimanne, E.S., Williams, C.E., Gluckman, P.D., 1995. Brain Res. Mol. Brain Res. 29, 81-91. Beilharz, E.J., Russo, V.C., Butler, G., Baker, N.L., Connor, B., Sirimanne, E.S., Dragunow, M., Werther, G.A., Gluckman, P.D., Williams, C.E., Scheepens, A., 1998. Brain Res. Mol. Brain Res. 59, 119-134. Benrabh, H., Lefauconnier, J.M., 1996. Neurochem. Res. 21, 1227-1235. Berelowitz, M., Szabo, M., Frohman, L.A., Firestone, S., Chu, L., Hintz, R.L., 1981. Science 212, 1279-1281. Bergstedt, K., Wieloch, T., 1993. J. Cereb. Blood Flow Metab. 13, 895-898. Binder, L.I., Frankfurter, A., Rebhun, L.I., 1985. J. Cell Biol. 101, 1371-1378.

279

Blair, L.A., Bence-Hanulec, K.K., Mehta, S., Franke, T., Kaplan, D., Marshall, J., 1999. J. Neurosci. 19, 1940-1951. Braak, H., Braak, E., 1991. Acta Neuropathol. 82, 239-259. Breese, C.R., D'Costa, A., Rollins, Y.D., Adams, C., Booze, R.M., Sonntag, W.E., Leonard, S., 1996. J. Comp. Neurol. 369, 388-404. Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S., Anderson, M.J., Arden, K.C., Blenis, J., Greenberg, M.E., 1999. Cell 96, 857-868. Busciglio, J., Yeh, J., Yankner, B.A., 1993. J. Neurochem. 61, 1565-1568. Butler, A.A., Yakar, S., Gewolb, I.H., Karas, M., Okubo, Y., LeRoith, D., 1998. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 121, 19-26. Cardone, M.H., Roy, N., Stennicke, H.R., Salvesen, G.S., Franke, T.F., Stanbridge, E., Frisch, S., Reed, J.C., 1998. Science 282, 1318-1321. Carson, M.J., Behringer, R.R., Brinster, R.L., McMorris, F.A., 1993. Neuron 10, 729-740. Cascieri, M.A., Chicchi, G.G., Bayne, M.L., 1991. In: Raizada, M.K., d LeRoith, D. (Eds.), Molecular Biology and Physiology of Insulin and Insulin-like growth factors, Vol. 293 Plenum, New York, pp. 23-30. Castro-Alamancos, M.A., Arevalo, M.A., Torres-Aleman, I., 1996. Neuroscience 70, 843-847. Cheng, B., Mattson, M.P., 1992. J. Neurosci. 12, 1558-1566. Clemmons, D.R., 1998. Mol. Cell Endocrinol. 140, 19-24. Crews, F.T., McElhaney, R., Freund, G., Ballinger, W.E. Jr., Raizada, M.K., 1992. J. Neurochem. 58, 1205-1210. Daly, R.J., 1998. Cell Signal 10, 613-618. Damiano, A.M., Patrick, D.L., Guzman, G.I., Gawel, M.J., Gelinas, D.F., Natter, H.M., Ingalls, K.K., 1999. Med. Care 37, 15-26. Denu, J.M., Lohse, D.L., Vijayalakshmi, J., Saper, M.A., Dixon, J.E., 1996. Proc. Natl. Acad. Sci. U.S.A. 93, 2493-2498. de Pablo, F., de la Rosa, E.J., 1995. Trends Neurosci. 18, 143-150. Dor& S., Pelletier, J.P., DiBattista, J.A., Tardif, G., Brazeau, P., MartelPelletier, J., 1994. Arthritis Rheum. 37, 253-263. Dorfi, S., Krieger, C., Kar, S., Quirion, R., 1996. Brain Res. Mol. Brain Res. 41, 128-133. Dor6, S., Kar, S., Quirion, R., 1997a. Proc. Natl. Acad. Sci. U.S.A. 94, 4772-4777. Dor~, S., Kar, S., Quirion, R., 1997b. Neuroscience 78, 373-383. Dor6, S., Kar, S., Quirion, R., 1997c. Trends Neurosci. 20, 326-331. Dor6, S., Kar, S., Chabot, J.-G., Quirion, R., 1999. Neuroscience 91, 1035-1043. Drechsel, D.N., Hyman, A.A., Cobb, M.H., Kirschner, M.W., 1992. Mol. Biol. Cell 3, 1141-1154. Fulton, D., Gratton, J.P., McCabe, T.J., Fontana, J., Fujio, Y., Walsh, K., Franke, T.F., Papapetropoulos, A., Sessa, W.C., 1999. Nature 399, 597-601. Galli, C., Meucci, O., Scorziello, A., Werge, T.M., Calissano, P., Schettini, G., 1995. J. Neurosci. 15, 1172-1179. Garrett, T.P., McKern, N.M., Lou, M., Frenkel, M.J., Bentley, J.D., Lovrecz, G.O., Elleman, T.C., Cosgrove, L.J., Ward, C.W., 1998. Nature 394, 395-399. Gluckman, P., Klempt, N., Guan, J., Mallard, C., Sirimanne, E., Dragunow, M., Klempt, M., Singh, K., Williams, C., Nikolics, K., 1992. Biochem. Biophys. Res. Commun. 182, 593-599. Goedert, M., Spillantini, M.G., Cairns, N.J., Crowther, R.A., 1992. Neuron 8, 159-168. Greenberg, S.G., Davies, P., 1990. Proc. Natl. Acad. Sci. U.S.A. 87, 5827-5831. Guan, J., Williams, C.E., Skinner, S.J., Mallard, E.C., Gluckman, P.D., 1996. Endocrinology 137, 893-898. Guthrie, K.M., Nguyen, T., Gall, C.M., 1995. J. Comp. Neurol. 352, 147-160. Harris, M.E., Hensley, K., Butterfield, D.A., Leedle, R.A., Carney, J.M., 1995. Exp. Neurol. 131, 193-202. Hoffmann, T.T., Schaebitz, W.R., Schwab, S., 1999. J. Cereb. Blood Flow Metab. 19, S 182. Hong, M., Lee, V.M., 1997. J. Biol. Chem. 272, 19547-19553.

280

S. Dor~ et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 273-280

Humbel, R.E., Rinderknecht, E., 1979. In: Giordane, G., Van Wyk, J.J., Minuto, F. (Eds.), Somatomedins and Growth, Vol. 23, Academic Press, New York, pp. 61-65. Isaksson, O.G., Ohlsson, C., Nilsson, A., Isgaard, J., Lindahl, A., 1991. Pediatr. Nephrol. 5, 451-453. Johansson, A.G., Lindh, E., Ljunghall, S., 1993. J. Intern. Med. 234, 553-560. Jones, J.I., Clemmons, D.R., 1995. Endocr. Rev. 16, 3-34. Kar, S., Baccichet, A., Quirion, R., Poirier, J., 1993. Neuroscience 55, 69-80. Kar, S., Seto, D., Dor6, S., Hanisch, U., Quirion, R., 1997. Proc. Natl. Acad. Sci. U.S.A. 94, 14054-14059. Klempt, M., Klempt, N.D., Gluckman, P.D., 1993. Brain Res. Mol. Brain Res. 17, 347-350. Lamothe, B., Baudry, A., Desbois, P., Lamotte, L., Bucchini, D., De Meyts, P., Joshi, R.L., 1998. Biochem. J. 335, 193-204. Lee, V.M., Balin, B.J., Otvos, L. Jr., Trojanowski, J.Q., 1991. Science 251,675-678. Lee, W.H., Javedan, S., Bondy, C.A., 1992. J. Neurosci. 12, 4737-4744. Lee, W.H., Wang, G.M., Seaman, L.B., Vannucci, S.J., 1996. J. Cereb. Blood Flow Metab. 16, 227-236. Lenoir, D., Honegger, P., 1983. Brain Res. 283, 205-213. LeRoith, D., Werner, H., Beitner-Johnson, D., Roberts, C.T. Jr., 1995. Endocr. Rev. 16, 143-163. Lewis, M.E., Neff, N.T., Contreras, P.C., Stong, D.B., Oppenheim, R.W., Grebow, P.E., Vaught, J.L., 1993. Exp. Neurol. 124, 73-88. Lienhard, G.E., 1994. Nature 372, 128-129. Loddick, S.A., Liu, X.J., Lu, Z.X., Liu, C., Behan, D.P., Chalmers, D.C., Foster, A.C., Vale, W.W., Ling, N., De Souza, E.B., 1998. Proc. Natl. Acad. Sci. U.S.A. 95, 1894-1898. Lonning, E., Lien, E.A., 1995. Crit. Rev. Oncol. Hematol. 21, 158-193. Loo, D.T., Copani, A., Pike, C.J., Whittemore, E.R., Walencewicz, A.J., Cotman, C.W., 1993. Proc. Natl. Acad. Sci. U.S.A. 90, 7951-7955. Luo, R.Z.T., Beniac, D.R., Fernandes, A., Yip, C.C., Ottensmeyer, F.P., 1999. Science 285, 1077-1080. Maurice, T., Lockhart, B.P., Privat, A., 1996. Brain Res. 706, 181-193. Meyerovitch, J., Farfel, Z., Sack, J., Shechter, Y., 1987. J. Biol. Chem. 262, 6658-6662. Mitchell, W., Kim, C.S., O'Tuama, L.A., Pritchard, J.B., Pick, J.R., 1982. Neurosci. Lett. 31, 37-40.

Nilsson, L., Sara, V.R., Nordberg, A., 1988. Neurosci. Lett. 88, 221-226. Pankov, Y.A., 1999. Biochemistry (Moscow) 64, 1-7. Pardridge, W.M., 1993. Annu. N. Y. Acad. Sci. 692, 126-137. Price, D.L., Sisodia, S.S., Gandy, S.E., 1995. Curr. Opin. Neurol. 8, 268-274. Reinhardt, R.R., Bondy, C.A., 1994. Endocrinology 135, 1753-1761. Roher, A.E., Lowenson, J.D., Clarke, S., Woods, A.S., Cotter, R.J., Gowing, E., Ball, M.J., 1993. Proc. Natl. Acad. Sci. U.S.A. 90, 10836-10840. Rosenstein, J.M., 1996. Exp. Neurol. 142, 66-79. Rotwein, P., Burgess, S.K., Milbrandt, J.D., Krause, J.E., 1988. Proc. Natl. Acad. Sci. U.S.A. 85, 265-269. Sandberg Nordqvist, A.C., von Holst, H., Holmin, S., Sara, V.P~. Bellander, B.M., Schalling, M., 1996. Brain Res. Mol. Brain Res. 38, 285-293. Sara, V.R., Carlsson-Skwirut, C., Drakenberg, K., Giacobini, M.B., Hakansson, L., Mirmiran, M., Nordberg, A., Olson, L., Reinecke, M., Stahlbom, P.A. et al., 1993. Annu. N. Y. Acad. Sci. 692, 183-191. Selkoe, D.J., 1998. Trends Cell Biol. 8, 447-453. Shemer, J., Adamo, M., Wilson, G.L., Heffez, D., Zick, Y., LeRoith, D., 1987. J. Biol. Chem. 262, 15476-15482. Sortino, M.A., Canonico, P.L., 1996. Endocrinology 137, 1418-1422. Tagami, M., Yamagata, K., Nara, Y., Fujino, H., Kubota, A., Numano, F., Yamori, Y., 1997. Lav. Invest. 76, 603-612. Tapanainen, P.J., Bang, P., Muller, H.L., Wilson, K., Rosenfeld, R.G., 1997. Horm. Res. 48, 227-234. Traxler, P., Furet, P., 1999. Pharmacol. Ther. 82, 195-206. Waters, S.B., Chen, D., Kao, A.W., Okada, S., Holt, K.H., Pessin, J.E., 1996. J. Biol. Chem. 271, 18224-18230. Yuen, E.C., Mobley, W.C., 1995. Mol. Med. Today 1,278-286. Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M.T., Pelaez, F., Ruby, C., Kendall, R.L., Mao, X., Griffin, P., Calaycay, J., Zierath, J.R., Heck, J.V., Smith, R.G., Moller, D.E., 1999. Science 284, 974-977. Zheng, W.-H., Kar, S., Quirion, R., 1999. Soc. Neurosci. Abst., (In press). Zhu, C.Z., Auer, R.N., 1994. J. Cereb. Blood Flow Metab. 14, 237-242. Ziylan, Y.Z., Lefauconnier, J.M., Ates, N., Bernard, G., Bourre, J.M., 1992. Mech. Ageing Dev. 62, 319-327.



Apoptosis induced by death receptors Pascal Schneider *, Jiirg Tschopp Institute of Biochemistry, University of Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland

Abstract

Death receptors belong to the TNF receptor family and are characterised by an intracellular death domain that serves to recruit adapter proteins such as TRADD and FADD and cysteine proteases such as Caspase-8. Activation of Caspase-8 on the aggregated receptor leads to apoptosis. Triggering of death receptors is mediated through the binding of specific ligands of the TNF family, which are homotrimeric type-2 membrane proteins displaying three receptor binding sites. There are various means of modulating the activation of death receptors. The status of the ligand (membrane-bound vs. soluble) is critical in the activation of Fas and of TRAIL receptors. Cleavage of membrane-bound FasL to a soluble form (sFasL) does not affect its ability to bind to Fas but drastically decreases its cytotoxic activity. Conversely, cross-linking epitope-tagged sFasL with anti-tag antibodies to mimic membrane-bound ligand results in a 1000-fold increase in cytotoxicity. This suggests that more than three Fas molecules need to be aggregated to efficiently signal apoptosis. Death receptors can also be regulated by decoy receptors. The cytotoxic ligand TRAIL interacts with five receptors, only two of which (TRAIL-R1 and -R2) have a death domain. TRAIL-R3 is anchored to the membrane by a glycolipid and acts as a dominant negative inhibitor of TRAIL-mediated apoptosis when overexpressed on TRAIL-sensitive cells. Intracellular proteins interacting with the apoptotic pathway are potential modulators of death receptors. FLIP resembles Caspase-8 in structure but lacks protease activity. It interacts with both FADD and Caspase-8 to inhibits the apoptotic signal of death receptors and, at the same time, can activate other signalling pathways such as that leading to NF-KB activation. 9 2000 Elsevier Science B.V. All rights reserved.

Keywords: Apoptosis; Tumor necrosis factor (TNF); FLIP; Death receptors; FasL

1. Characteristics of apoptosis Programmed cell death, also called apoptosis, is a physiologic event allowing a multicellular organism to smoothly get rid of old, superfluous, damaged or infected cells in a process which is clearly distinct from necrosis, the result of accidental cell death. Apoptosis is involved in a variety of processes such as the regression of defined tissues during development, the elimination of incompetent or self-reactive T-lymphocytes during positive and negative selection, or the elimination of non- or low-reactive B cells during affinity maturation. Cytotoxic T-lymphocytes, which are able to induce apoptosis in targets such as virus-infected cells, are eliminated by an apoptotic process called


activation-induced cell death once the infection has been overcome. Tissues like the skin or the intestinal mucosa, which experience rapid turnover and regeneration, have sites where massive programmed cell death does occur. Apoptosis is also a favoured response in cells exposed to stress stimuli and damaging conditions, therefore preventing the appearance and proliferation of potentially dangerous cells. However, deregulation of the apoptotic process leading to either increased or reduced cell death can contribute to various pathologic conditions such as autoimmune diseases, AIDS or cancer. At the morphological level, apoptosis is characterised by several of the following hallmarks: cell shrinking, chromatin condensation and cellular fragmentation into so-called apoptotic bodies. The latter are taken up by neighbouring or specialised cells, therefore avoiding the inflammatory response that would occur after spilling of intracellular material. These macroscopic changes are pre-


282

P. Schneider, J. Tschopp / Pharmaceutica Acta Helvetiae 74 (2000) 281-286

ceded by biochemical events such as the redistribution of membrane lipids, the loss of mitochondrial membrane potential, the activation of intracellular proteases (caspases), the proteolytic degradation of selected proteins in the cell and the fragmentation of DNA at internucleosomal sites. The central event in apoptotic cell death is the activation of caspases, a family of cysteine proteases with strict cleavage specificity at aspartic acid residues (Thornberry and Lazebink, 1998). Caspases pre-exist in the cell as inactive zymogen precursors composed of a prodomain followed by a large and a small catalytic subunits which, after proteolytic processing, fold into an active caspase able to specifically cleave a variety of cellular substrates, leading to the apoptotic phenotype. There are two main pathways leading to the activation of effector caspases: the first one is triggered in response to a variety of stress conditions such as UV irradiation, growth factor deprivation, abnormal mitosis or anti-cancer drugs, and proceeds via the recruitment and activation of Caspase-9 into a complex containing cytochrome c and Apaf-1. Pro- and anti-apoptotic members of the Bcl-2 family, which act on the mitochondria, regulate this pathway (Green and Reed, 1998). The second pathway utilises specialised membrane receptors, which are efficiently coupled to caspase activation, especially Caspase-8 (Ashkenazi and Dixit, 1998). These death-inducing receptors belong to the tumour necrosis factor receptor (TNFR) family and are activated upon engagement by their cognate ligands of the TNF family.

elongated structure (Fig. 1). Ligands of the TNF family are type-2 membrane proteins whose extracellular C-termini fold into [3-sheet "jelly roll" structures that homotrimerise to form the active ligands. Ligands, which can exert their functions in the membrane-bound form or after proteolytic processing to a soluble trimer, can bind three receptors at their monomer boundaries (Banner et al., 1993). The intracellular domains of ligand-aggregated receptors can, in turn, bind to specific signalling molecules. Fas recruits the adaptor protein FADD via D D - D D interaction. FADD, in turn, binds Caspase-8 via homotypic interaction involving "death effector domains". TNF-R1 can also recruit Caspase-8 via TRADD and FADD and, in addition, potently activates the NF-KB transcription factor via the DD-containing kinase RIP and c-Jun N-terminal kinase via TRAF2. Signalling through TRAMP/DR3 is similar to TNF-R1 (Ashkenazi and Dixit, 1998).

2. The TNF and TNFR families of ligands and receptors

3.1. Modulation of death receptors by ligands: the Fas/ FasL system and other examples

The TNF and TNFR families (Fig. 1) are mainly involved in the development and function of the immune system and of cells of lymphoid origin (Gravestein and Borst, 1998). For example, TNF plays a crucial role in inflammation, and the lymphotoxin system controls the development of peripheral lymphoid organs and splenic architecture. OX40 and 4-1BB play a co-stimulatory role in T cell proliferation whereas CD40 is a central player in B cell proliferation and immunoglobulin isotype switch. The R A N K / O P G system modulates osteoclast maturation and bone homeostasis and the Fas/FasL system fulfils an important role in the homeostasis and in the cytotoxic effector function of T cells. A subset of the TNF receptors, namely TNF-R1, Fas, TRAMP/DR3, TRAIL-R1, TRAILR2 and DR6 (Fig. 1), can signal cell death via an intracellular "death domain" (DD), and is therefore termed the death receptor subfamily. TNF receptors are type-1 membrane proteins characterised by cysteine-rich sequences in their extracellular domains that fold into modules stacking one on top of each other thus conferring to the receptor an

TNFa is synthesised as a membrane-bound protein that acts locally through cell to cell contact. Soluble TNFa (sTNFoL) is released from the cell surface as the result of metalloproteinases cleavage (McGeehan et al., 1994; Mohler et al., 1994). Although both cell surface and secreted TNFoL appear to be biologically active, deleterious physiological responses such as cachexia and endotoxic shock are mediated by the later. FasL is also processed and shed from the surface of human cells. Until recently, it was not clear whether the activities described for FasL were primarily due to its cell surface or secreted form. Elevated levels of sFasL were found in sera from patients with large granular lymphocytic leukemias and natural killer cell lymphomas (Tanaka et al., 1996). Since the administration of recombinant FasL or agonistic Fas antibodies into mice leads to liver failure and to rapid death of the animals (Ogasawara et al., 1993; Rensing-Ehl et al., 1995), it has been proposed that sFasL is implicated in the pathogenesis of various diseases such as hepatitis or AIDS.

3. Modulation of death receptors activity Apoptosis induced by death receptors can be controlled at several levels. Outside of the cell, the aggregation status of the ligand and the presence of soluble or membranebound decoy receptors have important regulatory roles. Anti-apoptotic intracellular proteins can block the apoptotic-signalling pathway or divert it into distinct responses. In addition, the overall sensitivity of a cell to apoptosis can be affected by engagement of receptors that do not themselves contain a death domain. The data presented below will exemplify some of these aspects.

P. Schneider, J. Tschopp / Pharmaceutica Acta Heluetiae 74 (2000) 281-286 80

56

~58

25 .....

37 ~

18

22

~37

20

283

24

2S

25

28

46

OX40L GITRL TNFa Fast.

VEGI

RANKL

"

4-II | B L

TWEAK

AI~I

TRAIL

fi

DcR3

BAR =

--

..,...-

4""

OPG

CD~

DRe

A1 NGFR

A1 ~ TRAILR$

B1 li', AI

A1

O1'

B2

F

~ A1 I TRAILR2 ~? t TRAILR1

A1

I

A1

A1 ,,,, Alt

A1 " Alt

B2

D27

A1 B2

E

A2

1

B1

E

A1

1

B1

E

B2

~3

221

154

A1 B2

PO.

A1

J A1

132

A1 B1

E

~ 18 145

r ;rTFI

1 A1 ~

1

=1

A2

411

"~ 382

193

~ 286

174

221

S8

187

188

L 45

N

42 IU

Fig. 1. TNF and TNFR family members. Trimeric ligands are shown at the top and receptors at the bottom of the figure. Positions of cysteine residues in the extracellular portion of receptors are indicated by horizontal bars; Basic structural modules (e.g., A1, A2, B1, B2) (Naismith and Sprang, 1998) are shown as shaded areas. The length of the intracellular domains is indicated by numbers. When present, death domains are represented by black boxes. Interactions that have been reported in the literature are indicated by arrows. The soluble receptor OPG contains two death domain motives in its C-terminal portion. Some of the ligands and receptors have several names: F a s L / A p o - l L / C D 9 5 L , VEGI/TL-1, TRAIL/Apo-2L, R A N K / O P G L / T R A N C E , TWEAK/Apo-3L, GITRL/AITRL, B A F F / T A L L - 1 / T H A N K / B l y S , DcR3/TR6, Fas/Apo-1/CD95, TRAILR1/DR4, TRAILR2/DR5/Killer/Apo-2, TRAILR3/DcR l, TRAILR4/DcR2, TARMP/Apo- 3 / D R 3 / W S L - 1/LARD, HVEM/ATAR, GITR/AITR.

293 cells transfected with FasL contained predominantly the unprocessed form of FasL, but a significant proportion of processed, soluble FasL was also recovered into the medium. Processing of FasL was mediated by a metalloprotease and occurred between Ser 126 and Leu 127, in the stalk linking the trimerisation domain of FasL to its transmembrane segment. As predicted, site directed mutagenesis of these residues to glutamic acid residues almost completely abolished FasL processing. Both naturally processed FasL and Flag-tagged, recombinant soluble FasL bound to recombinant Fas and to the surface of Fas positive cells. Surprisingly, however, these soluble FasLs were almost completely unable to induce

cell death into A20 or Jurkat cells, which are otherwise very sensitive to agonistic anti-Fas antibodies. These results suggested that the mere trimerisation of Fas by sFasL was not sufficient to efficiently trigger the death signal. Indeed, when we took advantage of the Flag tag (an octapeptide epitope recognised by a monoclonal antibody) to cross-link FasL, we observed a 1000-fold increase of FasL cytotoxic activity on a variety of cell lines and on primary mouse hepatocytes. A similar effect was observed when sFasL was administered to mice: the animals showed no sign of sickness if sFasL or cross-linking antibodies were injected alone, but died within 3 h when sFasL injection was followed by that of cross-linking anti-Flag

284


antibodies (Schneider et al., 1998). Massive apoptosis was detected in the liver of these mice, as observed in mice treated with agonistic anti-Fas antibodies (Ogasawara et al., 1993). This suggests that a cluster of several Fas trimers is required to signal apoptosis, and that cross-linking of soluble FasL trimers may mimic the membranebound form of FasL. Local cell-to-cell contact appears to be critical for T cell specific immunosurveillance, and indeed, FasL-mediated killing of virus-infected or tumor target cells is a highly specific process which assures that neighbouring cells or tissues are not affected. This specificity can only be guaranteed if the cytotoxic ligand remains associated with the lymphocytes. Considering the hepatic toxicity of FasL, circulating sFasL, if active, would be pathological. However, patients with elevated levels of sFasL (Tanaka et al., 1996) do not suffer from hepatitis, implying that sFasL in vivo is at best poorly active, in agreement with our results. We tested whether other members of the TNF family known to induce apoptosis also required cross-linking for their activity. Similar to FasL, sTRAIL was active at low concentrations only in the presence of cross-linking antibodies. In contrast, cross-linking of sTWEAK did not increase its cytotoxic activity on HT-29 cells, suggesting that this ligand can exert its apoptotic activity in the soluble form. Interestingly, the activity of sTNFo~ was differentially influenced by the presence of cross-linking antibodies, depending on the receptor triggered. Cross-linking only increased the action of TNFoL on TNF-R2 but not on TNF-R1 (Schneider et al., 1998). This is in line with results demonstrating that membrane-bound TNFoL is the prime activating ligand for TNF-R2-mediated responses (Grell et al., 1995). We also found that the proliferative effect of sCD40L on primary human B cells was considerably increased upon cross-linking. Taken together, these results suggest that several receptors are efficiently stimulated only by membrane-bound or cross-linked ligands. This probably provides a threshold ensuring that the signal is only transmitted under defined conditions, such as a cell-to-cell contact.

3.2. Modulation of death receptors by decoy receptors: the example of TRAIL-R3 The cytotoxic ligand TRAIL shows a broad tissue distribution (Wiley et al., 1995) and has the particularity to interact with five receptors (Fig. 1). Two of them (TRAILR 1 / D R 4 and TRAIL-R2/DR5) contain a death domain and are cytotoxic. The other three may act as decoy receptors. TRAIL-R3/DcR1 and TRAIL-R4/DcR2 are membrane-bound but lack a functional death domain, whereas OPG is a soluble receptor with dual specificity for TRAIL and RANKL/OPGL. In contrast to FasL, TRAIL is not cytotoxic to tissues despite the wide expression of TRAIL-R1 and -R2, suggesting that TRAIL receptor sig-

nailing is under the control of strong regulatory mechanisms. TRAIL-R3 cDNA encodes a short receptor, which is devoid of an intracellular domain and ends immediately after a carboxy-terminal stretch of 15 hydrophobic amino acids. This feature is reminiscent of a signal for the addition of a glycosyl-phosphatidylinositol (GPI) anchor and the sequence indeed fulfils all structural requirements for GPI addition at Ala 236 (Udenfriend and Kodukula, 1995). In mature GPI-anchored proteins, the carboxyterminal portion of the protein is exchanged for a preformed glycolipid, which anchors the protein to the membrane. Several lines of evidence indicate that TRAIL-R3 is indeed a GPI-anchored protein. First, TRAIL-R3 expressed in 293 cells partitioned quantitatively into the detergent phase of a Triton X-114 phase separation, as expected for a GPI-anchored protein. Second, TRAIL-R3 was biosynthetically labelled with [3H]inositol, which, in a protein, is highly specific of a GPI. The third evidence comes from the radiochemical analysis of TRAIL-R3. The hallmark of GPI-anchors is a non-acetylated glucosamine that can be deaminated with nitrous acid and reduced with sodiumborotritide to yield a radioactive anhydromannitol residue, with concomitant loss of the phosphatidylinositol. Subsequent dephosphorylation with hydrofluoric acid releases the neutral carbohydrate fraction of the GPI which, in the case of TRAIL-R3, co-migrated with authentic Man 3anhydromannitol, the minimal conserved carbohydrate structure present in all GPI anchors analysed so far. Taken together, these data demonstrate the presence of a GPI in TRAIL-R3. Finally, a number of GPI-anchored proteins can be solubilised by the action of phosphatidylinositolspecific phospholipase C (PI-PLC). This reaction is, however, not possible if the hydroxyl group on position 2 of the inositol is acylated. This is most likely the case for TRAIL-R3 expressed in 293 cells, because it was resistant to the action of PI-PLC. Because TRAIL-R3 can bind to TRAIL, yet is unable to transduce a death signal, it was expected that it could counteract the action of TRAIL in sensitive cells either by scavenging TRAIL or by forming inactive mixed receptors with TRAIL-R1/R2. MCF-7 cells respond to TRAIL or TNF by stopping proliferation and displaying morphological features of apoptotic cells. However, MCF-7 cells stably transfected with a dominant negative version of FADD became entirely resistant to the effects of TRAIL, TNF and agonistic anti-TRAIL-R2 antibodies, indicating that FADD-DN efficiently interacts with death signalling as previously described (Chinnaiyan et al., 1996; Schneider et al., 1997). MCF-7 cells stably transfected with TRAIL-R3 bound approximately 20 times more TRAIL on their surface than non-transfected cells, but only became susceptible to TRAIL with 100 times greater doses than those necessary to kill wild type cells. These TRAIL-R3 transfectants, however, still had functional apoptotic sig-


nalling pathways as demonstrated by their unaltered susceptibility to TNF and agonistic anti-TRAIL-R2 antibodies. These results point to the fact that TRAIL-R3 can indeed act as a dominant negative inhibitor of TRAIL action. Whether this reflects the role of TRAIL-R3 in vivo remains to be determined. However, experiments performed on melanoma cell lines revealed no correlation between TRAIL-R3 expression and resistance to TRAIL (Griffith et al., 1999; Zhang et al., 1999). Clearly, intracellular regulators of the pathway also play an essential role. In addition, Northern blot analysis indicated that TRAIL-R3 expression is less widespread than those of TRAIL-R1 and TRAIL-R2, suggesting that it may play a role in a restricted number of cell types.

3.3. Modulation of death receptors by other receptors: the example of TWEAK The widely expressed ligand TWEAK can induce apoptosis in a restricted number of cell lines (Chicheportiche et al., 1997). It also induces angiogenesis and proliferation of endothelial cells (Lynch et al., 1999). We found that TWEAK binds to the rhabdomyosarcoma Kym-1 cell line and efficiently kills it. This cytotoxicity could be prevented with either blocking anti-TNF antibodies, or with soluble TNF-R1 or with antagonistic anti-TNF-R1 antibodies, suggesting that the action of TWEAK was indirect and mediated by the induction of endogenous TNF. When Kym-1 cells were treated plus or minus TWEAK in the presence of blocking anti-TNF antibodies, cells survived and no difference was observed. However, TWEAK-treated cells were efficiently killed by sublethal doses of agonistic anti-TNF-R1 antibodies. This synergism indicates that TWEAK receptor is able to enhance TNF-Rl-mediated apoptosis in Kym-1 cells. TWEAK is therefore not cytotoxic on its own in this model system but only helps to provide factors allowing for autocrine/paracrine killing of the cells via TNF and TNF-R1 (Schneider et al., 1999). Sensitization of cells to TNFe~ can also occur upon stimulation of TNF-R2, CD30 or CD40, pointing to the fact that cross-talk between receptor may be a general mechanism (Grell et al., 1999).

3.4. Modulation of death receptors by intracellular factors: the example of FLIP Caspase-8 is recruited to FADD via interaction between death effector domains. We found that ORF E8 of the equine herpes virus-2 encoded a protein with two predicted death effector domains. This protein, called v-FLIP, is also present in several other viruses including human herpes virus-8. It interferes with the apoptotic pathway of FasL by binding to FADD and potently inhibits TRAIL-mediated cell death (Thome et al., 1997). The sequence information from v-FLIP led to the discovery of a mammalian homologue, cellular FLIP (c-FLIP, also called CASPER/I-

285

F L I C E / F l a m e / C A S H / C L A R P / M R I T or usurpin) which is expressed in several splice variants (Tschopp et al., 1998). The longer form of c-FLIP (c-FLIP/long) contains two death effector domains and an additional caspase-like domain with significant homology to Caspase-8 (and Caspase-10). In contrast to Caspase-8, however, c-FLIP/long lacks essential features that are required for substrate catalysis and is therefore devoid of proteolytic activity. c-FLIP/long binds to both FADD and Caspase-8 via its death effector domains. The caspase-like domain also binds to Caspase-8, suggesting that the intimate contact occurring between c-FLIP and Caspase-8 may interfere with the activity of the latter. Indeed, both c-FLIP/short and cFLIP/long inhibited cell death induced by several death receptors, c-FLIP/long appeared to be a better inhibitor than c-FLIP/short and both were especially effective against TRAIL-mediated apoptosis (Irmler et al., 1997). It is predicted that c-FLIP blocks apoptosis at the level of Caspase-8 by preventing the activation of downstream effectors such as Caspase-3. FLIP and other viral or cellular proteins involved in the intracellular regulation of death pathways, such as the IAPs, CrmA and Bcl-2 family members may contribute to the resistance of death receptor expressing cells to the action of death ligands (Tschopp et al., 1998). This could be the case of T cells during the early phase of activation, which are resistant to FasL despite abundant Fas expression. Expression of FLIP in various systems efficiently activates NF-kB transcription factor and the MAP kinase ERK pathway, suggesting that its physiological role may be more than just blocking apoptosis.

4. Conclusions

The activation of apoptosis via death receptors is a tightly regulated event. Soluble or membrane-bound decoy receptors can interfere with the death pathway by either scavenging the death ligand or by forming inactive mixed receptor complexes, thereby acting as dominant negative inhibitors. The aggregation state of the ligand is probably a crucial factor. Some receptors can efficiently signal cell death only if they are sufficiently aggregated in a complex comprising more than three receptors. This requirement counteracts undesired activation of the receptor by systemic soluble trimeric ligands but still allows specific activation of the receptor during a cell-to-cell contact. Preliminary experiments indicate that the efficiency of apoptosis signalling through TNF-R1 can be increased upon engagement of others receptors not directly linked to apoptosis. Further experiments are needed to determine if this is an exceptional or a more general mechanism. Finally, anti-apoptotic intracellular proteins that interfere with the apoptotic machinery can dictate the outcome of death receptor triggering.

286


References Ashkenazi, A., Dixit, V.M., 1998. Death receptors: signaling and modulation. Science 281, 1305-1308. Banner, D.W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.J., Broger, C., Loetscher, H., Lesslauer, W., 1993. Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell 73, 431-445. Chicheportiche, Y., Bourdon, P.R., Xu, H., Hsu, Y.M., Scott, H., Hession, C., Garcia, I., Browning, J.L., 1997. TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis. J. Biol. Chem. 272, 32401-32410. Chinnaiyan, A.M., Tepper, C.G., Seldin, M.F., O'Rourke, K., Kischkel, F.C., Hellbardt, S., Krammer, P.H., Peter, M.E., Dixit, V.M., 1996. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J. Biol. Chem. 271, 4961-4965. Gravestein, L.A., Borst, J., 1998. Tumor necrosis factor receptor family members in the immune system. Semin. Immunol. 10, 423-434. Green, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science 281, 1309-1312. Grell, M., Douni, E., Wajant, H., Lohden, M., Clauss, M., Maxeiner, B., Georgopoulos, S., Lesslauer, W., Kollias, G., Pfizenmaier, K., Scheurich, P., 1995. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 Kda tumor necrosis factor receptor. Cell 83, 793-802. Grell, M., Zimmermann, G., Gottfried, E., Chen, C.M., Grunwald, U., Huang, D.C., Lee, Y.H., Durkop, H., Engelmann, H., Scheurich, P., Wajant, H., Strasser, A., 1999. Induction of cell death by tumour necrosis factor (TNF) receptor 2, CD40 and CD30: a role for TNF-R1 activation by endogenous membrane-anchored. EMBO J. 18, 30343043. Griffith, T.S., Rauch, C.T., Smolak, P.J., Waugh, J.Y., Boiani, N., Lynch, D.H., Smith, C.A., Goodwin, R.G., Kubin, M.Z., 1999. Functional analysis of TRAIL receptors using monoclonal antibodies. J. Immunol. 162, 2597-2605. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J.L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L.E., Tschopp, J., 1997. Inhibition of death receptor signals by cellular FLIP. Nature 388, 190-195. Lynch, C.N., Wang, Y.C., Lund, J.K., Chen, Y.W., Leal, J.A., Wiley, S.R., 1999. TWEAK induces angiogenesis and proliferation of endothelial cells. J. Biol. Chem. 274, 8455-8459. McGeehan, G.M., Becherer, J.D., Bast, R.C. Jr., Boyer, C.M., Champion, B., Connolly, K.M., Conway, J.G., Furdon, P., Karp, S., Kidao, S. et al., 1994. Regulation of tumour necrosis factor-alpha processing by a metalloproteinase inhibitor. Nature 370, 558-561. Mohler, K.M., Sleath, P.R., Fitzner, J.N., Cerretti, D.P., Alderson, M.,

Kerwar, S.S., Torrance, D.S., Otten-Evans, C., Greenstreet, T., Weerawarna, K. et al., 1994. Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature 370, 218-220. Naismith, J.H., Sprang, S.R., 1998. Modularity in the TNF-receptor family. Trends Biochem. Sci. 23, 74-79. Ogasawara, J., Watanabe, F.R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T., Nagata, S., 1993. Lethal effect of the anti-Fas antibody in mice. Nature 364, 806-809. Rensing-Ehl, A., Frei, K., Flury, R., Matiba, B., Mariani, S.M., Weller, M., Aebischer, P., Krammer, P.H., Fontana, A., 1995. Local Fas/APO-1 (CD95) ligand-mediated tumor cell killing in vivo. Eur. J. Immunol. 25, 2253-2258. Schneider, P., Thome, M., Burns, K., Bodmer, J.L., Hofmann, K., Kataoka, T., Holler, N., Tschopp, J., 1997. TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-kappaB. Immunity 7, 831-836. Schneider, P., Holler, N., Bodmer, J.L., Hahne, M., Frei, K., Fontana, A., Tschopp, J., 1998. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J. Exp. Med. 187, 1205-1213. Schneider, P., Schwenzer, R., Haas, E., Muhlenbeck, F., Schubert, G., Scheurich, P., Tschopp, J., Wajant, H., 1999. TWEAK can induce cell death via endogenous TNF and TNF receptor 1. Eur. J. Immunol. 29, 1785-1792. Tanaka, M., Suda, T., Haze, K., Nakamura, N., Sato, K., Kimura, F., Motoyoshi, K., Mizuki, M., Tagawa, S., Ohga, S., Hatake, K., Drummond, A.H., Nagata, S., 1996. Fas ligand in human serum. Nature Medicine 2, 317-322. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J.L., Schroter, M., Scaffidi, C., Krammer, P.H., Peter, M.E., Tschop~, J., 1997. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386, 517-521. Thornberry, N.A., Lazebink, Y., 1998. Caspases: enemies within. Science 281, 1312-1316. Tschopp, J., Irmler, M., Thome, M., 1998. Inhibition of Fas death signals by FLIPs. Curr. Opin. Immunol. 10, 552-558. Udenfriend, S., Kodukula, K., 1995. Prediction of to site in nascent precursor of glycosylphosphatidylinositol protein. Methods in Enzymology 250, 571-583. Wiley, S.R., Schooley, K., Smolak, P.J., Din, W.S., Huang, C.P., Nicholl, J.K., Sutherland, G.R., Smith, T.D., Rauch, C., Smith, C.A., Goodwin, R.G., 1995. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673-682. Zhang, X.D., Franco, A., Myers, K., Gray, C., Nguyen, T., Hersey, P., 1999. Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma. Cancer Res. 59, 2747-2753.



Hijacked receptors D a v i d J. T r i g g l e * The Graduate School, 562 Capen Hall, State University of New York, 562 Capen Hall, Buffalo, NY 14260, USA

Abstract

Pharmacological receptors are typically defined by their selectivity of ligand recognition, including where appropriate stereoselectivity of interaction. It is increasingly clear that receptors may, in fact, be promiscuous species. This promiscuity arises at several levels of organization: two appear to be of particular importance. A given ligand-receptor complex may couple with different effectors and may generate quite different physiological responses: this is particularly common, although not uniquely so, for G protein-coupled receptors. Or a single receptor may recognize fundamentally different ligands often of significantly different characteristics: a number of viruses gain entry to cells through their interaction at receptors for neurotransmitters, peptides or hormones. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: Receptors; Co-evolution; Ligand-receptor interactions; Promiscuity; G protein-coupled receptors; Viral penetration; Chemokines; Chemokine receptors; Receptor coupling

1. Introduction

The concept of the specific receptor that binds a specific ligand, large or small, and that subsequently triggers a cascade of cellular events dates from the work of Paul Ehrlich and John Newton Langley and is firmly established as a fundamental principle of biological organization. Implicit in this developing understanding of receptor function was the concept of specificity of interaction early defined by Emil Fischer in his "lock-and-key" concept: ... I will say that enzyme and glucoside must fit together like lock and key in order to be able to exercise a chemical action on each other Emil Fischer, 1894.Advances over the past several decades have provided not only a better pharmacological understanding of drug-receptor interactions, but also have defined approaches to the quantitation of drug-receptor interactions, definitions and interpretations of structure-activity relationships and, through advances in molecular biology, have provided sequences and three-dimensional

* Tel.: +1-716-645-7315; fax: +1-716-645-2941; e-mail: [email protected]

models of receptors and of their interactions with ligands. The most dramatic recent example has been the determination of the three-dimensional structure of a bacterial K + channel and preliminary analyses of its ion translocation mechanisms: it can be anticipated with confidence that the next decade will see more membrane proteins yield to the power of structural techniques, including the very large class of G protein-coupled receptors. Important as these advances have been over the past century they leave some very important questions to be resolved, including that of receptor-ligand evolution. This is, in fact, the "chicken and the egg question" - - which came first, the receptor or the ligand? It is, in fact, plausible that ligand-receptor pairs have undergone a process of co-evolution in which an ancestral protein that may have been relatively promiscuous has progressively attained selectivity of ligand interaction (Moyle et al., 1994). An example of this is provided by lutotropin (luteinizing hormone, LH) and follitropin (follicle-stimulating hormone, FSH) whose actions are mediated by specific receptors. Moyle and his colleagues have demonstrated that short sequences of residues in the betasubunits of these receptors are responsible for the receptor selectivity and enable receptor differentiation by human chorionic gonadotropin. Thus, residues between the 1 l th and 12th cysteines restrict binding to the FSH receptor and residues between the 10th and 1 l th cysteines affect binding to the LH receptor. Chimeric constructs can be made


288

D.J. Triggle/ Pharmaceutica Acta Helvetiae 74 (2000) 287-290

that bind both LH and FSH suggesting that this may approximate the original promiscuous receptor. In principle, receptor promiscuity may have several origins. Two appear to be of particular importance. A common ligand-receptor complex may couple differentially to a variety of effectors to generate substantially different physiological responses or a single receptor may recognize very different ligands that mediate different physiological or pathological responses. A few specific examples of each will be discussed briefly.

3. Promiscuity of receptor recognition An increasing number of examples are known whereby receptors that recognize neurotransmitters also recognize much larger chemical species, notably viruses, and serve as entry points for virus penetration into the cell. Among the most widely studied such systems in recent years are the CD4 and chemokine receptors with which the HIV-1 and HIV-2 viruses interact with terminal consequences. A number of specific examples are given in Table 1. 3.1. Rabies virus

2. Promiscuity of receptor coupling Such promiscuity is, in fact, relatively common in receptor systems and is particularly well illustrated in the large G protein-coupled receptor class (Kenakin, 1995; Milligan, 1997; Kostensis et al., 1998). Here, for example, single hormones or neurotransmitters (ligands) bind to different receptor subtypes, receptors for quite distinct ligand classes can couple to a single pool of G proteins, a single receptor may couple to more than one effector (for example, adenyl cyclase and phospholipase C) and, finally, the extent to which receptors and G proteins are expressed differentially in tissues may determine tissue- and cellspecific receptor coupling. An interesting example is provided by FSH which is secreted in multiple isoforms, dependent upon physiological status and presumably reflecting the glycosylation pattern of the FSH alpha- and beta-subunits. These isoforms appear to interact in different ways with the FSH receptor and to activate distinct signaling pathways through interaction with different G proteins - - G i / G o and G s (Arey et al., 1997). Thus, hFSH produced sigmoidal stimulation of cAMP accumulation, whereas the deglycosylated form of hFSH induced bellshaped cAMP accumulation with differential sensitivity to pertussis and cholera toxins. Determination of the mechanisms by which individual G proteins or subsets of G proteins differentially couple to G protein-coupled receptors is obviously of fundamental significance to any understanding of the selectivity of cell signaling processes. This fidelity of interaction depends critically upon the ability of the receptors to discriminate between the structural features of different GoL subunits. Multiple regions of this subunit may be involved in determining selective receptor coupling. In the GoLq and GoL~ subunits, the N-terminus carries a distinct and conserved six-amino acid extension - - MTLESI(M) and wher~ this is deleted these subunits couple to several different G s- and Gi/o-COupled receptors (Kostensis et al., 1998). Such systems may be thought of as constructed on a "mix-and-match" basis in which differential coupling of the several components of the receptor-coupler-effector system can occur. Such promiscuity of organization permits substantial of biological response and efficiency of information use.

The rabies virus has long been recognized and feared for its neurological effects and in 1981, Lentz et al. suggested that the virus gained entry to the central nervous system through initial interaction at the nicotinic acetylcholine receptors of the neuromuscular junction and subsequent transmission through axoplasmic flow (Lentz et al., 1982). This ability to interact with acetylcholine receptors may originate from sequence homology between the virus glycoprotein and snake oL-neurotoxins, an overlap that is most striking in the highly conserved regions (Lentz, 1985). 3.2. Herpes simplex virus

The herpes simplex virus Type I presents a widely distributed infective profile. HSV-1 enters cells through the fibroblast growth factor receptor (Kaner et al., 1990). There is no viral entry into cells that do not express these receptors and entry of the virus is inhibited by peptide receptor antagonists. 3.3. Salmonella typhi

Typhoid fever is caused by the entry of the pathogen S. typhi into gastrointestinal epithelial cells. This entry pro-

cess is apparently mediated through the cystic fibrosis transmembrane regulator, a chloride channel of the ATP cassette family of proteins, since cells that expressed the mutant nonfunctional 508phenylanine deletion did not take up as much pathogen as wild-type cells (Pier et al., 1998). Table 1 Some hijacked receptors Receptor

Virus/bacterium

ICAM n-AChR Integrin VLA-2 Complement receptor-2

Human rhinovirus Rabies virus Echovirus-1 Epstein-Barr virus Vaccinia virus

EGF receptor PAF receptor LDL receptor

Salmonella typhimurium S. pneumoniae

Duffy blood group antigen

Plasmodium vivax

Rous sarcoma virus (A) Human rhinovirus 2


Similarly, the homozygous expression of this mutant channel in mice resulted in an absence of uptake of S. typhi into the gastrointestinal mucosal cells, and heterozygous mice took up 86% less pathogen than did wild-type mice. Given the high prevalence of cystic fibrosis in the Caucasian population, it is tempting to speculate that it has persisted because it provides an evolutionary advantage in childhood namely a resistance to typhoid fever.

3.4. Mycobacterium leprae M. leprae is the causative agent of leprosy that invades the schwann cells of the peripheral nervous system where it causes irreversible damage to peripheral nerves. Despite its widely feared nature leprosy is a curable disease, but its infectivity has not been determined, oL-Dystroglycan is a peripheral membrane protein that interacts with laminin-2 in the basal lamina and is involved in a variety of cellular events including receptor clustering and morphogenesis and whose mis-expression gives rise to some muscular dystrophies. In Schwann cells, the complex of c~-dystroglycan and laminin-2 serves as the receptor for M. leprae entry into these cells (Rambukkana et al., 1998). 3.5. Streptococcus pneumoniae S. pneumoniae remains a major cause of pneumonia, but is remarkably widespread in quiescent and apparently non-infective form. Cellular infection occurs when inflammatory activation of cells causes surface cellular changes that permit bacterial entry. In particular, the PAF receptor is rapidly up-regulated upon cellular activation and this receptor serves as an entry point for the bacterium (Cundell et al., 1995). 3.6. The human immunodeficiency virus (HIV) The HIV presents a particularly interesting case since it requires dual receptor occupancy for its entry into immune cells. The resolution of this process carried out for the past decade is of profound scientific and clinical importance. The CD4 antigen is a glycoprotein expressed dominantly on the cell surface of T lymphocytes, but also to a reduced extent on macrophages. CD4 + T cells interact with targets bearing MHC class II molecules and it is the occupation of these T cell receptors by the MHC molecules bearing antigen fragments that is the activating stimulus. By the mid-1980s, several lines of evidence indicated directly and indirectly that these T cell receptors were also the receptors for HIV: mAbs directed against CD4 inhibited HIV activity in a variety of conditions and, of course, cells bearing CD4 receptors were depleted during the AIDS infection (reviewed in Sattentau and Weiss, 1988; Robey and Axel, 1990). It was, however, very rapidly found that the presence of the CD4 receptor alone was not sufficient

289

to permit HIV fusion and entry and that at least one other process or co-receptor must be involved. This co-receptor is one or more members of the chemokine receptor family themselves members of the 7-transmembrane G protein-coupled receptor family (reviewed in Bates, 1996; Cairns and D'Souza, 1998; Wells et al., 1998; Proudfoot et al., 1999). In particular, the macrophage-tropic lines of HIV-1 use the CCR5 receptor and the T-tropic HIV-1 lines use CXCR4 as well as CCR5 as major co-receptors for entry. Detailed structural analyses are now available that outline the mechanisms by which this dual receptor occupancy permits HIV-1 fusion and entry (Kwon et al., 1998; Moore and Binley, 1998). There are two important implications to the role of the chemokines as a co-receptor for HIV-1 infection. First, the G protein receptor family has already yielded many clinically available drugs and thus the opportunity exists for the development of ligands for the CXCR4 and CCR5 receptors that may serve as HIV antagonists. Some progress has already been made along these lines (Bright et al., 1998; Baba et al., 1999; Nishiyama et al., 1999). The second is that a mutation exists in the CCR5 receptor a 32-basepair deletion that results in a non-expressed truncated receptor (Liu et al., 1996; Paxton et al., 1996; Samson et al., 1996). Individuals bearing this mutation, almost exclusively of Caucasian origin, in homozygous form show considerable resistance to HIV-I infection. About 1% of the population is homozygous and approximately 15% of the population are heterozygous for this defect. Since individuals with this mutation appear to be of normal physiological health it is plausible that antagonists of this receptor will not exert major physiological effects.

4. Conclusion

"Hijacked" receptors present an interesting set of issues in receptor recognition and also opportunities for new directions in drug design. Molecules that affect virus and bacterial attachment and entry to cells may well constitute new small molecule approaches to infectious diseases sorely needed in today's world of increasing drug resistance in infectious diseases.

References

Arey, B.J., Stevis, P.E., Deecher, D.C., Shen, E.S., Frail, D.E., NegroVilar, A., Lopez, F.J., 1997. Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms. Mol. Endocrinol. 11, 517-526. Baba, M., Nishimura, O., Kanzaki, N., Okamoto, M., Sawada, H., Izawa, Y., Shiraishi, M., Aramaki, Y., Okonogi, K., Ogawa, Y., Meguro, K., Fujino, M., 1999. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc. Nat. Acad. Sci. U.S.A. 96, 5698-5703.


290

Bates, P., 1996. Chemokine receptors and HIV-1: an attractive pair?. Cell 86, 1-3. Bright, C., Brown, T.J., Halley, F., Lockey, P., McLay, I.M., Moore, U., Porter, B., Williams, R.J., 1998. Identification of a non-peptidic RANTES antagonist. Bioorg. Med. Chem. Lett. 8, 771-774. Cairns, J.S., D'Souza, M.P., 1998. Chemokines and HIV-1 second receptors: the therapeutic connection. Nature Med. 4, 563-568. Cundell, D.R., Gerard, N.P., Gerard, C., Idanpaan-Helkkla, I., Tuomanen, E.I., 1995. Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature 377, 435-438. Kaner, R.J., Baird, A., Mansukhani, A., Basilico, C., Summers, B.D., Florkiewicz, Hajjar, D.P., 1990. Fibroblast growth factor receptor factor receptor is a portal of cellular entry for herpes simplex virus type 1. Science 248, 1410-1413. Kenakin, T., 1995. Agonist-receptor efficacy: I. Mechanisms of efficacy and receptor promiscuity. Trends Pharmacol. Sci. 16, 188-192. Kostensis, E., Zeng, F.Y., Wess, J., 1998. Functional characterization of a series of mutant G protein alphaq subunits displaying promiscuous receptor coupling properties. J. Biol. Chem. 273, 17886-17892. Kwon, P.D., Wyatt, R., Robinson, J., Sweet, R.W., Sodroski, J., Hendrickson, W.A., 1998. Structure of an HIV gpl20 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648-659. Lentz, T.L., 1985. Rabies virus receptors. Trends Neurosci. 7, 360-364. Lentz, T.L., Burrage, T.G., Smith, A.L., Crick, J., Tignor, G.H., 1982. Is the acetylcholine receptor a rabies virus receptor?. Science 215, 182-184.

Liu, R., Paxton, W.A., Choe, S., Ceradini, D., Martin, S.R., Horuk, R., MacDonaldm, E., Stuhlmann, H., Koup, R.A., Landau, N.R., 1996. Homozygous defect in HIV-1 co-receptor accounts for resistance of some multiply exposed individuals to HIV-1 infection. Cell 86, 367-377. Milligan, G., 1997. Is promiscuity of G protein interaction an issue in the classification of receptors?. Ann. N. Y. Acad. Sci. 812, 126-132.

Moyle, W.R., Campbell, R.K., Myers, R.V., Bernard, M.P., Han, Y., Wang, R.K., 1994. Co-evolution of ligand-receptor pairs. Nature 368, 251-255. Nishiyama, Y., Murakami, T., Kurita, K., Yamamoto, N., 1999. Lowmolecular weight anti-HIV peptides for the amino-terminal sequence of RANTES: possible lead compounds for coreceptor-directed antiHIV agents. Bioorg. Med. Chem. Lett. 9, 1357-1360. Paxton, W.A., Martin, S.R., Tse, D., O'Brien, T.R., Skurnick, J., VanDevanter, N.L., Padian, N., Braun, J.F., Kotler, D.P., Wolinsky, S.M., Koup, R.A., 1996. Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposure. Nature Med. 2, 412-417. Pier, G.P., Grout, M., Zaidi, T., Meluleni, G., Mueschenborn, S.S., Banting, G., Ratcliff, R., Evans, M.J., Colledge, W.H., 1998. Salmonella typhi uses CFTR to enter intestinal epithelial cells. Nature 393, 79-92. Proudfoot, A.EI., Wells, T.N.C., Clapham, P.R., 1999. Chemokine receptors u future therapeutic targets for HIV?. Biochem. Pharmacol. 57, 451-463. Rambukkana, A., Yamada, H., Zanazzi, G., Mathus, G., Salzer, J.L., Yurchenco, P.D., Campbell, K.P., Fischetti, V.A., 1998. Role of a-dystroglycan as a Schwann cell receptor for Mycobacterium leprae. Science 282, 2076-2081. Robey, E., Axel, R., 1990. CD4: collaborator in immune recognition and HIV infection. Cell 60, 697-700. Samson, M., Libert, F., Doranz, B.J., Rucker, J., Liesnard, C., Farber, C.-M., Saragosti, S., Lapoumerouille, C., Cognaux, J. et al., 1996. resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR5 chemokine receptor gene. Nature 382, 722-725. Sattentau, Q.J., Weiss, R.A., 1988. The CD4 antigen: physiological ligand and HIV receptor. Cell 52, 631-633. Wells, T.N.C., Proudhoot, A.EI., Power, C.A., Marsh, M., 1998. Chemokine receptors the new frontier for AIDS research. Chem. Biol. 3, 603-609.



ICAM-1 receptors and cold viruses Jordi Bella ~'*, Michael G. Rossmann

b

a School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK b Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA

Abstract Human rhinoviruses (HRVs), the single most important etiologic agent of common colds, are small viruses composed of an icosahedral protein shell that encapsidates a single, positive RNA strand. Multiplication of HRVs occurs in the cytoplasm of the host cell. To produce infection, HRVs must first attach to specific cellular receptors embedded in the plasma membrane. Ninety percent of HRVs immunogenic variants use as receptor intercellular adhesion molecule-1 (ICAM-1), a cell surface glycoprotein that promotes intercellular signaling in processes derived from inflammation response. As HRV receptor, ICAM-1 positions the virus to within striking distance of the membrane, and then triggers a conformational change in the virus that ultimately results in delivery of the viral RNA genome into the cytoplasm, across a lipid bilayer. The interaction between ICAM-1 and HRVs has been analyzed by the combination of crystal structures of HRVs and ICAM-1 fragments with electron microscopy reconstructions of the complexes. The resulting molecular models are useful to address questions about receptor recognition, binding specificity, and mechanisms by which ICAM-1 induces virus uncoating. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: Rhinovirus; ICAM-1; Virus-receptor; Electron microscopy

More than 200 different viruses are known to cause common colds, infections usually mild in nature that exhibit such symptoms as sneezing, nasal congestion, and decreased energy level. Human rhinoviruses (HRVs), are the most important etiologic agents for common colds (Rueckert, 1996), accounting for 30% to 50% of all adult colds and 10% to 25% of colds in children. Other coldproducing viruses are coronaviruses, adenoviruses, coxsackieviruses, echoviruses, orthomyxoviruses, paramyxoviruses, respiratory syncytial virus, and enteroviruses. Each type of virus produces infections with slightly different patterns of symptoms and severity, and some are also responsible for other, more severe illnesses. Rhinoviruses belong to the p i c o r n a v i r i d a e family: small icosahedral viruses with an average diameter of 300 A and a molecular mass of approximately 8.5 • 10 6 Da. Like all picornaviruses, HRVs are made of a protein capsid that encases a single-stranded, positive-sense RNA molecule of about 7000 bases. The capsid is built from 60 copies of viral proteins 1, 2, 3 and 4 (VP1, VP2, VP3, and VP4). VP1, VP2 and VP3 assemble on the exterior to form the protein shell, and VP4 resides in the interior of the capsid,

* Corresponding author. Telefax: j [email protected]

+44-161-275-5467;

e-mail:

in contact with the viral RNA. About a hundred different HRV serotypes have been identified, each characterized by its own specific antigens. Three-dimensional structures of several HRV capsids have been determined to atomic detail by using X-ray crystallography (Rossmann et al., 1985; Kim et al., 1989; Oliveira et al., 1993; Zhao et al., 1996). These structures show the precise arrangement of the different viral proteins in the capsid. In all of them, a 20-.~ deep depression, or " c a n y o n " , encircles each five-fold vertex.

1. Rhinovirus receptors and the canyon hypothesis Most viruses initiate infection of susceptible cells by first attaching to specific cell surface receptors. Receptors have been identified for many animal, insect, and bacterial viruses, and only plant viruses seem to pose an exception. In animal viruses, the receptor determines, in part, host range and tissue tropism. Viruses are able to subvert a wide variety of cell-surface molecules as receptors. Some viruses are able to recognize very specific proteins, whereas others use sugar moieties or common chemical groups for attachment. Many viral receptors act simply as molecular tethers that concentrate virus particles on the cell surface


292

J.

Bella, M.G. Rossmann / Pharmaceutica Acta Helvetiae 74 (2000) 291-297

and direct them towards disassembly pathways. For some viruses, however, the cell receptor plays an active role in the uncoating of the viral genome, that is the translocation of the RNA from the interior of the capsid to the host cell cytoplasm. Not all the 102 characterized rhinovirus serotypes share the same receptor. Ninety percent, the major group, utilize the cell surface glycoprotein intercellular adhesion molecule-1 (ICAM-1) as receptor (Greve et al., 1989; Staunton et al., 1989). A minor group of about 10 HRV serotypes uses members of the low-density lipoprotein receptor (LDLR) family for cell binding (Hofer et al., 1994; Marlovits et al., 1998), and one individual serotype, HRV87, binds to a still uncharacterized receptor (Uncapher et al., 1991). ICAM-1 is also shared as receptor by several members of the Coxsackievirus-A family, another group of picornaviruses causing upper respiratory infections (Colonno et al., 1986; Shafren et al., 1997). Amino acid residues at the bottom of the canyon are more conserved among HRVs than those that are exposed elsewhere on the viral surface (Rossmann and Palmenberg, 1988; Chapman, 1993). Hypervariable surface residues coincide with the binding sites of neutralizing monoclonal antibodies, and mutations at these sites allow the virus to escape the neutralization effect. On the basis of these residue conservation patterns, the canyon was proposed as the receptor binding site (Rossmann et al., 1985; Rossmann, 1989). It was also suggested that HRVs could hide their receptor binding site inside canyons or depressions, inaccessible to bulkier antibodies, while mutating the external residues outside the canyon to elude the host's immune surveillance. These postulates form the basis of what became known as the canyon hypothesis (Rossmann, 1989). Confirmation of the canyon as receptor binding site was provided by mutational analysis of specific residues in the canyon (Colonno et al., 1988), and by the cryo-electron microscopy (cryo-EM) reconstruction of a complex of HRV 16 with a two-domain fragment of ICAM-1 (Olson et al., 1993).

2. I n t e r c e l l u l a r a d h e s i o n m o l e c u l e - 1

ICAM-1 is a transmembrane glycoprotein with an extracellular part composed of several immunoglobulin (Ig) domains arranged end-to-end. Ig domains are characteristic building blocks of antibodies. The structure of an Ig domain can be described as two antiparallel [3-sheets packed tightly against each other, connected through several loops following a well-defined topology, and linked by one or two disulfide bridges (Harpaz and Chothia, 1994). ICAM-1 has five Ig domains (Fig. 1), a transmembrane region, and a short cytoplasmic domain. Other members of the ICAM family are ICAM-2, ICAM-3, ICAM-4 and ICAM-5. All ICAMs show the same domain organization, sequence homology between domains, and are func-

Coxsackie A? PFIE

HRV LFA-1 (HIV-1)

N S$

Jl D1

SS

Fibrinogen

S

J D3

S

284-------t I . . . ,

Mac-1

385 453

~ D5

lillllLllllllI iiillllLllllllllll ltlllll ]lllllllltl]i

Illlllllllllltllltll

477

c~-Actinin

C

505

Fig. 1. Domain structure of ICAM-1. Each Ig domain is represented schematically by a circle closed by one or two disulfide bonds. Amino acid numbers indicate the beginning and end of each domain. Approximate locations of relevant binding sites are shown. Lollipop-shaped structures indicate N-linked glycosylationsites. tionally and structurally related. ICAMs differ in the number of Ig domains, cell type, and expression regulation. ICAM-1 promotes intercellular signaling, mainly in processes derived from response to inflammation. It permits antigen-independent adhesion between lymphocytes and their targets, and has a major role in leukocyte transmigration from the blood to the tissues in inflammatory sites (van de Stolpe and van der Saag, 1996). Two membrane-bound integrin receptors, leukocyte function-associated antigen (LFA- 1), and macrophage- 1 antigen (Mac- 1) are the normal ligands for ICAM-1. Binding sites for these receptors have been identified by mutagenesis techniques and competition experiments in domains D1 and D3, respectively (Fig. 1). ICAM-1 also serves as a receptor for soluble fibrinogen, its binding site still controversial, as well as for the extracellular matrix factor hyaluronan (van de Stolpe and van der Saag, 1996). These interactions probably mediate leukocyte adhesion to vascular endothelium. The cytoplasmic domain of ICAM-1 interacts with the cytoskeleton-binding protein oL-actinin (Carp6n et al., 1992), determining cell surface distribution of ICAM-1 and recruitment to points of interaction with other cells.

293

J. Bella, M.G. Rossmann / Pharmaceutica Acta Helvetiae 74 (2000) 291-297

3. ICAM-1 as pathogen receptor

Rather uniquely, ICAM-1 is also subverted as receptor by human pathogens in at least three different ways. Major group rhinoviruses and A-type coxsackieviruses use ICAM-1 to release their RNA into the host cell cytoplasm. Erythrocytes infected by the malarial parasite Plasmodium falciparum, are able to bind ICAM-1 in the surface of endothelial cells (Berendt et al., 1992; Ockenhouse et al., 1992), and use this cytoadherence to sequester themselves in deep vascular beds, including the brain, minimizing exposure of the parasite to immune surveillance. Finally, human immunodeficiency virus- 1 (HIV- 1), uses ICAM- 1 as a coreceptor (Bastiani et al., 1997; Fortin et al., 1997; Rizzuto and Sodroski, 1997). HIV-1 acquires several host cell membrane proteins when it buds from infected cells, making it possible for ICAM-1 to be incorporated into the envelope of the virions. This results in an increase of subsequent virus-cell interactions, enhancement of virus infectivity, and extension of the host cell range. Mutational studies have identified the binding sites for HRVs and PFIE at the tip of the first domain, the one that is the most distal from the membrane, next to the LFA-1 binding site (Staunton et al., 1990; McClelland et al., 1991; Register et al., 1991). Binding sites for HRV and PFIE overlap partially. Binding of coxsackievirus A to ICAM-1 is likely to be similar to that of HRVs, but may involve in some cases a second receptor (Shafren et al., 1997). Many cell surface proteins use tandems of Ig domains to build their extracellular region. Together with the ICAMs, they constitute the Ig superfamily. Other members of the Ig-superfamily are subverted as receptors by HIV-1, poliovirus, coxsackievirus B, or murine encephalomyocarditis virus. Interestingly, no pathogen has been reported to bind specifically to ICAMs other than ICAM-1.

4. Three-dimensional structures of ICAM-1 fragments

Intact ICAM-1 is not amenable for structural studies. Instead, most efforts have been directed to the structural determination of soluble fragments. The structure of the two amino-terminal domains of ICAM-1 (D1D2) has been determined recently by X-ray crystallography (Bella et al., 1998; Casasnovas et al., 1998). As expected, each domain adopts an Ig fold. Binding sites on domain D1 can be mapped on the crystal structure (Fig. 2). Residues identified by mutational analysis as important for HRV binding belong to the three loops DE, BC, and FG, located at the very tip of domain D1. The interaction between ICAM-1 and LFA-1 probably requires a broad surface centered around Glu34, a residue crucial for binding located in strand C. By analogy, domain D1 could be described as a hand with three stretched fingers and a folded thumb. The tips of the three fingers (loops DE, BC, and FG) would be

FG

BC

D1

N175 Nl18

D2

C

l~

G

NI~

Fig. 2. Ribbon diagram of the first two domains of ICAM-1. Strands are labeled A, B, C, etc., following the convention for Ig domain nomenclature (Harpaz and Chothia, 1994). Loops are named by the strands they connect. Only functionally relevant loops mentioned in the text are labeled. Glycan models show the position of the four glycosylation sites in domain D2, linked to four asparagine residues. Only the first one or two sugars in each glycan can be identified in ICAM-1 D1D2 crystal structures (Bella et al., 1998; Casasnovas et al., 1998).

responsible for recognition and binding to the major group of rhinoviruses, whereas the folded thumb would be the LFA-1 recognition site. Residues important for binding ICAM-1 to malarial-infected erythrocytes map to strand B and loop CD, defining another interacting surface that differs in location from both rhinovirus and LFA-1 binding sites. The major group of HRVs does not recognize ICAM-1 from species other than human, with the probable exception of chimpanzees, neither the homologous molecules ICAM-2 or ICAM-3. The structure of the two extracellular domains of ICAM-2 has also been determined by X-ray crystallography (Casasnovas et al., 1997). ICAM-2 D1 superimposes on ICAM-1 D 1 quite well, except for significant differences in conformation located in the three loops DE, BC, and FG (Bella et al., 1998). Comparison of ICAM amino acid sequences at these loops shows major differences in the disposition of proline and charged residues, suggesting a crucial role for the three loops, the

294


"fingers" of the hand, in recognition and binding between ICAM-1 and the major group of rhinoviruses.

5. ICAM-I and the uncoating of viral RNA Attachment of major group HRVs to ICAM-1 initiates entry in the host cell and translocation of the viral RNA across the cellular membrane (Rueckert, 1996). During this uncoating process, conformational changes take place in the viral capsid. HRVs undergo several progressive transformations when bound to cells. Infectious virions are thought to uncoat through intermediate particles characterized by the loss of VP4 and the externalization of the hydrophobic N-termini of VP1, which in turns makes the capsid hydrophobic. Intermediate particles further evolve into empty particles by release of RNA. Empty particles and most intermediate particles are noninfective, abortive products, and only a small fraction of intermediate particles attaches to the membrane and delivers the viral RNA into the cytoplasm (Rueckert, 1996). Soluble ICAM-1 fragments have been used to reproduce in vitro some of the events described above (Greve et al., 1991; Hoover-Litty and Greve, 1993). Mixing HRVs with ICAM-1 soluble fragments produces complexes that reversibly dissociate at low temperature. Increase in temperature or receptor concentration converts these complexes into subparticles analogous to the ones seen in cell binding studies. For instance, HRV14, HRV3, and HRV16 are all converted to empty capsids when incubated with soluble ICAM-1, although the rate of uncoating depends strongly on temperature and serotype (Hoover-Litty and Greve, 1993).

6. Studying the ICAM-1/rhinovirus interaction by a combination of cryo-EM and X-ray crystallography Reversible complexes between HRVs and soluble fragments of ICAM-1 make possible the study of their interaction by structural techniques. No atomic resolution structure of a complex between virus and receptor is available yet, but low-resolution EM reconstructions of complexes of D1D2 fragments of ICAM-1 with HRV14 or HRV16 demonstrate that, indeed, the two-domain ICAM-1 fragment binds into the central part of the canyon (Fig. 3). ICAM-1 fragments have an approximate dumbbell shape in these reconstructions, and are oriented roughly perpendicular to the viral surface, extending to a radius of about 205 A. Three-dimensional structures for HRV 14, HRV 16, and ICAM-1 D1D2 are available, and they can be combined to fit molecular models into the EM reconstructions (Kolatkar et al., 1999). Given the elongated shape of the ICAM-1 D1D2 model, ambiguity exists in its correct orientation around the major D1D2 axis. That problem can be solved by comparing EM

Fig. 3. Cryo-EM reconstruction of a complex between HRV16 and a low-glycosylation form of ICAM-1 D1D2 (Bella et al., 1998). There are 60 copies of the receptor fragment, visible as radial projections on the viral surface.

reconstructions from complexes of HRV16 with ICAM-1 D1D2 fragments with different degrees of glycosylation. The second domain of ICAM-1 has four N-linked carbohydrates (Fig. 1), three of which have been removed in a genetically engineered version of ICAM-1 D1D2 with reduced glycosylation (Bella et al., 1998). The reconstruction of the complex between HRV16 and this modified ICAM-1 D1D2 (Fig. 3), can be subtracted from the reconstruction between HRV16 and intact, fully glycosylated ICAM-1 D1D2 (Olson et al., 1993). The resulting difference map identifies the positions of the glycosylation sites as "lumps" on the surface of the density corresponding to the D2 domain, and allows to remove the orientation ambiguity (Fig. 4). Carbohydrates are intrinsically flexible molecules and can adopt a myriad of different conformations. This inherent disorder results in only the first two or three sugars from each glycosylation site being visible in crystal structures of glycoproteins, including ICAM-1 D1D2. Analogously, the lumps observed in the EM reconstructions only cover these few sugars that are closer to the protein, and therefore more ordered. Glycosylated models of ICAM-1 D1D2 have been fit into the EM reconstructions, and the fitting refined to maximize the agreement between the density map and the atomic models (Kolatkar et al., 1999). In these models, the three loops BC, DE, and FG penetrate deep into the canyon and the short CD loop of ICAM-1 lies against VP2 of HRV 14 or HRV 16, on the so-called "south" rim of the canyon (in an analogy to the Grand Canyon and the usual representation of the rhinovirus structure, where the fivefold axis is at the top or "north"). Analysis of the charge distribution on the interacting surfaces in both complexes


Fig. 4. Fitting of an atomic model for ICAM-1 D1D2 into the cryo-EM reconstruction of its complex with HRV14. Protein and carbohydrate are represented as C~ and C 1-C 4 tracings, respectively.

shows a remarkable complementarity. The same pattern of electrostatic interactions occurs both for HRV14 and HRV16, in spite of lack of conservation of some key residues. Thus, complementarity is not necessarily maintained as a result of residue conservation but rather by compensating changes of amino acids between serotypes.

295

of the receptor attached to the virus plus a conformational change on the virus surface, resulting in a tighter, higher affinity binding between ICAM-1 and HRVs. This second step could result from conformational changes in VP1 in which the region around the hydrophobic pocket would act as a hinge (Fig. 5). Such movement would induce a destabilization in the virus capsid and perhaps the opening of a channel in the five-fold vertex through which externalization of VP4, the N-terminus of VP1, and eventually RNA could occur (Kolatkar et al., 1999). In this way, interaction between ICAM-1 and HRVs would initiate uncoating of the viral RNA. In this hypothetical mechanism, the first step would be insensitive to the contents in the hydrophobic pocket. The presence of an antiviral compound in the pocket would impart rigidity to the hinge region and prevent the second step to occur. As a consequence uncoating would be inhibited. Conversely, empty pockets would impart conformational flexibility to the hinge region, and ultimately allow movement of polypeptide chains during the uncoating process. Crystal structures of HRVs show uncharacterized pocket molecules, probably lipidic in nature, filling the hydrophobic pocket in a manner similar to the antiviral compounds in HRV14 (Smith et al., 1986; Kim et al., 1989; Oliveira et al., 1993). It has been suggested that pocket-binding antiviral drugs displace such "pocket factors", and that competition between pocket factors and receptors regulates the viral stability (Rossmann, 1994). For example, ICAM-1 would be able to eject a weakly bound pocket molecule during the second step of the mechanism shown in Fig. 5, thus emptying the hydropho-

7. Possible uncoating mechanism The mechanisms by which ICAM-1 binding to HRV triggers virus destabilization and uncoating are not understood. A hydrophobic pocket inside VP1 lies directly beneath the canyon floor. This pocket was shown to be the binding site for certain antiviral compounds that inhibit the replication of HRVs and related picornaviruses (Fox et al., 1986; Smith et al., 1986). Experimental evidence has shown that antiviral drugs bound to this hydrophobic pocket " l o c k " the viral particle in a state in which neither VP4 nor the N-terminus of VP1 can be externalized (Lewis et al., 1998), thus preventing uncoating. The footprint of ICAM-1 on the surface of HRV14 or HRV16 does not include the area beneath which the hydrophobic pocket is located, but it is immediately contiguous to it. Nevertheless, there is experimental evidence that the presence of antiviral compounds in the hydrophobic pocket affects cell binding for many of the major group HRVs. A two-step mechanism for the binding of ICAM-1 to HRVs can reconcile these conflicting observations. In a first step, complexes between HRVs and ICAM-1 form in a reversible manner, as they can be observed in cryo-EM reconstructions. The second step involves a displacement

ICAM-1

VP1 N-term VP4 IP' RNA ICAM-1

VP3 p-cylinder

RNA Fig. 5. Scheme of a two-step binding mechanism between ICAM-1 and major group HRVs. The first step, on the left, is observed in the cryo-EM reconstructions of HRV-ICAM-1 fragments. On the right, the second (hypothesized) step involves a conformationalchange in the virus surface. The five-fold channel may open as both walls and floor of the canyon bind to domain D1 of ICAM-1. The pocket region is postulated as the hinge area, and needs to be empty in order to provide conformational flexibility.

296


bic pocket and initiating uncoating. In contrast, a tightly bound antiviral compound would not be removed.

8. Summary The study of the interaction between major group HRVs and their receptor, ICAM-1, suggests an hypothesis for how ICAM-1 might initiate uncoating of the viral RNA. Initial contact between the virus and receptor occurs mostly through capsid residues in the canyon and four loops at the tip of domain D1 of ICAM-1, at the end distal from the membrane. This interaction appears to have an important electrostatic component. High-affinity binding probably requires a second step in which other areas of the viral surface and the receptor may come into contact. This second step probably weakens the interactions between the capsid protomers and triggers the uncoating mechanism, perhaps through channels opened in the icosahedral fivefold axes. Capsid-binding antiviral compounds can effectively block the uncoating process by imparting rigidity to the viral capsid and preventing the high affinity binding step between ICAM-1 and HRVs. Many details need still to be addressed, and parallels sought with other picornavirus-receptor interactions. Lacking crystallographic resolution images of the virus-receptor complexes, cryoEM reconstructions assisted with fitting of molecular models of the individual components will prove very valuable to demonstrate precisely how the virus and receptor interact, and to provide clues about the sequence of events that goes from initial cell surface attachment to release of the RNA into the host cell cytoplasm.

References Bastiani, L., Laal, S., Kim, M., Zolla-Pazner, S., 1997. Host cell-dependent alterations in envelope components of human immunodeficiency virus type 1 virions. J. Virol. 71, 3444-3450. Bella, J., Kolatkar, P.R., Marlor, C.W., Greve, J.M., Rossmann, M.G., 1998. The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand. Proc. Natl. Acad. Sci. U. S. A. 95, 4140-4145. Berendt, A.R., McDowall, A., Craig, A.G., Bates, P.A., Sternberg, M.J.E., Marsh, K., Newbold, C.I., Hogg, N., 1992. The binding site on ICAM-1 for Plasmodium falciparum-infected erythrocytes overlaps, but is distinct from, the LFA-1-binding site. Cell 68, 71-81. Carp6n, O., Pallai, P., Staunton, D.E., Springer, T.A., 1992. Association of intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskeleton and oL-actinin. J. Cell Biol. 118, 1223-1234. Casasnovas, J.M., Springer, T.A., Liu, J.-H., Harrison, S.C., Wang, J.-H., 1997. Crystal structure of ICAM-2 reveals a distinctive integrin recognition surface. Nature 387, 312-315. Casasnovas, J.M., Stehle, T., Liu, J.H., Wang, J.H., Springer, T.A., 1998. A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1. Proc. Natl. Acad. Sci. U. S. A. 95, 4134-4139. Chapman, M.S., 1993. Comparison of surface properties of picornaviruses: strategies for hiding the receptor site from immune surveillance. Virology 195, 745-756. Colonno, R.J., Callahan, P.L., Long, W.J., 1986. Isolation of a m o n o -

clonal antibody that blocks attachment of the major group of human rhinoviruses. J. Virol. 57, 7-12. Colonno, R.J., Condra, J.H., Mizutani, S., Callahan, P.L., Davies, M.-E., Murcko, M.A., 1988. Evidence for the direct involvement of the rhinovirus canyon in receptor binding. Proc. Natl. Acad. Sci. U. S. A. 85, 5449-5453. Fortin, J.F., Cantin, R., Lamontagne, G., Tremblay, M., 1997. Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity. J. Virol. 71, 3588-3596. Fox, M.P., Otto, M.J., Mc Kinlay, M.A., 1986. The prevention of rhinovirus and poliovirus uncoating by WIN 51711, a new antiviral drug. Antimicrob. Agents Chemother. 30, 110-116. Greve, J.M., Davis, G., Meyer, A.M., Forte, C.P., Yost, S.C., Marlor, C.W., Kamarck, M.E., McClelland, A., 1989. The major human rhinovirus receptor is ICAM-1. Cell 56, 839-847. Greve, J.M., Forte, C.P., Marlor, C.W., Meyer, A.M., Hoover-Litty, H., Wunderlich, D., McClelland, A., 1991. Mechanisms of receptor-mediated rhinovirus neutralization defined by two soluble forms of ICAM- 1. J. Virol. 65, 6015-6023. Harpaz, Y., Chothia, C., 1994. Many of the immunoglobulin superfamily domains in cell adhesion molecules and surface receptors belong to a new structural set which is close to that containing variable domains. J. Mol. Biol. 238, 528-539. Hofer, F., Gruenberger, M., Kowalski, H., Machat, H., Huettinger, M., Kuechler, E., Blaas, D., 1994. Members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus. Proc. Natl. Acad. Sci. U. S. A. 91, 1839-1842. Hoover-Litty, H., Greve, J.M., 1993. Formation of rhinovirus-soluble ICAM-1 complexes and conformational changes in the virion. J. Virol. 67, 390-397. Kim, S., Smith, T.J., Chapman, M.S., Rossmann, M.G., Pevear, D.C., Dutko, F.J., Felock, P.J., Diana, G.D., McKinlay, M.A., 1989. Crystal structure of human rhinovirus serotype 1A (HRV1A). J. Mol. Biol. 210, 91-111. Kolatkar, P.R., Bella, J., Olson, N.H., Betor, C.M., Baker, T.S., Rossmann, M.G., 1999. Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor. EMBO J. 18, 6249-6259. Lewis, J.K., Bothner, B., Smith, T.J., Siuzdak, G., 1998. Antiviral agent blocks breathing of the common cold virus. Proc. Natl. Acad. Sci. U. S. A. 95, 6774-6778. Marlovits, T.C., Abrahamsberg, C., Blaas, D., 1998. Very-low-density lipoprotein receptor fragment shed from HeLa cells inhibits human rhinovirus infection. J. Virol. 72, 10246-10250. McClelland, A., deBear, J., Yost, S.C., Meyer, A.M., Marlor, C.W., Greve, J.M., 1991. Identification of monoclonal antibody epitopes and critical residues for rhinovirus binding in domain 1 of intercellular adhesion molecule 1. Proc. Natl. Acad. Sci. U. S. A. 88, 7993-7997. Ockenhouse, C.F., Betageri, R., Springer, T.A., Staunton, D.E., 1992. Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-l, and human rhinovirus. Cell 68, 63-69. Oliveira, M.A., Zhao, R., Lee, W.-M., Kremer, M.J., Minor, I., Rueckert, R.R., Diana, G.D., Pevear, D.C., Dutko, F.J., McKinlay, M.A., Rossmann, M.G., 1993. The structure of human rhinovirus 16. Structure 1, 51-68. Olson, N.H., Kolatkar, P.R., Oliveira, M.A., Cheng, R.H., Greve, J.M., McClelland, A., Baker, T.S., Rossmann, M.G., 1993. Structure of a human rhinovirus complexed with its receptor molecule. Proc. Natl. Acad. Sci. U. S. A. 90, 507-511. Register, R.B., Uncapher, C.R., Naylor, A.M., Lineberger, D.W., Colonno, R.J., 1991. Human-murine chimeras of ICAM-1 identify amino acid residues critical for rhinovirus and antibody binding. J. Virol. 65, 6589-6596. Rizzuto, C.D., Sodroski, J.G., 1997. Contribution of virion ICAM-1 to human immunodeficiency virus infectivity and sensitivity to neutralization. J. Virol. 71, 4847-4851.


Rossmann, M.G., 1989. The canyon hypothesis. Hiding the host cell receptor attachment site on a viral surface from immune surveillance. J. Biol. Chem. 264, 14587-14590. Rossmann, M.G., 1994. Viral cell recognition and entry. Protein Sci. 3, 1712-1725. Rossmann, M.G., Palmenberg, A.C., 1988. Conservation of the putative receptor attachment site in picornaviruses. Virology 164, 373-382. Rossmann, M.G., Arnold, E., Erickson, J.W., Frankenberger, E.A., Griffith, J.P., Hecht, H.-J., Johnson, J.E., Kamer, G., Luo, M., Mosser, A.G., Rueckert, R.R., Sherry, B., Vriend, G., 1985. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 317, 145-153. Rueckert, R.R., 1996. Picornaviridae: the viruses and their replication. In: Fields, B.N., Knipe, D.M., Howley, P.M., et al. (Eds.), Fields Virology. Lippincott-Raven Publishers, Philadelphia, 1,609-654. Shafren, D.R., Dorahy, D.J., Greive, S.J., Burns, G.F., Barry, R.D., 1997. Mouse cells expressing human intercellular adhesion molecule-1 are susceptible to infection by coxsackievirus A21. J. Virol. 71,785-789.

297

Smith, T.J., Kremer, M.J., Luo, M., Vriend, G., Arnold, E., Kamer, G., Rossmann, M.G., McKinlay, M.A., Diana, G.D., Otto, M.J., 1986. The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating. Science 233, 1286-1293. Staunton, D.E., Merluzzi, V.J., Rothlein, R., Barton, R., Marlin, S.D., Springer, T.A., 1989. A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses. Cell 56, 849-853. Staunton, D.E., Dustin, M.L., Erickson, H.P., Springer, T.A., 1990. The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-I and rhinovirus. Cell 61,243-254. Uncapher, C.R., DeWitt, C.M., Colonno, R.J., 1991. The major and minor group receptor families contain all but one human rhinovirus serotype. Virology 180, 814-817. van de Stolpe, A., van der Saag, P.T., 1996. Intercellular adhesion molecule-1. J. Mol. Med. 74, 13-33. Zhao, R., Pevear, D.C., Kremer, M.J., Giranda, V.L., Kofron, J.A., Kuhn, R.J., Rossmann, M.G., 1996. Human rhinovirus 3 at 3.0 A resolution. Structure 4, 1205-1220.




Viral-encoded G-protein coupled receptors" new targets for drug research? Martine J. Smit *, Henk Timmerman, Dennis Verzijl, Rob Leurs Leiden / Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Vrije Universiteit, Amsterdam, Netherlands

Keywords." Viral-encoded G-protein coupled receptor; Herpes virus; CMV; KSHV; GPCR

1. G-protein coupled receptors and oncogenic signalling The large multigene family of G-protein coupled receptors (GPCRs) plays a fundamental role in cellular communication. GPCRs couple extracellular signals as diverse as light, odors, hormones and neurotransmitters to distinct intracellular signals and are estimated to be the target of +__60% of the currently available drugs. Upon activation by agonists, GPCRs transfer the signal to the family of hetero-trimeric G proteins, which in turn can either stimulate or inhibit the activity of a wide variety of cellular effectors via the release of the a and [3~/-subunits (Bourne, 1997). It has become clear that GPCRs are involved in proliferative signalling through activation of the MAP kinase pathways (van Biesen et al., 1996; Gutkind, 1998). There is also accumulating evidence that they can harbor oncogenic potential. Various GPCRs, including 5HT~c, C~B and muscarinic m l, 3 and 5 receptors have been found to induce oncogenic transformation in an agonist-dependent manner (see for references Gutkind, 1998). Interestingly, some GPCRs display spontaneous activity (constitutive activity) in the absence of neurotransmitter or hormone stimulation thereby activating intracellular signalling pathways (Leurs et al., 1998). Expression of constitutively active GPCRs (e.g., CAM oL~B, 5HTzc) can induce oncogenic transformation in vitro (Allen et al., 199 l; Westphal and Sanders-Bush, 1996) and tumor formation in nude mice (Allen et al., 1991), indicating the potential hazard of

* Corresponding author. Tel.: +31-20-4447572;fax: +31-20-4447610; e-mail: [email protected]

constitutively active GPCRs. A variety of human diseases, including some proliferative disorders, have indeed been ascribed to the constitutive activity of specific GPCRs due to naturally occurring mutations (Spiegel, 1996).

2. Viral-encoded GPCRs Recently, it has been suggested that viral infections play an important role in cancer or other chronic diseases (Danesh et al., 1997). Evidence for their involvement comes partly from epidemiological studies, focusing on the detection of viruses in (cancer)patients. Herpesviruses, papillomaviruses and hepadnaviruses (Hepatitis B) are all associated with transformation of cells and, along with other factors, initiate oncogenesis (Table 1). CMV has been reported to play a role in artherosclerosis (Persoons et al., 1994). Various viruses encode proteins with sequence homology to host proteins which are known to be involved in host defense functions. Viruses pirate and modify key immunoregulatory molecules, by use of molecular mimicry, to elude the immune system (Murphy, 1997). Viruses also encode proteins that exploit or alter their host cells, replicate or induce migration for virus dissemination. Interestingly, DNA viruses such as the Herpesviruses (Cytomegalovirus (CMV), human herpesvirus (HHV-6 and 7), herpesvirus Saimiri (HVS) and Kaposi's sarcoma-associated Herpesvirus (KSHV)) all express GPCRs (Table 2). The viral-encoded GPCRs show highest homology to the family of chemokine receptors. The members of this family can be divided in three subclasses based on their ability to bind to different classes of chemokines. One


300

M.J. Smit et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 299-304

Table 1 Viruses implicated in human cancers Virus family

Tumor types

Associated human cancer

Herpesviruses

lymphomas, carcinomas, sarcomas

Pappiloma virus Retroviruses Hepatitis B virus

pappiloma and carcinomas hematopoietic cancers, sarcomas, carcinomas, leukemias Hepatocellular carcinoma

Kaposi sarcoma(HHV-8) Body cavity lymphoma(HHV-8) Multiple myeloma(HHV-8) Burkitt's lymphoma(EBV) Nasopharyngeal carcinoma(EBV) gastric cancer pappilomas, cervical carcinoma Adult-T-cell leukemia Hepatocellular carcinoma

distinguishes the CC, CXC, C and CX3C chemokines based on the arrangement of the first two of four conserved cysteine residues. The chemokine receptors respond to chemokines that are released at sites of inflammation and play a major role in the regulation and trafficking of immune cells (Horuk, 1994). They are also involved in angiogenesis and act as crucial cellular entry factors for the HIV virus (Fauci, 1996; Baggiolini, 1998). The function of the viral-encoded GPCRs is currently not clear and little is known about the signalling pathways activated by these viral GPCRs.

3. Viral GPCR encoded by KSHV KSHV, also termed human herpesvirus 8 (HHV-8), is a ~/herpesvirus that is implicated as a transforming agent in the pathogenesis of Kaposi's sarcoma (KS) and other proliferative disorders such as primary effusion B-cell lymphomas (Ganem, 1997). KS is the most common malignancy in HIV-infected individuals, but has also been detected in non HIV-infected individuals. KS is a highly angiogenic multicentric tumor associated with abundant

Table 2 Viral-encoded GPCRs Virus KSHV/HHV-8 HCMV MCMV RCMV HHV-6 HHV-7 HVS EHV-2 EBV RRV Swine pox virus capri-pox virus

CXCR

vasculature and spindle cell proliferation. It is believed that spindle cells are the driving force of KS pathogenesis (Ganem, 1997). However, it is unknown what exactly initiates and sustains spindle cell growth. The KSHV genome displays an unusual degree of genetic piracy compared to other herpesviruses. Many of its viral genes encode proteins whose cellular homologues are known to be involved in growth control, signal transduction and other regulatory processes. We are in particularly, interested in the protein encoded by open reading frame 74, named ORF74 or KSHV GPCR, which is a chemokine receptor homologue. KSHV GPCR shows most resemblance to the chemokine receptor CXCR2 (IL-8 receptor) but binds both CC and CXC chemokines, in contrast to CXCR2 and other chemokine receptors. Only the Duffy antigen receptor, a non-signalling receptor, is known to bind both CC and CXC chemokines (Horuk et al., 1996). Remarkably, the KSHV GPCR stimulates signalling pathways linked to cell proliferation in a constitutively active (agonist-independent) manner (Arvanitakis et al., 1997). The KSHV GPCR couples to the family of Gtx q proteins and not to GoL15 or GoL16, that are commonly used

CCR

UL33

other

US27/US28

UL33 M33 R33 U12 U12

UL78 M78

ORF74/KSHV GPCR

ORF74/ECRF3 ORF74

U51 (induces CCR7) U51 (induces CCR7) E6 induces CCR7

ORF74 K2R Q2/3L

M.J. Smit et al. / Pharmaceutica Acta Helvetiae 74 (2000) 299-304

by other chemokine receptors. Hence, this may allow the receptor to signal in a broader range of cell types. The KSHV GPCR can activate J N K / S A P K and p38MAPK signalling pathways (Bais et al., 1998). Moreover, KSHV GPCR signalling induces cellular transformation, tumorigenicity and induces a switch to an angiogenic phenotype in an agonist-independent manner (Bais et al., 1998; Rosenkilde et al., 1999). Cellular transformation is accompanied by the secretion of vascular endothelial growth factor (VEGF), an angiogenesis and KS's spindle cell growth factor, suggesting that the constitutively active KSHV GPCR contributes to sustained growth of spindle cells. Recently, Ganem and colleagues found that KSHV GPCR is primarily expressed during lytic replication (Kirshner et al., 1999). It is therefore unlikely that the viral GPCR is responsible for spindle cell proliferation in KS tumors since most lytically infected cells eventually die. The paracrine release of the potent angiogenic factor VEGF by KSHV GPCR from a small population of cells, might be sufficient to trigger dramatic neovascularization. The agonist-independent signalling of KSHV GPCR can be inhibited by CXC chemokines that lack the Nterminal ELR motif, such as IP- 10, SDF- 1oL and vMIPII, which thus act as inverse agonists. Chemokines that contain the ELR motif, such as IL-8 and GRO-oL, stimulate KSHV GPCR signalling over constitutive levels (Rosenkilde et al., 1999). Addition of the ELR motif to chemokines acting as neutral antagonists converted them into agonists. IP10-ELR, however, remained an inverse agonist, suggesting that another structural determinant is involved in inverse agonistic activity. A N-terminal deletion mutant of KSHV GPCR (A2-11) failed to be inhibited by IP-10, implying that the N-terminus of KSHV GPCR is important for inverse agonistic action. However, loss of binding of IP-10 to the receptor could not be ruled out. Since IP-10 is also angiostatic and acts as inverse agonists at KSHV GPCR it might be considered as a therapeutic agent to inhibit KS development (Sgadari et al., 1996). However, IP-10 is an agonist of the CXCR3 receptor, which plays a fundamental role in inflammatory processes (Tensen et al., 1999). An IP-10 like protein which selectively binds to KSHV GPCR and that acts as an inverse agonist might be a likely candidate for the treatment of KS. The constitutively activity of KSHV GPCR can also be modulated by Zn 2+, through binding to a metal ion site introduced in the receptor by substituting two Arg residues involved in IL-8 binding by two His residues (Rosenkilde et al., 1999). These findings imply the use of non-peptide compounds as potential therapeutic agents in the treatment of KS. Cellular transformation can also be inhibited by GPCR-specific receptor kinases (GRKs), known to phosphorylate GPCR and blunt signalling (Premont et al., 1995), further indicating that transformation is KSHV GPCR-mediated.

301

Taken together, the enhanced proliferation of KSHV GPCR transfectants and their tumorigenic activity in nude mice supports a role for this receptor in tumorigenesis.

4. Viral GPCRs encoded by cytomegalovirus Human cytomegalovirus (HCMV) or human herpesvirus 5 (HHV-5) is a [3 herpesvirus, whose infection is widespread (50-95% seroprevalence). The HCMV genome consists of 200 open reading frames and persists in a latent form in the host. Infection with CMV is usually asymptomatic but can cause mononucleosis syndrome in normal hosts. In immunocompromised or immunologically immature individuals HCMV infection can have severe manifestations. CMV has been detected repeatedly in arterial tissue of humans suffering from severe arteriosclerosis (Persoons et al., 1994). Transformation of arterial smooth muscle cells by HCMV, causing focal proliferation and inducing changes in cellular metabolism, is considered to be one of the several mechanisms that explain the participation of HCMV in arteriogenesis. In view of these findings, the HCMV encoded GPCRs US27, US28, UL33 and UL78 may be considered potential candidates involved in the pathogenesis of arteriosclerosis. Besides, others role for these HCMV-encoded GPCRs ffave emerged. Currently, most information is available on US28. This receptor displays 70% and 52% homology in its N-terminus with the CCR1 and CCR2 receptors, respectively (Gao and Murphy, 1994), and appears to be a broad spectrum receptor for CC chemokines. Interestingly, US28 has a higher affinity for the CC chemokines than the cellular homologue CCR1. Hence, it may sequester chemokines from the environment evading the immune system at sites of infection and contribute to the latent presence of the virus. Importantly, US28 is a functional receptor as increases in intracellular Ca 2+ are recorded in response to CC chemokines (Gao and Murphy, 1994). Infection of fibroblasts with HCMV was found to alter production of second messengers and regulation of transcription factors, suggesting the involvement of one or more of these HCMV-encoded GPCRs in signalling. US28 couples to both Got i and Got l6 following chemokine stimulation. Coupling of US28 to more than one G protein enables the receptor to activate a wider variety of cells, including cells of hematopoietic origin expressing GoL~6Activation of US28 leads also to stimulation of MAP kinase signalling pathways, suggesting that HCMV may be able to activate proliferative signalling within the host cells through expression of US28. In this context, one might speculate that US28 may contribute to the observed transformation of smooth muscle cells infected by HCMV. Moreover, expression of US28 may allow infected cells to migrate to CC chemokine secreting tissues and aid to

302


virus dissemination. For e.g., neutrophil granulocytes, which normally respond only to CXC chemokines become sensitive to CC chemokines after HCMV infection, enabling them to migrate to CC chemokine secreting tissues (Grundy et al., 1998). US28 also binds with high affinity to CX3C fractalkine, implying that HCMV-infected cells may migrate to fractalkine-expressing cells (Kledal et al., 1998). US28 has also been suggested to play a role in cell fusion and virus entry. This receptor binds the membranebound fractalkine and could therefore be involved in fusion with fractalkine-expressing cells as part of the cell-cell transfer mechanism for HCMV (Bazan et al., 1997). None of the tested CC chemokines can hinder binding of US28 to fraktalkine (Kledal et al., 1998) and are therefore not able to block the hypothetical virus transfer between cells. However, US28 alone is not sufficient to permit cell-cell fusion or the hypothetical virus entry. In addition, US28 similar to a range of human chemokine receptors has been shown to act as a HIV coreceptor (Pleskoff et al., 1998). There are no data available on the function of US28 in the viral life cycle, since a mouse or rat US28 homologue is lacking, making it impossible to develop an animal model system. In contrast to US28, little is known about US27 which also displays significant homology to CC chemokine receptors.

6. UL78 family Another group of viral-encoded GPCRs comprises HCMV UL78, RCMV R78 and MCMV M78 (Gompels et al., 1995; Nicholas, 1996; Rawlinson et al., 1996). The positions of the UL78 like genes are conserved within the [3-herpesvirus genome, whereas the sequences are less conserved. The amino acid sequence of these receptors have a fairly modest homology to chemokine receptors or other cellular GPCRs. No ligands are currently known to bind to these receptors. R78 has recently been found to be important for RCMV replication in vitro and in the pathogenesis of viral infection in vivo (Beisser et al., 1999). Two recombinant viruses, a R78 null mutant (RCMVAR78) and a mutant encoding R78, with its putative C-terminal tail deleted, showed reduced replication in vitro compared to the wild-type. These data imply that a functional receptor is required for replication. In addition, rat embryo fibroblasts developed a syncytium-like appearance upon infection with the recombinant viruses. Vink and colleagues postulated that a functional R78 plays a role in stabilizing the cell membrane of infected cells (Beisser et al., 1999). Furthermore, a lower rate of mortality was observed in rats infected with recombinant virus.

7. ViraI-GPCRs encoded by HHV-6 and HHV-7 5. UL33 family A distinct set of chemokine-like receptors encoded by CMV is the UL33 family which comprises human (UL33), rat (R33) and murine (M33) chemokine receptor homologues. Sequences and position of these genes within their genomes are conserved. UL33 has been detected in both membranes of HCMV-infected cells and envelopes of HCMV virions (Margulies et al., 1996). Unlike US27 and US28, UL33 has a short N-terminus which displays little homology with N-termini of chemokine receptors. Since the N-terminus of chemokine receptors is important for recognition and binding of chemokines, it is possible that UL33 and its rodent homologues are not involved in chemokine binding. So far, research is hampered by the fact that no ligands have been identified for this family of receptors. UL33, R33 and M33 appear non-essential for viral growth in vitro. However, in vivo both R33 and M33 genes were shown to be essential for replication, in salivary glands of infected rat and mice, respectively (DavisPoynter et al., 1997; Beisser et al., 1998). The salivary gland is the major site for replication and virus persistence. In addition, a markedly higher survival was observed in a group of immunocompromised rats infected with recombinant RCMV lacking the R33 gene than with WT RCMV infected rats (Beisser et al., 1998).

Human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) are [3-herpesviruses that have been associated with febrile illnesses and the childhood disease exanthem subitum, as well as infectious mononucleosis for HHV-6 (Levy, 1997). HHV-6 activates other herpesviruses such as Epstein-Barr virus (EBV), HCMV and human papillomavirus. HHV-6 and 7 express U12 and U51 genes that are most related to GPCRs of the HCMV UL33 family and U12 displays highest homology with CCR3. HHV-6 U12 appears to be a functional receptor capable of binding the CC chemokines MIPlot, MIPI[3, RANTES and MCP-1 (Isegawa et al., 1998) and of increasing intracellular C a 2+ levels. No functional data are available on U51. The role of this receptor might be different from other viral-encoded receptors since it shows more resemblance to cellular opioid receptors than to chemokine receptors.

8. Viral GPCRs encoded by Herpesvirus saimiri (HVS) HVS is a T-lymphotropic ~/-herpesvirus that causes a fatal lymphoproliferative disease in several non-human primates. HVS is closely related to the human B-lymphotropic ~/-herpesvirus Epstein-Barr virus. ECRF3, also referred to as HVS ORF74, is a gene displaying homology to CCR1 and CXCR1 and 2, CMV-encoded US28 and KSHV


GPCR (Nicholas et al., 1992). A marked sequence homology was observed with the N-terminus of CXCR2. ECRF3 was shown to bind CXC chemokines IL-8, GROo~/MGSA and NAP-2. Little information is available on this protein, probably because the virus does not infect humans.

9. Viral GPCRs encoded by poxviruses The family of poxviruses from sheep, goat or swine, was also found to encode putative GPCR homologues; Q2/3L, ORF K2R, ooORF C3L (Massung et al., 1993; Cao et al., 1995). In contrast to other viral-encoded GPCRs C3L is predicted to have 6 transmembrane domains. Only genomic data are available but similar roles as found for other viral-encoded GPCR are anticipated.

303

cellular chemokines to exploit the host cell. In addition, viruses may even use constitutively active GPCRs such as KSHV GPCR to alter the basal proliferative state of the cell and serve as oncogenes (Arvanitakis et al., 1997). Since GPCRs have proven to be successful drug targets in the past and their role in viral infections is becoming apparent, viral GPCRs are emerging targets for the development of drug research. It is therefore of utmost importance to further define their binding sites, signalling pathways and involvement in viral infection.

Acknowledgements M.J. Smit is supported by the Royal Netherlands Academie of Arts and Sciences.

10. Use of cellular GPCRs by viruses

References

In the last decade it has become apparent that the human immunodeficiency virus (HIV) uses endogenous chemokine receptors, in particular CXCR4 and CCR5, as coreceptors for cell entry (Fauci, 1996). The Duffy antigen receptor, belonging to the family of chemokine receptors, has been shown to facilitate invasion by the malaria parasite, plasmodium vivax (Chaudhuri et al., 1993). Viruses such as HHV-6 an 7 and Epstein-Barr virus (EBV) are known to induce expression of CCR7, earlier defined as the EBI1 receptor (Yoshida et al., 1997; Campbell et al., 1998). CCR7 has been suggested to be involved in migration of infected cells, especially since the ligands for this receptor, ELC (EBIl-ligand chemokine) and SLC (secondary lymphoid tissue chemokine) show strong constitutive expression in various lymphoid tissues. EBV has also been shown to induce expression of CXCR5, previously referred to as BLR1, in Burkitt's lymphoma cells and B lymphocytes (Gunn et al., 1998). Since GPCRs have been linked to proliferation a role for this receptor in the pathogenesis of Burkitt's lymphoma is expected.

Allen, L.F., Lefkowitz, R.J., Caron, M.G., Cotecchia, S., 1991. G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the alpha 1B-adrenergic receptor enhances mitogenesis and tumorigenicity. Proc. Natl. Acad. Sci. U.S.A. 88, 1135411358. Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M.C., Cesarman, E., 1997. Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation. Nature 385, 347-350. Baggiolini, M., 1998. Chemokines and leukocyte traffic. Nature 392, 565-568. Bais, C., Santomasso, B., Coso, O., Arvanitakis, L., Raaka, E.G., Gutkind, J.S., Asch, A.S., Cesarman, E., Gerhengorn, M.C., Mesri, E.A., 1998. G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator. Nature 391, 86-89. Bazan, J.F., Bacon, K.B., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D.R., Zlotnik, A., Schall, T.J., 1997. A new class of membrane-bound chemokine with a CX3C motif. Nature 385, 640644. Beisser, P.S., Vink, C., Van Dam, J.G., Grauls, G., Vanherle, S.J., Bruggeman, C.A., 1998. The R33 G protein-coupled receptor gene of rat cytomegalovirus plays an essential role in the pathogenesis of viral infection. J. Virol. 72, 2352-2363. Beisser, P.S., Grauls, G., Bruggeman, C.A., Vink, C., 1999. Deletion of the R78 G protein-coupled receptor gene from rat cytomegalovirus results in an attenuated, syncytium-inducing mutant strain. J. Virol. 73, 7218-7230. Bourne, H.R., 1997. How receptors talk to trimeric G proteins. Curr. Opin. Cell Biol. 9, 134-142. Campbell, J.J., Bowman, E.P., Murphy, K., Youngman, K.R., Siani, M.A., Thompson, D.A., Wu, L., Zlotnik, A., Butcher, E.C., 1998. 6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3beta receptor CCR7. J. Cell Biol. 141, 1053-1059. Cao, J.X., Gershon, P.D., Black, D.N., 1995. Sequence analysis of HindIII Q2 fragment of capripoxvirus reveals a putative gene encoding a G-protein-coupled chemokine receptor homologue. Virology 209, 207-212. Chaudhuri, A., Polyakova, J., Zbrzezna, V., Williams, K., Gulati, S., Pogo, A.O., 1993. Cloning of glycoprotein D cDNA, which encodes the major subunit of the Duffy blood group system and the receptor

11. Conclusion Taken together, it is becoming clear that viral-encoded GPCRs may play an important role in viral infection and are likely implicated in chronic diseases. Expression of viral-encoded GPCRs, may help viruses elude the host immune response, render infected cells sensitive to chemokines, promote virus dissemination or transform the host cell. Recent animal studies have showed that (functional) viral-encoded GPCRs are essential for viral replication. The binding pattern of viral-encoded GPCRs does not correspond to that of their cellular homologues, implying that viruses have optimized pirated GPCRs to interact with

304


for the Plasmodium vivax malaria parasite. Proc. Natl. Acad. Sci. U.S.A. 90, 10793-10797. Danesh, J., Newton, R., Beral, V., 1997. Epidemiology. A human germ project? Nature 389, 21-24. Davis-Poynter, N.J., Lynch, D.M., Vally, H., Shellam, G.R., Rawlinson, W.D., Barrell, B.G., Farrell, H.E., 1997. Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus. J. Virol. 71, 1521-1529. Fauci, A.S., 1996. Host factors and the pathogenesis of HIV-induced disease. Nature 384, 529-534. Ganem, D., 1997. KSHV and Kaposi's sarcoma: the end of the beginning? Cell 91, 157-160. Gao, J.L., Murphy, P.M., 1994. Human cytomegalovirus open reading frame US28 encodes a functional beta chemokine receptor. J. Biol. Chem. 269, 28539-28542. Gompels, U.A., Nicholas, J., Lawrence, G., Jones, M., Thomson, B.J., Martin, M.E., Efstathiou, S., Craxton, M., Macaulay, H.A., 1995. The DNA sequence of human herpesvirus-6: structure, coding content, and genome evolution. Virology 209, 29-51. Grundy, J.E., Lawson, K.M., MacCormac, L.P., Fletcher, J.M., Yong, K.L., 1998. Cytomegalovirus-infected endothelial cells recruit neutrophils by the secretion of C-X-C chemokines and transmit virus by direct neutrophil-endothelial cell contact and duringneutrophil transendothelial migration. J. Infect. Dis. 177, 1465-1474. Gunn, M.D., Ngo, V.N., Ansel, K.M., Ekland, E.H., Cyster, J.G., Williams, L.T., 1998. A B-cell-homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1. Nature 391, 799803. Gutkind, J.S., 1998. The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J. Biol. Chem. 273, 1839-1842. Horuk, R., 1994. Molecular properties of the chemokine receptor family. Trends Pharmacol. Sci. 15, 159-165. Horuk, R., Martin, A., Hesselgesser, J., Hadley, T., Lu, Z.H., Wang, Z.X., Peiper, S.C., 1996. The Duffy antigen receptor for chemokines: structural analysis and expression in the brain. J. Leukocyte Biol. 59, 29-38. Isegawa, Y., Ping, Z., Nakano, K., Sugimoto, N., Yamanishi, K., 1998. Human herpesvirus 6 open reading frame U12 encodes a functional beta-chemokine receptor. J. Virol. 72, 6104-6112. Kirshner, J.R., Staskus, K., Haase, A., Lagunoff, M., Ganem, D., 1999. Expression of the open reading frame 74 (G-protein-coupled receptor) gene of Kaposi's sarcoma (KS)-associated herpesvirus: implications for KS pathogenesis. J. Virol. 73, 6006-6014. Kledal, T.N., Rosenkilde, M.M., Schwartz, T.W., 1998. Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28. FEBS Letters 441,209-214. Leurs, R., Smit, M.J., Alewijnse, A.E., Timmerman, H., 1998. Agonistindependent regulation of constitutively active G-protein-coupled receptors. Trends Biochem. Sci. 23, 418-422. Levy, J.A., 1997. Three new human herpesviruses (HHV6, 7 and 8). Lancet 349, 558-563. Margulies, B.J., Browne, H., Gibson, W., 1996. Identification of the

human cytomegalovirus G protein-coupled receptor homologue encoded by UL33 in infected cells and enveloped virus particles. Virology 225, 111-125. Massung, R.F., Jayarama, V., Moyer, R.W., 1993. DNA sequence analysis of conserved and unique regions of swinepox virus: identification of genetic elements supporting phenotypic observations including a novel G protein-coupled receptor homologue. Virology 197, 511-528. Murphy, P., 1997. Pirated genes in Kaposi's sarcoma. Nature 385, 296-299. Nicholas, J., 1996. Determination and analysis of the complete nucleotide sequence of human herpesvirus. J. Virol. 70, 5975-5989. Nicholas, J., Cameron, K.R., Honess, R.W., 1992. Herpesvirus saimiri encodes homologues of G protein-coupled receptors and cyclins. Nature 355, 362-365. Persoons, M.C., Daemen, M.J., Bruning, J.H., Bruggeman, C.A., 1994. Active cytomegalovirus infection of arterial smooth muscle cells in immunocompromised rats. A clue to herpesvirus-associated atherogenesis? Circ. Res. 75, 214-220. Pleskoff, O., Treboute, C., Alizon, M., 1998. The cytomegalovirus-encoded chemokine receptor US28 can enhance cell-cell fusion mediated by different viral proteins. J. Virol. 72, 6389-6397. Premont, R.T., Inglese, J., Lefkowitz, R.J., 1995. Protein kinases that phosphorylate activated G protein-coupled receptors. FASEB J. 9, 175-182. Rawlinson, W.D., Farrell, H.E., Barrell, B.G., 1996. Analysis of the complete DNA sequence of murine cytomegalovirus. J. Virol. 70, 8833-8849. Rosenkilde, M.M., Kledal, T.N., Brauner-Osborne, H., Schwartz, T.W., 1999. Agonists and inverse agonists for the herpesvirus 8-encoded constitutively active seven-transmembrane oncogene product, ORF-74. J. Biol. Chem. 274, 956-961. Sgadari, C., Angiolillo, A.L., Cherney, B.W., Pike, S.E., Farber, J.M., Koniaris, L.G., Vanguri, P., Burd, P.R., Sheikh, N., Gupta, G., Teruya-Feldstein, J., Tosato, G., 1996. Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo. Proc. Natl. Acad. Sci. U.S.A. 93, 13791-13796. Spiegel, A.M., 1996. Defects in G protein-coupled signal transduction in human disease. Annu. Rev. Physiol. 58, 143-170. Tensen, C.P., Flier, J., Van Der Raaij-Helmer, E.M., Sampat-Sardjoepersad, S., Van Der Schors, R.C., Leurs, R., Scheper, R.J., Boorsma, D.M., Willemze, R., 1999. Human IP-9: a keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3) J. Invest. Dermatol. 112, 716-722. van Biesen, T., Luttrell, L.M., Hawes, B.E., Lefkowitz, R.J., 1996. Mitogenic signaling via G protein-coupled receptors. Endocr. Rev. 17, 698-714. Westphal, R.S., Sanders-Bush, E., 1996. Differences in agonist-independent and -dependent 5-hydroxytryptamine2C receptor-mediated cell division. Mol. Pharmacol. 49, 474-480. Yoshida, R., Imai, T., Hieshima, K., Kusuda, J., Baba, M., Kitaura, M., Nishimura, M., Kakizaki, M., Nomiyama, H., Yoshie, O., 1997. Molecular cloning of a novel human CC chemokine EBIl-ligand chemokine that is a specific functional ligand for EBI1, CCR7. J. Biol. Chem. 272, 13803-13809.



Chemokine receptors" interaction with HIV-1 and viral-encoded chemokines Silvano Sozzani a,,, Paola Allavena a, Annunciata Vecchi a, Jo Van Damme Alberto Mantovani ~,c

b,

lstituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea 62, 20157 Milan, Italy b Rega Institute for Medical Research, University of Leuven, Louvain, Belgium c Department of Biotechnology, Section of General Pathology, University of Brescia, Brescia, Italy a

Abstract

Chemokines are a superfamily of proteins that play a central role in immune and inflammatory reactions and in viral infections. About 50 different chemokines divided in four subfamilies are known, CXC, CC, C, and CX3C. Chemokine receptors can function as entry/fusion co-receptors for human immunodeficiency virus (HIV)-I infection, and regulation of receptor expression by cytokines may be relevant for viral infection. Posttranslational processing of chemokines can profoundly affect their interaction with receptors. The serine protease CD26/dipeptidyl-peptidase IV (CD26/DPP IV) removes NHz-terminal dipeptides from several chemokines and profoundly affect their biological activity. Kaposi's sarcoma (KS)-associated herpes virus 8 encodes for three chemokine-like proteins that show homology with MIP cluster of CC chemokines. These viral chemokines possess a partial agonist activity for certain chemokine receptors and may function as receptor antagonists. This biological activity could represent a strategy developed by the virus to subvert immunity impairing the generation of an effective anti-viral immune response. 9 2000 Elsevier Science B.V. All rights reserved. Keywords: Chemokine; Receptors; Chemotaxis; Dendritic cells; HIV-I; Virus

1. Introduction

Chemokines are a superfamily of small proteins which play a crucial role in immune and inflammatory reactions and in viral infection (Hedrick and Zlotnik, 1996; Baggiolini et al., 1997; Rollins, 1997). Most chemokines cause migration of leukocytes, but these molecules also affect angiogenesis, proliferation of hematopoietic precursors, and viral responses. Based on a cysteine motif, a CXC, CC, C and CX3C family have been identified (Fig. 1). CXC (or oL) chemokines are active on neutrophils and lymphocytes while CC (or [3) chemokines exert their action on multiple leukocyte subtypes, including monocytes, basophils, eosinophils, T-lymphocytes, dendritic cells (DC) and NK cells, but they are generally inactive on PMN. Eotaxins

* Corresponding author. Fax: +0039-02-354-6277; e-mail: sozzani@irfmn,mnegri,it

(CC) represent the chemokines with the most restricted spectrum of action being selectively active on eosinophilic and basophilic granulocytes (Schall, 1994; Ben-Baruch et al., 1995; Baggiolini et al., 1997; Mantovani et al., 1998). Lymphotactin and fractalkine are the only proteins so far described with a C and CX3C motif, respectively (Kelner et al., 1994; Bazan et al., 1997; Pan et al., 1997). They both act on lymphoid cells (T-lymphocytes and NK cells) and fractalkine is also active on monocytes and NK cells (Kelner et al., 1994; Bianchi et al., 1996; Bazan et al., 1997; Imai et al., 1997; Pan et al., 1997). Chemokines bind to seven transmembrane domain proteins coupled to GTP-binding proteins with homology to the family of chemotactic receptors. Five receptors for C-X-C chemokines (CXCR1 to 5) and nine for C-C chemokines (CCR1 to 9) were recently cloned (Table 1). These receptors show a promiscuous pattern of ligand recognition and are differentially expressed and regulated in leukocytes (Baggiolini et al., 1997; Izumi et al., 1997; Nibbs et al., 1997; Rollins, 1997; Legler et al., 1998). The


306

S. Sozzaniet al. ,/Pharmaceutica Acta Helvetiae 74 (2000) 305-312

CD4 Env

~

Target

obtained by Ficoll (Biochrom, Berlin, FGR) and Percoll (Pharmacia, Uppsala, Sweden) gradients (Sozzani et al., 1994). Purity was > 90% as assessed by immunofluorescence and FACS analysis for cell surface expression of CD14. Monocyte-derived DC (mono-DC) and CD34 + cell-derived DC (CD34+-DC) were obtained culturing in vitro monocyte and cord blood purified CD34 + cells exactly as previously described (Sozzani et al., 1997a). 2.2. Chemotaxis assay

Target cell

Monocyte migration was evaluated using a chemotaxis microchamber technique (Neuroprobe, Pleasanton, CA) using polycarbonate filters (5 txm pore size; Neuroprobe), as previously described (Sozzani et al., 1994). The chamber was incubated at 37~ in air with 5% CO 2 for 90 min. At the end of the incubation, filters were removed, and stained with Diff-Quik (Baxter, Rome, Italy). Five high power oil-immersion fields were counted. 2.3. Study of chemokine receptor expression

2. Eperimental procedures

For Northern blot analysis, RNA was extracted by the guanidium thiocyanate method, blotted and hybridized as described (Sica et al., 1997). Probes were labeled by Megaprime DNA labeling system (Amersham, Buckinghamshire, UK) with oL3zp-dCTP (3000 Ci/mmol, Amersham). Membranes were prehybridized at 42~ in Hybrisol (Oncor, Gaithersburg, MD), hybridized overnight with 1 • 106 c p m / m l of 32p-labeled probe, and washed twice before being autoradiographed using Kodak XAR-5 films and intensifier screens at - 8 0 ~

2.1. Isolation of leukocytes

2.4. HIV assays

Peripheral blood mononuclear cells were obtained from buffy coats of healthy blood donors. Monocytes were

Monocytes were plated in 24-well plates (Falcon, Becton-Dickinson Labware, Lincoln Park, NJ) at 0.5 • 106

Fig. 1. A schematic, simplified view of human immunodeficiency virus (HIV)-1 interaction with CD4 and chemokines. receptor for fractalkine and lymphotactin were also characterized (Imai et al., 1997; Yoshida et al., 1998).

Table 1 Chemokine receptors and their ligands. Mo, monocytes, DC, dendritic cells; DC (CD34), DC derived from CD34 cells in vitro; PMN, neutrophils; Eo, eosinophils; Ba, basophils; Th, T helper; Tc, T cytotoxic; (act.), activated Receptor Main ligands Main cells Mo, T, NK, DC, PMN CCR1 MCP-3, RANTES, MIP-1c~, MIP-5 Mo, T (act.), NK (act.) CCR2 MCP-s Eo, Ba, T (Th2) CCR3 Eotaxin, MCP-3, RANTES, MIP-5 T (Th2, Tc2), NK, DC CCR4 TARC, MDC Mo, T (Thl, Tcl), DC CCR5 MIP-1[3, MIP-1oL,RANTES T, DC (CD34) CCR6 MIP-3oL/LARC/Exodus T, Mo CCR7 ELC/MIP-3[3, SLC T (Th2), Mo CCR8 1309 DC CCR9 Teck PMN CXCR1 IL-8, granulocytechemotacticprotein (GCP)-2 PMN CXCR2 IL-8, gro, NAP-2, GCP-2 T (Thl) CXCR3 IP10, MIG, ITAC widely expressed CXCR4 SDF-1 B CXCR5 BCA-1 NK, T XCR1 Lymphotactin Mo, NK, T CX3CR1 Fractalkine

S. Sozzani et al. / Pharmaceutica Acta Helvetiae 74 (2000) 305-312

cells/ml in RPMI 1640 (Bio Whittaker, Verviers, Belgium) supplemented of 10% FCS (Hyclone Europe, OudBeijerland, The Netherlands). Monocytes were exposed to CC chemokines for 30 min prior to HIV infection. The macrophage-tropic BaL or the T-tropic strain NL4.3 strains of HIV-1 were added to the cultures at the multiplicity of infection of 0.1. Culture supernatants were collected at fixed intervals, stored at - 8 0 ~ until tested for a conventional analysis of their MgZ+-dependent reverse transcriptase (RT) activity (Weissman et al., 1995).

2.5. Purification of CD26/dipeptidyl-peptidase IV (CD26 /DPP IV) and cleavage of chemokines Human membrane bound CD26/DPP IV was obtained from prostasome (prostate-derived organelles which occur freely in seminal plasma) and soluble C D / D P P IV was isolated from total seminal plasma. The enzyme was purified to homogeneity by anion exchange chromatography and affinity chromatography on immobilized adenosine deaminase. A 100 to 1000 M excess of chemokines was incubated overnight at 37~ with CD26/DPP IV. Chemokines were separated from the enzyme by an acetonitrile gradient on a C-8 Aquapore RP-300 column (1 • 50 mm) (Perkin-Elmer) and NHz-terminally sequenced by Edman degradation on a pulsed liquid phase 477A/120A protein sequencer, or used for biological assays (Proost et al., 1999).


3.1. Chemokine receptors and HIV The HIV is a human pathogenic retrovirus causing a syndrome characterized by a profound immunodeficiency leading to death the great majority of infected individuals. HIV displays a peculiar affinity for cells that express the CD4 molecule on their membrane, namely, a subset of T-lymphocytes with "helper" function and mononuclear phagocytes. However, the membrane expression of CD4 is not enough for HIV infection, and the existence of a putative co-receptor for viral entry has been postulated for several years. It was only in the late 1995 that chemokine receptors were shown to function as fusion co-receptors for HIV-1 infection. The CC chemokine receptor CCR5 has been shown to act as major co-receptor for macrophage tropic (R5) HIV-1 strains. Otherwise, CXCR4 is the major co-receptor used by the T-cell tropic HIV strains (X4). In parallel, it was also shown that the respective chemokine receptor ligands, RANTES, MIP- 1r and MIP- 1[3 for CCR5 and SDF-1 for CXCR4, acted as inhibitors of the binding of HIV to the chemokine co-receptor molecules, without interfering with its interaction with CD4. Indeed, it was demonstrated that interaction between gpl20 and CD4 is

307

the first obligatory step in viral entry, inducing conformational changes indispensable for its subsequent binding to the chemokine receptor (Dimitrov, 1997). In keeping with this model, synthetic truncated molecules capable of binding but not of transducing signals of cell activation can inhibit HIV infection. This finding opened an interesting area of research aimed at the discovery of muted chemokines or low molecular weight organic compounds that could act as chemokine receptor antagonists and could be used to inhibit HIV infection. Other chemokine receptors were shown to function as fusion co-receptors. In particular, the usage of some HIV isolate of CCR2, CCR3 and CCR8, as well as of orphan receptors (STRL33/ BONZO, and GPR1) or viral-encoded receptors (i.e., the CMV encoded US28) were also reported (Locati and Murphy, 1999). The great majority of the HIV strains isolated early after infection are CCR5-monotropic viruses, and the importance of this chemokine receptor for HIV infection has been impressively highlighted by the observation that individuals carrying an homozygous deletion of 32 bp in the CCR5 gene are virtually protected from HIV infection (Samson et al., 1996). Their cells were still infectable in vitro by X4 HIV but were totally resistant to R5 HIV. This gene variant causes the retention of a truncated protein in the cytoplasm without expression at the cell surface. This gene mutation is apparently compatible with a normal life. All in all, these finding indicate that the expression of the cell surface of chemokine receptors is required for HIV infection. Thus, regulation of chemokine receptor expression may represent an important factor that regulates infection and viral spreading during HIV disease progression.

3.2. Regulation of chemokine receptor expression in phagocytes Leukocyte infiltration into tissues is regulated by local production of chemotactic signals. Chemokine receptors are expressed on different types of leukocytes. Some receptors are restricted to certain cells (e.g., CXCR1 on PMN, and CCR3 on eosinophils and basophils), while others, such as CCR1 and CCR2, are expressed on different types of leukocytes. In addition, chemokine receptors are constitutively expressed on some cells, whereas they are inducible in others. Regulation of chemokine receptors is emerging as an alternative mechanism to control the level and the specificity of leukocyte migration. IL-2activated, but not resting, T-lymphocytes and NK cells migrate in response to MCPs. CXCR3 is expressed only in IL-2-activated T-lymphocytes and IL-2 upregulates CCR6 expression (Baggiolini et al., 1997). In PMN, IL-8 receptors can be upregulated or downregulated by G-CSF and LPS, respectively (Lloyd et al., 1995). It was recently described that inflammatory and anti-inflammatory agonists regulated in opposite ways CC chemokine receptor expression in human monocytes. LPS

308


and other microbial agents caused a rapid and drastic reduction of CCR2 mRNA levels (Sica et al., 1997). IFN-~/, a potent activator of mononuclear phagocytes, also inhibited CCR2 expression in a rapid (1 h) and selective manner by a reduction of the half life of mRNA. Its effect was synergistic with the action of other proinflammatory molecules, such as IL-1, TNF and LPS (Penton-Rol et al., 1998). Conversely, incubation of human monocytes with IL-10 increased the expression of CCR1, 2 and 5 as evaluated by Northern blot analysis. No major variations in the expression of CXCR2 were detectable while CXCR4 mRNA levels were reduced (Sozzani et al., 1998b). The estimated half life of CCR5 mRNA was doubled after exposure to IL-10. In contrast, the rate of nuclear transcription of the gene, as investigated by nuclear run off analysis, was not affected. Accordingly, IL-10-treated monocytes responded better to CC chemokines in terms of chemotactic migration and intracellular calcium transients and the effect was best observed when sub optimal agonist concentrations were used. IL-10-treated monocytes were also more easily infected by the macrophage tropic HIV strain BAL (Sozzani et al., 1998b). This result is consistent with the use of CCR5 as major fusion co-receptor by B AL. Selective expression and regulation of chemokine receptors can be intimately associated with the biological program, and function, of specialized leukocyte subsets. This is the case for DC and T h l / T h 2 polarized lymphocytes.

expressed in lymphoid organs, were strongly upregulated, with a maximal effect at 24 h. Upregulation of CCR7 in DC migrating to secondary lymphoid organs appear of biological relevance, since in situ hybridization analysis has shown that ELC/MIP-3[3 is specifically expressed in T-cell rich areas of tonsils and spleen, where mature DC home, becoming interdigitating DC (Dieu et al., 1998; Ngo et al., 1998). In contrast, MIP3oL is produced at sites of inflammation (e.g., inflamed epithelial crypts of tonsils) where immature DC are recruited (Dieu et al., 1998). Chemokine receptor expression in DC has been recently investigated in relation to HIV infection, as these cells may represent an important port of entry of the virus and a vehicle for HIV-1 transmission (Cameron et al., 1992; Graziosi and Pantaleo, 1998). In vitro cultured DC and circulating blood DC express both CCR5 and CXCR4 the two main co-receptors for M-tropic (R5) and T-tropic (X4) strains of the virus, respectively (Granelli-Piperno et al., 1996; Ayehnie et al., 1997; Sozzani et al., 1997b). Immature DC selectively replicate R5 HIV (Granelli-Piperno et al., 1998), and immature LC express only CCR5 and select R5 HIV strains in vivo (Zaitseva et al., 1997; Reece et al., 1998). LC and immature DC are present at mucosal sites of virus transmission and selective entry and replication of R5 HIV could explain the restriction of virus phenotype during viremia before seroconversion. 3.4. Regulation of chemokine receptors in Thl and Th2 lymphocytes

3.3. Regulation of chemokine receptors in DC DC have the unique capacity to initiate primary and secondary immune responses. They take-up antigens in peripheral tissues and migrate to lymphoid organs where they present processed peptides to T-cells. During migration DC undergo maturation from a "immature" to a "mature" functional phenotype, characterized by the expression of co-stimulatory molecules, cytokine production and high ability to stimulate T-cell proliferation. Immature DC express a number of functional chemokine receptors including CCR1, CCR5, CCR6, CXCR4 (Sozzani et al., 1999). Most of these receptors bind "inflammatory" chemokines. These chemokines are induced during inflammation and immune response and very likely regulate the recruitment of new DC in peripheral organs. Maturation of DC can be induced in vitro by a variety of factors, including LPS, and the inflammatory cytokines TNF and IL-1 or the engagement of CD40 (Bancherau and Steinman, 1998). Exposure of DC to inflammatory agonists, such as LPS, IL-1, and TNF, or culture in the presence of CD40 ligand, induced a rapid inhibition of chemotactic response to inflammatory chemokines, such as MIP-loL, MIP-I[3, MIP-3oL RANTES, RANTES, MCP-3 and fMLP (Dieu et al., 1998; Sozzani et al., 1998a). Concomitantly, the expression of CCR7 and the migration to its ligand ELC/MIP-3[3, a chemokine constitutively

T-cells can be subdivided in polarized type I and type II cells, depending on the spectrum of cytokines which they are able to produce. Thl cells are characterized by production by of TNF and IFN-~/and activate immunity based on macrophage activation and effector functions. At the other extreme of the spectrum, Th2 cells are characterized by IL-4 and IL-5 production and elicit immune responses based on the effector function of mastocytes and eosinophils. The latter cell types are typically involved in allergic inflammation. Recent results indicate that chemokines are part of the Thl and Th2 paradigm. It was found that polarized Thl and Th2 populations differentially express chemokine receptors. In particular, Thl cells characteristically express high levels of CCR5 and CXCR3 whereas Th2 cells express CCR4, CCR8 and to a lesser degree, CCR3. In accordance with receptor expression, polarized T h l / T h 2 cells (as well as CD8 § T-cells with a similar cytokine profile), differentially respond to appropriate agonists for these receptors, including, for Thl cells, MIP-I[3 and IP-10 and for Th2 cells MDC, 1309 and eotaxin (Sallusto et al., 1997; Bonecchi et al., 1998). Production of IP-10 and similar CXCR3 agonists such as ITAC, is induced by IFN-~/. Conversely, production of eotaxin and/or MDC is induced by IL-4 and IL-13, typical Th2 cytokines. Thus, chemokines, and chemokine receptors are probably an


essential part of an amplification circuit of polarized Thl and Th2 responses. 3.5. Role of C D 2 6 / D P P IV in chemokine processing

DPP IV, also known as CD26, was originally identified as a marker of activated memory T-lymphocytes (Morimoto and Schlossman, 1998; DeMeester et al., 1999). However, CD26 is expressed in several cell types, including endothelial, and epithelial cells. CD26 is a cell membrane associated protein that is also found in a soluble form in seminal fluid, urine and plasma. This protein exerts through its extracellular domain a unique peptidase activity. It cleaves dipeptides from the NH z-terminus of proteins having a Pro or Ala residue at the penultimate position. The presence of a Pro in the NHz-terminal region protects proteins from proteolytic degradation by aminopeptidases. Although several cytokines including interleukines (e.g., IL-I[3, IL-2, IL-5, IL-6, and IL-10) are potential substrates, they are not processed by CD26, probably because of their large molecular weight (DeMeester et al., 1999). Chemokines of both CXC and CC subfamilies are naturally posttranslationally modified. For instance, two predominant isoforms of the neutrophils chemoattractant IL-8 have been described. This truncation is due to the action of several proteases, but not by the action of CD26. Several chemokines possess a proline residue in the NH 2 region in position 2 (Table 1). However, some of them like MCP-1, -2, -3, and -4 are protected from CD26 degradation by a pyroglutamate at the NHz-terminus that protects the protein by degradation. On the contrary, other chemokines can be effectively processed by the enzyme, and the biological output of this cleavage is unpredictable. GCP-2, a CXC chemokine, is cleaved, but its biological activity remains unaffected. However, for most chemokines (RANTES, SDF-1, MDC, eotaxin) truncation by CD26 is accompanied by reduced receptor binding and signaling and by impaired chemotactic responses (Table 2). For all these chemokines a Pro and not Ala residue at the penultimate position is involved. An exception, is MDC that is processed beyond this cleavage site. Very often, CD26-processed chemokines not only loose their biological activity but also start to function as Table 2 Biological relevance of chemokine processing by CD26 Family Chemokine NH2-sequence Cleavage Change in activity CXC CC

SDF- 1 GCP-2 MCP-1 MCP-2 MCP-3 RANTES

KPVSLSYR GPVSAVLT QPDAINAP QPDSVSIP QPVGINTS SPYSSDT

+ + +

Eotaxin MDC

GPASVPTT GPYGANM

+

+

(i.e. decrease) ant. =

(i.e. decrease) ant. CCR1 (i.e. increase) CCR3 (i.e. decrease) ant. (i.e. decrease) ant. CCR4 = mono

309

receptor antagonists being able to inhibit the biological activity of intact proteins. This is the case for SDF-1 and eotaxin. Alternatively, the processed chemokine may have a more complex biological behaviour. For instance, when RANTES is processed by CD26 it looses activity, and actually becomes a receptor antagonist, with respect to the two of the three receptors that this protein uses (CCR1 and CCR3). On the contrary, the affinity of truncated RANTES for CCR5 results increased (DeMeester et al., 1999). Similarly, truncated MDC loose the ability to interact with CCR4, the only described receptor for this chemokines. However, truncated MDC completely retains its ability to activate monocytes and NK cells, suggesting that this protein can interact with as still unknown MDC receptor (Struyf et al., 1998; Proost et al., 1999). In order to verify whether chemokine processing by CD26 occurs and how this process affects their biological activity, protein purification needs to be performed. At the moment, antibodies that can specifically distinguish the processed chemokine are not available, and the ELISA which is commercially available can detect both truncated and full-length proteins. 3.6. Viral-encoded chemokines and chemokine receptors

Viruses have developed a wide variety of responses to the host immune system. DNA viruses, such herpes viruses and pox viruses have the ability to encode genes that can manipulate the host immune response. Many of these genes encode for cytokines, cytokine receptors, included chemokines and chemokine receptors (Wells and Schwartz, 1997). For instance, human cytomegalovirus encodes for three G-protein-coupled receptors, US27, US28 and UL33, some of them known to be able to bind chemokines. The advantage of virus in having these receptors expressed is unclear, but recently, it was proposed that they could serve as scavenger receptors for chemokines (Bodaghi et al., 1998). Human herpes virus 8 (HHV8), also known as Kaposi's sarcoma (KS) virus, is associated with KS, body cavity-based lymphoma and Castelman's disease (Chang et al., 1994; Boshoff et al., 1995a,b; Dupin et al., 1995). The HHV8 genome includes three open reading frames coding for proteins with considerable ( ~ 40%) identity to human CC chemokines and one coding for a chemokine receptor, ORF74 (Arvanitakis et al., 1997). KS is an opportunistic tumor characterized by prominent angiogenesis and leukocyte infiltration (Armes, 1989; Sciacca et al., 1994), including T-cells and monocytes, vMIPII has been shown to interact with multiple chemokine receptors as an antagonist or as an agonist (Boshoff et al., 1997; Kledal et al., 1997). It binds CCR3 (Boshoff et al., 1997), and with CCR8 (Sozzani et al., 1998c) two receptors expressed preferentially on polarized Th2 cells (Gerber et al., 1997; Sallusto et al., 1997; Bonecchi et al., 1998; Loetscher et al., 1998; Sozzani et al., 1998c), and shows activity on eosinophils (Boshoff et al., 1997), monocytes, and Th2 cells (Sozzani et al., 1998c). Hence, the ability of vMIP-II

310

S. Sozzani et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 305-312

to act on chemokine receptors, such as CCR3 and CCR8 which is expressed on Th2 cells, underlies the ability of vMIP-II to selectively attract these cells into KS lesions and may represent a strategy of the virus to subvert host immunity (Sozzani et al., 1998c). 3. 7. Concluding remarks Chemokines constitute a large family of about 50 proteins that interact with about 20 different receptors. Chemokines play a crucial biological role in inflammation, immunity and viral infection. In spite of certain degree of redundancy, the chemokine system presents several levels of specificity in terms of receptor interaction, target cells and biological functions. The relevance of certain chemokines in specific immune responses is also suggested by the expression of selected chemokine-like proteins or chemokine-receptors by viruses as way to escape host response. Regulation of receptor expression, in addition to agonist production, is likely a crucial point in the regulation of the chemokine system. An emerging paradigm indicates that at least some pro- and anti-inflammatory molecules exert reciprocal and opposing influences on chemokine agonist production and receptor expression. We speculate that the divergent effect of certain proinflammatory signals on agonist vs. receptor expression may serve to retain mononuclear phagocytes at sites of inflammation, to prevent their reverse transmigration, and possibly, to limit excessive recruitment. In addition, selective expression of certain chemokine receptors by different leukocyte subsets and their peculiar regulation may play an important role in controlling the spreading of HIV infection in vivo. A better understanding of the physiological role of chemokines in directing the traffic of different leukocyte subsets that play a crucial role for the activation and orientation of specific immunity may provide a basis for less empirical design of chemokine-based therapeutic strategies.

Acknowledgements This work was supported by Istituto Superiore Sanit?a (AIDS 30B.87, and 40B.63) and by Associazione Italiana Ricerca sul Cancro, and by National Research Council (CNR) Finalized Project Biotechnology.

References Armes, J., 1989. A review of Kaposi's sarcoma. Adv. Cancer Res. 53, 73-95. Arvanitakis, L., GerasRaaka, E., Varma, A., Gershengorn, M.C., Cesarman, E., 1997. Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation. Nature 385, 347-350.

Ayehnie, S., Garcia-Zepeda, E.A., Hoxie, J.A., Horuk, R., Kupper, T.S., Luster, A.D., Ruprecht, R.M., 1997. Human immunodeficiency virus-1 entry into purified blood dendritic cells through CC and CXC chemokine coreceptors. Blood 90, 1379-1386. Baggiolini, M., Dewald, B., Moser, B., 1997. Human chemokines: an update. Annu. Rev. Immunol. 15, 675-705. Bancherau, J., Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature 392, 245-252. Bazan, J.F., Bacon, K.B., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D.R., Zlotnik, A., Schall, T.J., 1997. A new class of membrane-bound chemokine with a CX3C motif. Nature 385, 640644. Ben-Baruch, A., Michiel, D.F., Oppenheim, J.J., 1995. Signals and receptors involved in recruitment of inflammatory cells. J. Biol. Chem. 270, 11703-11706. Bianchi, G., Sozzani, S., Zlotnik, A., Mantovani, A., Allavena, P., 1996. Migratory response of human NK cells to lymphotactin. Eur. J. Immunol. 26, 3238-3241. Bodaghi, B., Jones, T.R., Zipeto, D., Vita, C., Sun, L., Laurent, L., Arenzanaseisdedos, F., Virelizier, J.L., Michelson, S., 1998. Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells. J. Exp. Med. 188, 855-866. Bonecchi, R., Bianchi, G., Bordignon, P.P., D'Ambrosio, D., Lang, R., Borsatti, A., Sozzani, S., Allavena, P., Gray, P.A., Mantovani, A., Sinigaglia, F., 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Thl) and Th2. J. Exp. Med. 187, 129-134. Boshoff, C., Schulz, T.F., Kennedy, M.M., Graham, A.K., Fisher, C., Thomas, A., McGee, J.O.'D., Weiss, R.A., O'Leary, J.J., 1995a. Kaposi's sarcoma-associated herpesvirus infects endothelial and spindle cells. Nat. Med. 1, 1274-1278. Boshoff, C., Whitby, D., Hatziioannou, T., Fisher, C., Walt, J., Hatzakis, A., Weiss, R., Schulz, T., 1995b. Kaposi's-sarcoma-associated herpesvirus in HIV-negative Kaposi's sarcoma. Lancet 345, 1043-1044. Boshoff, C., Endo, Y., Collins, P.D., Takeuchi, Y., Reeves, J.D., Schweickart, V.L., Siani, M.A., Sasaki, T., Williams, T.J., Gray, P.W., Moore, P.S., Chang, Y., Weiss, R.A., 1997. Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines. Science 278, 290294. Cameron, P.U., Freudenthal, P.S., Barker, J.M., Gezelter, S., Inaba, K., Steinman, R.M., 1992. Dendritic cells exposed to human immunodeficiency virus type- 1 transmit a vigorous cytopathic infection to CD4 + T cells. Science 257, 383-387. Chang, Y., Cesarman, E., Pessin, M.S., Lee, F., Culpepper, J., Knowles, D.M., Moore, P.S., 1994. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 266, 18651869. DeMeester, I., Korom, S., Van Damme, J., Scharp6, S., 1999. CD26, let it cut or cut it down. Immunol. Today, in press. Dieu, M.C., Vanbervliet, B., Vicari, A., Bridon, J.-M., Oldham, E., Ait-Yahia, S., Briere, F., Zlotnik, A., Lebecque, S., Caux, C., 1998. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J. Exp. Med. 188, 373-386. Dimitrov, D.S., 1997. How do viruses enter cells? The HIV coreceptors teach us a lesson of complexity. Cell 91,721-730. Dupin, N., Grandadam, M., Calvez, V., Gorin, I., Aubin, J.T., Havard, S., Lamy, F., Leibowitch, M., Huraux, J.M., Escande, J.P., Agut, H., 1995. Herpesvirus-like DNA sequences in patients with mediterranean Kaposi's sarcoma. Lancet 345, 761-762. Gerber, B.O., Zanni, M.P., Uguccioni, M., Loetscher, M., Mackay, C.R., Pichler, W.J., Yawalkar, N., Baggiolini, M., Moser, B., 1997. Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils. Curr. Biol. 7, 836-843. Granelli-Piperno, A., Moser, B., Pope, M., Chen, D.L., Wei, Y., Isdell, F., O'Doherty, U., Paxton, W., Koup, R., Mojsov, S., Bhardwaj, N.,


Clarklewis, I., Baggiolini, M., Steinman, R.M., 1996. Efficient interaction of HIV-1 with purified dendritic cells via multiple chemokine coreceptors. J. Exp. Med. 184, 2433-2438. Granelli-Piperno, A., Delgado, E., Finkel, V., Paxton, W., Steinman, R.M., 1998. Immature dendritic cells selectively replicate macrophagetropic (M-tropic) human immunodeficiency virus type 1, while mature cells efficiently transmit both M- and T-tropic virus to T cells. J. Virol. 72, 2733-2737. Graziosi, C., Pantaleo, G., 1998. The multi-faceted personality of HIV. Nat. Med. 12, 1318-1320. Hedrick, J.A., Zlotnik, A., 1996. Chemokines and lymphocyte biology. Curr. Opin. Immunol. 8, 343-347. Imai, T., Hieshima, K., Haskell, C., Baba, M., Nagira, M., Nishimura, M., Kakizaki, M., Takagi, S., Nomiyama, H., Schall, T.J., Yoshie, O., 1997. Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91,521-530. Izumi, S., Hirai, K., Miyamasu, M., Takahashi, Y., Misaki, Y., Takaishi, T., Morita, Y., Matsushima, K., Ida, N., Nakamura, H., Kasahara, T., Ito, K., 1997. Expression and regulation of monocyte chemoattractant protein-1 by human eosinophils. Eur. J. Immunol. 27, 816-824. Kelner, G.S., Kennedy, J., Bacon, K.B., Kleyensteuber, S., Largaespada, D.A., Jenkins, N.A., Copeland, N.G., Bazan, J.F., Moore, K.W., Schall, T.J., Zlotnik, A., 1994. Lymphotactin: a cytokine that represents a new class of chemokine. Science 266, 1395-1399. Kledal, T.N., Rosenkilde, M.M., Coulin, F., Simmons, G., Johnsen, A.H., Alouani, S., Power, C.A., Luttichau, H.R., Gerstoft, J., Clapham, P.R., Clarklewis, I., Wells, T.N.C., Schwartz, T.W., 1997. A broadspectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus. Science 277, 1656-1659. Legler, D.F., Loetscher, M., Roos, R.S., Clarklewis, I., Baggiolini, M., Moser, B., 1998. B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CXCR5. J. Exp. Med. 187, 655-660. Lloyd, A.R., Biragyn, A., Johnston, J.A., Taub, D.D., Xu, L.L., Michiel, D., Sprenger, H., Oppenheim, J.J., Kelvin, D.J., 1995. Granulocytecolony stimulating factor and lipopolysaccharide regulate the expression of interleukin 8 receptors on polymorphonuclear leukocytes. J. Biol. Chem. 270, 28188-28192. Locati, M., Murphy, P.M., 1999. Chemokines and chemokine receptors: biology and clinical relevance in inflammation and AIDS. Annu. Rev. Med. 50, 425-440. Loetscher, P., Uguccioni, M., Bordoli, L., Baggiolini, M., Moser, B., Chizzolini, C., Dayer, J.-M., 1998. CCR5 is characteristic of Thl lymphocytes. Nature 391,344-345. Mantovani, A., Allavena, P., Vecchi, A., Sozzani, S., 1998. Chemokines and chemokine receptors during activation and deactivation of monocytes and dendritic cells and in amplification of Thl versus Th2 responses. Int. J. Clin. Lab. Res. 28, 77-82. Morimoto, C., Schlossman, S.F., 1998. The structure and function of CD26 in the T-cell immune response. Immunol. Rev. 161, 55-70. Ngo, V.N., Tang, H.L., Cyster, J.G., 1998. Epstein-Barr virus-induced molecule 1 ligand chemokine is expressed by dendritic cells in lymphoid tissues and strongly attracts naive T cells and activated B cells. J. Exp. Med. 188, 181-191. Nibbs, R.J.B., Wylie, S.M., Yang, J.Y., Landau, N.R., Graham, G.J., 1997. Cloning and characterization of a novel promiscuous human beta-chemokine receptor D6. J. Biol. Chem. 272, 32078-32083. Pan, Y., Lloyd, C., Zhou, H., Dolich, S., Deeds, J., Gonzalo, J.A., Vath, J., Gosselin, M., Ma, J.Y., Dussault, B., Woolf, E., Alperin, G., Culpepper, J., Gutierrezramos, J.C., Gearing, D., 1997. Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation. Nature 387, 611-617. Penton-Rol, G., Polentarutti, N., Luini, W., Borsatti, A., Mancinelli, R., Sica, A., Sozzani, S., Mantovani, A., 1998. Selective inhibition of expression of the chemokine receptor CCR2 in human monocytes by IFN-% J. Immunol. 160, 3869-3873.

311

Proost, P., Struyf, S., Schols, D., Opdenakker, G., Sozzani, S., Allavena, P., Mantovani, A., Augusuns, K., Bal, G., Haemers, A., Lambeir, A.M., Scharp6, S., Van Damme, J., De Meester, I., 1999. Truncation of macrophage-derived chemokine by CD26/dipeptidyl-peptidase IV beyond its predicted cleavage site affect chemotactic activity and CC chemokine receptor 4 interaction. J. Biol. Chem. 274, 3988-3999. Reece, J.C., Handley, A.J., Anstee, E.J., Morrison, W.A., Crowe, S.M., 1998. HIV-1 selection by epidermal dendritic cells during transmission across human skin. J. Exp. Med. 187, 1623-1631. Rollins, B.J., 1997. Chemokines. Blood 90, 909-928. Sallusto, F., Mackay, C.R., Lanzavecchia, A., 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277, 2005-2007. Samson, M., Libert, F., Doranz, B.J., Rucker, J., Liesnard, C., Farber, C.M., Saragosti, S., Lapoumeroulie, C., Cognaux, J., Forceille, C., Muyldermans, G., Verhofstede, C., Burtonboy, G., Georges, M., Imai, T., Rana, S., Yi, Y.J., Smyth, R.J., Collman, R.G., Doms, R.W., Vassart, G., Parmentier, M., 1996. Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382, 722-725. Schall, T.J., 1994. The chemokines. In: Thomson, A. (Ed.), The Cytokine Handbook. Academic Press, London, p. 419. Sciacca, F.L., Stiirzl, M., Bussolino, F., Sironi, M., Brandstetter, H., Zietz, C., Zhou, D., Matteucci, C., Peri, G., Sozzani, S., Benelli, R., Arese, M., Albini, A., Colotta, F., Mantovani, A., 1994. Expression of adhesion molecules, platelet-activating factor, and chemokines by Kaposi' s sarcoma cells. J. Immunol. 153, 4816-4825. Sica, A., Saccani, A., Borsatti, A., Power, C.A., Wells, T.N.C., Luini, W., Polentarutti, N., Sozzani, S., Mantovani, A., 1997. Bacterial lipopolysaccharide rapidly inhibits expression of C-C chemokine receptors in human monocytes. J. Exp. Med. 185, 969-974. Sozzani, S., Zhou, D., Locati, M., Rieppi, M., Proost, P., Magazin, M., Vita, N., Van Damme, J., Mantovani, A., 1994. Receptors and transduction pathways for monocyte chemotactic protein-2 and monocyte chemotactic protein-3 - - similarities and differences with MCP-1. J. Immunol. 152, 3615-3622. Sozzani, S., Longoni, D., Bonecchi, R., Luini, W., Bersani, L., D'Amico, G., Borsatti, A., Bussolino, F., Allavena, P., Mantovani, A., 1997a. Human monocyte-derived and CD34+ cell-derived dendritic cells express functional receptors for platelet activating factor. FEBS Lett. 418, 98-100. Sozzani, S., Luini, W., Borsatti, A., Polentarutti, N., Zhou, D., Piemonti, L., D'Amico, G., Power, C.A., Wells, T.N., Gobbi, M., Allavena, P., Mantovani, A., 1997b. Receptor expression and responsiveness of human dendritic cells to a defined set of CC and CXC chemokines. J. Immunol. 159, 1993-2000. Sozzani, S., Allavena, P., D'Amico, G., Luini, W., Bianchi, G., Kataura, M., Imai, T., Yoshie, O., Bonecchi, R., Mantovani, A., 1998a. Cutting edge: differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties. J. Immunol. 161, 1083-1086. Sozzani, S., Ghezzi, S., Iannolo, G., Luini, W., Borsatti, A., Polentarutti, N., Sica, A., Locati, M., Mackay, C., Wells, T.N.C., Biswas, P., Vicenzi, E., Poli, G., Mantovani, A., 1998b. Interleukin-10 increases CCR5 expression and HIV infection in human monocytes. J. Exp. Med. 187, 439-444. Sozzani, S., Luini, W., Bianchi, G., Allavena, P., Wells, T.N.C., Napolitano, M., Bernardini, G., Vecchi, A., D'Ambrosio, D., Mazzeo, D., Sinigaglia, F., Santoni, A., Maggi, E., Romagnani, S., Mantovani, A., 1998c. The viral chemokine macrophage inflammatory protein-II is a selective Th2 chemoattractant. Blood 92, 4036-4039. Sozzani, S., Allavena, P., Vecchi, A., Mantovani, A., i999. The role of chemokines in the regulation of dendritic cell trafficking. J. Leukocyte Biol. 66, 1-9. Struyf, S., Proost, P., Sozzani, S., Mantovani, A., Wuyts, A., De Clercq, E., Schols, D., Van Damme, J., 1998. Cutting edge: enhanced antiHIV-1 activity and altered chemotactic potency of NH2-terminally

312

S. Sozzani et al./Pharmaceutica Acta Helvetiae 74 (2000) 305-312

processed macrophage-derived chemokine (MDC) imply an additional MDC receptor. J. Immunol. 161, 2672-2675. Weissman, D., Poli, G., Fauci, A.S., 1995. IL-10 synergizes with multiple cytokines in enhancing HIV production in cells of monocytic lineage. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 9, 442-449. Wells, T.N.C., Schwartz, T.W., 1997. Plagiarism of the host immune system: lessons about chemokine immunology from viruses. Curr. Opin. Biotechnol. 8, 741-748.

Yoshida, T., Imai, T., Kakizaki, M., Nishimura, M., Takagi, S., Yoshie, O., 1998. Identification of single C motif-1 lymphotactin receptor XCR1. J. Biol. Chem. 273, 16551-16554. Zaitseva, M., Blauvelt, A., Lee, S., Lapham, C.K., Klaus-Kovtun, V., Motowski, H., Manischewitz, J., Golding, H., 1997. Expression and function of CCR5 and CXCR4 on human Langerhans cells and macrophages: implication for HIV primary infection. Nat. Med. 3, 1369-1375.



General topics and perspectives H. T i m m e r m a n

*

Dpt. Farmacochemie, Leiden/Amsterdam Center for Drug Research, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, Netherlands

The session deals especially with recent developments in the field of the receptor machinery of G-protein coupled receptors, GPCRs. Nowadays, the general structure of GPCRs, being protein embedded in cellular membranes, with seven intramembrane domains (a-helices) is generally accepted. So is the coupling to G-proteins and, upon activation, the subsequent activation of the G-protein, followed by the next activation process, the effector mechanism, being ionchannels, enzymes, membranes etc. Much less is known about the first interaction between ligand and receptor. It seems to become clearer and clearer that the productive lock and key theory needs some adjustment. Receptors are dynamic molecules, being able to adapt several conformations. The simple concept of an agonist capable of activating a receptor and of a competitive antagonist which binds to the receptor without activating it, however, thereby blocking the possibility of the agonist to activate the same receptor has been proven to be too simple. In this session, Maggio (Pisa, Italy) presented with a number of elegant experiments that a receptor is able to form with units of other receptors new receptors, with unique properties. This receptor dimerization has been shown to take place under experimental conditions, but may be as well occur under physiological or pathological circumstances. Maggio illustrates these possibilities with muscarinic M 2 and M 3 receptors, but the processes may be more general.

* Fax: +31-20-6461479.

During recent years, unexplained observations found an explanation in the acceptance that GPCRs might have a basic activity, that is, they may produce the second messenger in the absence of the given agonist. This constitutive activity is often but not exclusively seen in mutants (e.g., by genetic causes) of receptors; this so-called receptor polymorphism is in certain cases the mechanism which causes a certain disease. Receptors having this basic or constitutive activity might be (further) activated or their activity may be reduced. The first effect is caused by the classical agonists; the second process needs the involvement of inverse agonists. It has been found that several, but not all, receptor antagonists behave rather as inverse agonists: they reduce the basic activity of a receptor; in classical experiments, these compounds present themselves as antagonists. Besides agonists and reverse antagonists, the so-called neutral antagonists have been shown to antagonize both the agonists and the inverse agonists. Leurs (Amsterdam, The Netherlands) presented several examples of agonists, inverse agonists and neutral antagonists, as explained the way by which these ligands obtain their activity. He focused also on the constitutive activity of receptors in general, showing that constitutive activity may be the cause of certain pathologies. He illustrated his observations with the histamine H 2 antagonists, showing that most H 2 blockers, such as cimetidine, ranitidine and famotidine are rather H 2 inverse agonists; however, burinamide, the first H 2 blocker ever, is a neutral antagonist. In a subsequent contribution, Simonds (Bethesda, USA) showed the complexity of the machinery of the GPCRs. It is generally accepted that the GoL unit of the G-protein mediates the effects following receptor stimulation. In several cases, however, the G[3y units exert comparable functions. Simonds showed that the G[35 isoform especially has physiological roles, activating different pathways. Simonds also introduced functions of the G-protein signaling proteins (RGS), new players in the downstream


314

H. Timmerman / Pharmaceutica Acta Helvetiae 74 (2000) 313-314

processes upon receptor stimulations, having a special role in accelerating the GTPase reactions. The new findings presented by Simonds leave on the one hand a number of questions unanswered, but may be at the other represent new possibilities to interfere with GPCR-mediated processes.

Neumeyer (Rochester, USA) introduced a series of new kappa opioid antagonists. These compounds might offer certain advantages as agents to be used to treat cocaine addicts. The new derivatives interfere with dopamine pathways in the CNS, thereby altering neurochemical and behavioural effects of cocaine.


Pharmaceutica Acta Helvetiae 74 (2000) 315-326 www.elsevier,com/locate / pharmactahelv

Pharmacological evidence of muscarinic receptor heterodimerization S. Chiacchio, M. Scarselli, M. Armogida, R. Maggio Department of Neuroscience, University of Pisa, Via Roma 55-56126 Pisa, Italy

Keywords: Heterodimerization; Tripitramine; Pirenzepine; Muscarinic; Chimeric

1. Introduction G protein-coupled receptors are transmembrane proteins that mediate a variety of signaling processes, such as neurotransmission, hormonal response, olfaction and light transduction. Muscarinic receptors are members of this family and molecular cloning has revealed the existence of five different subtypes (Kubo et al., 1986; Bonner et al., 1987, 1988; Peralta et al., 1987) which show a high degree of sequence homology but differ in their ligand binding and functional properties, as well as in their tissue distribution (Hulme et al., 1990). They are predicted to be composed of seven hydrophobic transmembrane domains (TMDs I-VII) connected by alternating cytoplasmic and extracellular loops, an extracellular amino-terminal domain and an intracellular carboxyl-terminal segment. While they are generally considered as closely-packed structures, an increasing amount of evidence indicates that they can behave structurally in a fashion analogous to multiple subunit receptors. The subunit characteristic of the receptor could be at the basis of receptor interaction with the formation of new functional structures, the simplest of which is a dimer. The property of receptors to cross-interact at the molecular level suggests that when different subtypes of muscarinic receptors are co-expressed in the same cells, they might interact to form heterodimers with new pharmacological properties. In this chapter, we will describe recent work dealing with the heterodimerization of muscarinic M2 and M3 receptors.

2. G protein-coupled receptors consist of multiple autonomous folding domains Previous studies with bacteriorhodopsin, a light-driven proton pump, have demonstrated that this protein can be

* Corresponding author.

functionally reconstituted from individual receptor fragments resulting from proteolytic cleavage of various loop regions (Liao et al., 1983; Popot et al., 1987; Kahn and Engelman, 1992). Following these pioneer experiments these findings have been described for G protein-coupled receptors. Kobilka et al. (1988) demonstrated that split [32-adrenergic receptor behave in terms of binding activity and function like the wild type [32-adrenergic receptor. Afterwards, this phenomenon was demonstrated for other G protein-coupled receptors: muscarinic (Maggio et al., 1993a; SchiSneberg et al., 1995; Jakubik and Wess, 1999), vasopressin (SchiSneberg et al., 1996) and dopamine receptors (Barbier et al., 1996). In particular, Maggio et al. (1993a) showed that when truncated M 2 or M 3 receptors (containing TMDs I-V, named M 2- or M 3-trunc) were co-expressed in COS-7 cells with gene fragments coding for the corresponding C-terminal receptor portions (containing TMDs VI and VII, named M 2- or M3-tail), muscarinic receptors with ligand-binding properties and functional activity similar to the wild-type receptors were obtained. As the separate transfection of each individual fragment was not able to show any binding, it was supposed that the two receptor domains interact with each other to form a macromolecular complex with the same characteristics as the wild-type receptor. Furthermore, experiments performed with the M 3 muscarinic receptor have shown that it can be split not only at the level of the third cytoplasmic loop but also in the second intra- and third extra-cytoplasmic loops and it can retain its binding properties (SchiSneberg et al., 1995). These data imply that individual receptor fragments have the ability to fold independently and to recognize each other in order to form a functional receptor complex. Interaction has been demonstrated to occur also between fragments originating from different receptors; cotransfection of M2-trunc with M3-tail (Fig. 1) results in the formation of a split chimeric receptor with a high-affinity binding for N-[3H]methylscopolamine (the other chimeric

0031-6865/00/$ - see front matter 9 2000 Published by Elsevier Science B.V. All rights reserved. PII: S003 1-6865(99)00041-2

316

S. Chiacchio et al. / Pharmaceutica Acta Helvetiae 74 (2000) 315-326

the agonist carbachol with an affinity that is intermediate between those found for the two generating M 2 and M 3 receptors, indicating that the binding pocket created by the structural assembly of the two receptor fragments retains the conformation of muscarinic receptors (Maggio et al., 1993a). In agreement with these findings, the affinity of the nonspecific muscarinic antagonist N-[3H]methyl scopolamine for the split chimeric M2-trunc/M3-tail receptor was in the order of magnitude found for all the other muscarinic receptors (Table 1). While it is predictable that nonspecific muscarinic antagonists will not distinguish the chimeric receptor from the wild-type M 2 and M 3 receptors, selective antagonists should probably recognize the structural differences. For

combination, M 3 - t r u n c + M 2 - t a i l did not s h o w any binding) (Maggio et al., 1993a). In a recent paper (Barbier et al., 1998) we studied the antagonist binding characteristics of the split M z-trunc/M3-tail receptor with the purpose of identifying compounds capable of discriminating between this split chimera and the wild-type M 2 and M 3 muscarinic receptors.

3. Antagonist binding profile of the split chimeric M2trunc / M 3-tail receptor The split chimeric M2-trunc/M3-tail receptor (Fig. 1) binds the physiological neurotransmitter acetylcholine and

N

Ni i[ "

I

c I

M3

M2

............. U .l

UiH '

lj,N

c

M3 - s h o r t

N

M2- trunc

M3 - tail

M2

(Asn404-~ Ser)

TRANSMEMBRANE DOMAIN VII

TRANSMEMBRANE DOMAIN VI

Hm2 Rm3

T ILAILLAFI TLSAILLAF

$T

ITWAPYNVMVL

INT

T

IGYWLCYINSTINPACYA

KTYWNLGYWLC

I ITWTPYNIMVLVNT

T

NTVWT

T

T

T

$T

Y I N S TVNP

T

VCYA

T

Fig. 1. Schematic representation of the wild-type human M 2 and rat M 3 receptors, the M2-trunc and M3-tail fragments, and the mutants M3-short and M2(Asn404 ~ Ser) muscarinic receptors. The truncated fragment, M2-trunc, contains the amino-terminal domain, the first five hydrophobic transmembrane regions and the initial portion (56 amino acids) of the third cytoplasmic (i3) loop of the wild-type muscarinic M 2 receptor. The M3-tail fragment contains the final portion of the i3 loop (105 amino acids), the last two hydrophobic transmembrane regions and the carboxyl-terminal segment of the wild-type M 3 muscarinic receptor. The short construct (M3-short) represents a receptor in which 196 amino acids of the i3 loop have been deleted; the remaining loop is 43 amino acids long. The point mutant M2(Asn404 ~ Ser) has the asparagine 404 replaced with serine. The amino acid differences in the transmembrane domains VI and VII of the human muscarinic M 2 and the rat muscarinic M 3 receptors are presented.


Table 1 Antagonist binding affinities of several compounds for the chimeric Mz-trunc/M3-tail receptor compared with the wild-type M 2 and M 3 muscarinic receptors The affinities estimate were derived from N-[3H]methulscopolamine displacement experiments and represent the mean +_S.E.M. Compound

M2 (Ki; nM)

M2-trunc/ M 3-tail (K i" nM)

M3 (Ki; nM)

[3H]NMS (K d)

0.22_+0.02 3.41 +0.50 231 _+20.8 14.3 +_1.46 0.26_+0.02 1.02 _+0.07 11.8 +_1.31 1.21 _+0.16 0.19+0.01 10.4__ 0.93 137 _+7.30 2.00+0.17 4.69 + 0.72 278+32.6 57.1 +_6.71 491 + 51.4 135+ 15.3 150+ 12.9 60.9+ 8.32

0.10_+0.01 1.23_+0.11 86.3 +_5.33 52.7 +_3.19 0.34 _+0.02 1.49 +_0.09 30.9 _+4.29 3.43 +_0.18 0.36+_0.04 13.4 +_1.05 297 _+19.8 2.91 +_0.31 14.2 _+3.21 687_+52.0 566_+43 38.9 _+5.48 52.7 _+6.30 148 + 15.4 40.6_+3.65

0.08_+0.01 0.42+_0.03 19.4 _+1.93 207 +_11.9 35.1 _+3.32 32.4 +_2.81 581 _+36.6 58.0 _+6.23 2.24 _+0.41 26.5 +_3.56 1071 _+97.5 1.92 +0.20 91.2 _+8.25 230_+19.6 195+ 13.1 108 _+6.54 27.3+4.88 198 _+21.0 12.2 +_1.07

4-DAMP p-F-HHSiD Methoctramine Tripitramine CC 8 CC 9 Dipitramine ML 116 ML 105 ML 187 ML 121 AO 47 ML 183 CC 21 Pirenzepine Rociverine (IxM) SS 62, CH3I SS 63, CH3I

this reason, we started our pharmacological analysis with two selective muscarinic M 3 receptor antagonists: 4-DAMP and p-F-HHSiD. These compounds respectively showed eight- and 12-fold higher affinity for M 3 respect to M 2 and an intermediate affinity for the split chimeric receptor (Table 1). If we consider that five of the TMDs that constitute the chimeric receptor derive from the M 2 receptor, we can assume that the carboxyl-terminal domain of M 3 increases the affinity for 4-DAMP and p-F-HHSiD. Methoctramine is a well-known muscarinic M 2 selective antagonist that in our experiments showed a 15-fold higher affinity for M 2 as compared to M 3. The affinity found for the split chimeric M z-trunc/M3-tail receptor was intermediate (Table 1). A recently synthesized muscarinic M 2 receptor ligand, tripitramine (Fig. 2), has shown a higher selectivity for M 2 compared to methoctramine (Maggio et al., 1994). Tripitramine has a 135-fold higher affinity for M 2 with respect to M3; surprisingly the split chimeric Mz-trunc/M3-tail receptor binds tripitramine as well as M 2 (Table 1). In this case it seems that the carboxyl-terminal segment of M 3 has not affected the binding of the antagonist tripitramine. In order to better understand how this compound binds to the chimeric receptor, we tested a series of tripitramine analogues (Fig. 2, Table 1). CC8 is a tripitramine analogue in which the PBD (ll-acetyl-5,11-dihydrobenzole[e]pyrido[3,2-b][1,4]diazepin-6-one) groups have been replaced by in PBD (ll-acetyl-6,11-dihydro-benzo[ b]pyrido[2,3-e][1,4]diazepin-5-one) groups; this corn-

317

pound maintains the same affinity ratio between M 2 and M z-trunc/M3-tail receptor, but it is four-fold less potent compared to tripitramine. The addition to CC8 of a fourth inPBD group to form CC9 results in a drastic loss of affinity for all the three receptors, indicating that the steric hindrance of the added group is unfavorably accepted by the muscarinic binding pocket. Subtraction of a PBD from tripitramine to form dipitramine decreases the affinity for M 2 and M 2-trunc/M 3-tail respectively of 4- and 10-times, indicating that this group actively interacts with the binding site of these two receptors to increase the affinity. It is important to notice that in order to maintain an affinity ratio of more than one order of magnitude between M 3 on the one hand, and M z-trunc/M3-tail on the other hand, it is important to have at least two PBDs bound to the molecule. Consequently, the selectivity of ML116, ML 105, ML187, ML121 and AO47 that have only one PBD is considerably reduced. Modification in the polymethylene chain variably affects the binding; for example ML 116, which has a butyryl instead of an acetyl moeity between the tricyclic system and the terminal amino group, maintains a high affinity for both M 2 and M z - t r u n c / M 3-tail, while the reduction of the distance between the two central amino groups from eight to six carbons, as in AO47, reduces the affinity for M 2 and M2-trunc/M3-tail. A more pronounced diminution of affinity is obtained by the transformation of two amine functions into two amide groups (compare ML187 with ML116 and ML183 with dipitramine). Truncation of the polymethylene tetra-amine chain and addition of a 2methoxybenzyl group, as in ML121, also reduce the affinity for M 2 and M2-trunc/M3-tail. All these tripitramine analogues showed a difference in affinity between M 2 and Mz-trunc/M3-tail of not more than three-fold (Table 1), suggesting that the PBDs probably recognize either on M 2 and on the split chimeric M 2-trunc/M 3-tail receptors identical amino acid determinants in the binding pockets. This is explained by the high homology existing between the two receptors. If we exclude the C-terminal portion of the third cytoplasmic loop (probably not involved in antagonist binding) only a few amino acids in the TMDs VI and VII differentiate M 2 from M 2-trunc/M 3-tail (Fig. 1). The structure of CC21 is closely related to that of methoctramine, rather than tripitramine, as confirmed also by the fact that CC21 was only four-fold less potent than, and equipotent to, methoctramine at M 2 and M 3 receptors, respectively. From a chemical point of view, methoctramine and CC21 structures can be easily superimposed, the only difference is, being part of the tricyclic moiety of CC21, namely a phenyl group and the hydroxyl function, which is lacking in methoctramine. It can be derived that the significant loss of affinity with respect to methoctramine displayed by CC21 toward the Mz-trunc/M3-tail receptor can probably be ascribed to the tricyclic moiety of CC21, which may interact with the chimeric receptor differently as compared to M 2 and M 3 receptors.

318


CH:~:)~N~ N H

H

H• O

Methoctramine

O~~

O

H

H O

N

Tripitramine

~

N

~

c! ~

I

~N

O H

~ O O ~N/ ~

S NIN

CH3 ~

CC 8

CH3

~ I H

o

O~1~~ 0

"N

Cl 'la I ~ N ~ ~ N ~ N

N ~%~, I / ~ NN ,~(pN. ~ ~~ 0

ll

-:

H

cc9

N~ ~ 1 ~

H Dipitramine (ML 141)

03/'

N~

cHa

OHm'

N

O H

0

"H

H O "N

ML116 Gila I o ~ ~ N ~ ~ ~ N ~ N CH3 Fig. 2. Chemical structures.

H

12t

319

S. Chiacchio et a l . / Pharmaceutica Acta Helvetiae 74 (2000) 315-326

H

"N

"~..~"~ H

0

O

ML187

o#~NH~

ML 105

"~~-~

~ ~ N

o

iy~ 0

N

O

AO 47

ML 121

I

CH3

fi H

0.3

O

~

~

N

~

N ~ I CH3

N

,~ ~

N.

CH3'-'O" v

H"N O OH

~

N

CH3

2

CC21

~ HN

0

CH3 I

O

~

~ O

N

O

Dipitramine amide (ML 183)

H

o~'-I O,, I r Pirenzepine

Rociverine

o./.~

~ SS 63

SS 62 Fig. 2 (continued).

/~N ~

L

I~1

320


To continue our pharmacological characterization we tested two muscarinic M 1 receptor antagonists: pirenzepine, a well-known high affinity ligand, and rociverine (the enantiomer (1S,ZS)S-1, Fig. 2), a low affinity compound that we have characterized on CHO-K1 cell lines stably transfected with the five muscarinic receptors (Barbier et al., 1995). We selected rociverine because it has a pattern of selectivity for the muscarinic receptors very similar to pirenzepine. While rociverine showed a similar affinity for the three receptors tested, pirenzepine was the only substance in our study that showed a higher affinity for the split chimeric M z-trunc/M3-tail receptor compared to M 2 and M3; the affinity ratios of M 2trunc/M3-tail vs. M 2 and M 3 were 12- and three-fold, respectively (Table 1). This result was quite unexpected considering the fact that pirenzepine, like tripitramine and many of its congeners mentioned above, contains a PBD group. In this case it is clear that the piperazine ring of pirenzepine determines the specificity for the split chimeric M 2-trunc/M 3-tail receptor. The last two compounds that we tested, SS 62, CH3I and SS 63, CH 31 (Fig. 2), have been shown to differentiate clearly between guinea pig atrium and guinea pig ileum when tested in functional experiments, but when they were tested on cell lines stably transfected with muscarinic receptors they completely lost their selectivity (Angeli et al., 1993). The affinity we found for our split chimeric M z-trunc/M3-tail receptor was intermediate between those found for M 2 and M3 (Table 1). The pharmacological profile of the split chimeric receptor was clearly different from that of M 2 and M 3 wild-type receptors; while most of the compounds tested showed affinities intermediate between those of M 2 and M 3, tripitramine and pirenzepine showed a high affinity for the split chimeric Mz-trunc/M3-tail receptor, therefore, they were used to demonstrate M z / M 3 wild type receptor interaction.

4. Functional studies with the split chimeric M 2-trunc/ M3-tail receptor In functional experiments we studied whether or not the split chimeric Mz-trunc/M3-tail receptor retains in part the functional activity of the wild type M 2 and M 3 receptors. Carbachol stimulation of COS-7 cells co-transfected with the split chimeric M z-trunc/M3-tail receptor neither inhibited forskolin-stimulated cAMP accumulation nor induced inositol monophosphate accumulation (Fig. 3a and b). It is important to note that carbachol stimulation of COS-7 cells co-transfected with fragments originating from the same receptor, M 2-trunc/Mz-tail and M 3-trunc/M 3tail resulted respectively in the inhibition of adenylate

150

50 40 E=

100

-*,,4

"-

30 20

50

10

o
Ser) I

I

I

4020-

O

O

00.1

N

I

1 10 100 1000 Tripitramine concentration (nM)

104

~-

0 0.1

co-exmssion of M3-short with Mz-trunc l

I

I

I

1 10 100 1000 Tripitramine concentration (nM)

104

Fig. 4. Competition studies between the antagonists pirenzepine and tripitramine and the radioligand N-[3H]methylscopolamine (filled symbols) in membranes prepared from COS-7 cells co-transfected with M : + M3-tail (a), M 3 + Mz-trunc (b), M 3 + Mz(Asn404 ~ Ser) (c) and M3-short + M:-trunc (d). The open symbols in panel a, b and c represent curves obtained in membranes from COS-7 cells separately transfected with " M 2 and M3-tail", " M 3 and Mz-trunc" and " M 3 and Mz(Asn404 ~ Ser)", respectively. The open symbols in panel d represents the curve obtained with the M3-short receptor transfected alone.

M:(Asn404 ~ Ser) and M 3 in terms of plasmid DNA amount was 3.3 p~g and 0.7 txg, respectively. With this ratio of plasmid DNA we had 17% of the total binding in the high-affinity state, while the rest was in the low-affinity state (Table 2). These results were not observed on membranes obtained from COS-7 cells separately transfected with M z(Asn404 ~ Ser) and M 3 and then pooled together (Fig. 4c). From these experiments we can conclude that a molecular interaction occurs between Mz(Asn404 ~ Ser) and M 3 when they are expressed together in the same cells, and that a receptor with a high affinity for tripitramine is formed. As M z(Asn404 ~ Ser) and M 2 wild-type have only one amino acid difference, they probably have the same steric impediment, and consequently steric hindrance does not seem to play against the formation of the chimeric M 2-trunc/M 3-tail receptor.

The comparison of these data with those obtained with the chimeric (Xzc/M 3 and M3/oLzc receptors deserves comment. In the latter case there is no interaction between the N- and C-terminal receptor parts inside the chimeric OLzc/M 3 and M3/OLzc receptors because they originate from two different receptor types, therefore all the fragments are in some way forced to cross-interact. In the case of the wild-type M 2 and M 3 receptors, the intramolecular interaction of the N- and C-terminal parts inside the receptors competes with the intermolecular interaction with the foreign fragments. For example, in the co-transfection of M 2 with M3-tail, the trunc part of M 2 will have a far greater probability of hitting the tail part of M 2 than the foreign M3-tail fragment. Since we have calculated in our experiments that up to 34% of the total binding could be found in the chimeric receptor form, it is likely that the relative affinity between the trunc part of M 2 and the


323

Table 2 Antagonist binding parameters of pirenzepine and tripitramine for the wild-type, fragmented and mutant muscarinic receptors co-expressed in COS-7 cells COS-7 cells were co-transfected ( U ) or separately transfected ( - ) with plasmid DNAs coding for M 2 and M 3 wild-type receptors and for different muscarinic receptor mutants. A total amount of 4 txg DNA was transfected (the relative amount of each plasmid is given in parentheses). IC 50 values were obtained in displacement curves against 200 pM N-[3H]methylscopolamine. Competition curves were fitted to one- or two-site binding models; to determine whether the data were best fitted by a one- or two-site model, the residual sums of squares were compared by Scheffe's F-test. Data are presented as mean _+S.E. of at least four experiments, each performed in duplicate. Small numbers in square brackets represent the ICs0 s corrected for the affinity of N-[3H]methylscopolamine to the wild type M 2 and M 3 receptors and the split chimeric Mz-trunc/M3-tail receptor (see Table 1). ND = Non Detectable. Transfected receptors

% of high-affinity binding

IC50 H (nM)

IC50 L (nM)

Pirenzepine M 2 (1.6 Ixg) O M 3 -tail (2.4 ~g) M 2 (1.6 Ixg)-M3-tail (2.4 Ixg)

34 +_4 ND

114 +_8 [38 +_2.6] -

1572 +_56 [748 +_27] 1462 +_61 [696+_ 29]

Tripitramine M 3 (0.5~g)O M2-trunc (3.5 I~g) M 3 (0.5 Ixg)-M2-trunc (3.5 Ixg) M 2 (Asn404 ~ Ser)(3.3 ~ g ) U M 3 (0.7 Ixg) M 2 (Ash404 ~ Ser)(3.3 IXg)-M 3 (0.7 Ixg) M3-short (0.5 IXg)U Mz-trunc (3.5 Ixg) M3-short (4 ~g)

22 +_6 ND 17 +_5 ND ND ND

2.22 _+0.29 [1.05 +0.13] 1.18 +_0.22 [0.56+0.1] -

402 _+33 215+_21 408 +_25 225 +_31 307 +_23 255 _+29

M3-tail receptor fragment is high enough to overcome this disadvantage. Furthermore, it is possible that ligands, depending on their relative affinities for the chimeric and the wild-type receptors, can favour the intra- or the intermolecular interaction.

6. Importance of the third cytoplasmic loop in receptor / receptor interaction Previous findings indicated that receptor interaction is prevented by the shortening of the third cytoplasmic loop (Maggio et al., 1996). Chimeric O~2c/M 3 and M3/o~2c receptors with the deletion of a large portion of the third cytoplasmic loop were no longer able to cross-interact and bind M 3 muscarinic and oL2c adrenergic receptor ligands when co-expressed in the same cells. Furthermore, the activity of a functionally impaired M 3 muscarinic receptor mutant (in which 16 amino acids of the N-terminal part of the third cytoplasmic loop were substituted with the corresponding M 2 sequence, M 3 / M 216aa) was rescued by the co-expression with a healthy M3-trunc fragment bearing the correct sequence; conversely the deletion of 196 amino acids from the third cytoplasmic loop of M3/Mzl6aa, though leaving intact the binding characteristics of the receptor, completely prevented the functional rescue operated by M3-trunc. Experiments performed with a short form of the wild-type M 3 muscarinic receptor (M3-short, Fig. 1) in which 196 amino acids of the third cytoplasmic loop had been removed, demonstrated that the large deletion of the third cytoplasmic loop leaves unvaried the binding characteristics of the receptor, the phosphatidyl inositol hydrolysis activity and the ability of the receptor to internalize (Maggio et al., 1996). We co-transfected this

[115 _+9.4] [61.4+6.0] [116+_ 7.1] [64.3 +_8.8] [87.7 +_6.6] [72.8 +- 8.3]

M3-short receptor with the M2-trunc receptor fragment, and then we tested tripitramine in a displacement curve against N-[3H]methylscopolamine. While with the cotransfection of the muscarinic M 3 wild-type receptor with the M z-trunc fragment we obtained a biphasic curve (see above), in this case we observed a monophasic curve, expression of a single population of binding sites (the Hill coefficient was not significantly different from unity, Fig. 4d). The affinity of tripitramine was equal to the M 3-short receptor when it was transfected alone (Table 2). These data clearly confirm that the recruitment of the carboxyl terminal receptor portion of M 3 by the Mz-trunc fragment (a fundamental requirement for the formation of the chimeric M z-trunc/M3-tail receptor) directly depends on the length of the third cytoplasmic loop.

7. Western blotting There is the possibility that the wild-type M 2 and M 3 receptors could be split proteolytically and generate truncated receptor fragments. This could occur especially in the third cytoplasmic loop, which is extremely long in all the muscarinic receptors. If this is the case, the data we generated could be explained by fragment reassembling rather then receptor interaction. In order to exclude this possibility we used tagged (c-myc-M 2 and HA-M 3) muscarinic receptors and we performed SDS-polyacrylamide gel electrophoresis and Western blotting of the purified receptors. Western blotting showed no sign of fragmentation of either of the two receptors; the two proteins migrated as a doublet under the 79 kDa marker (Fig. 5). Presumably, the doublet reflects glycosylated (the larger

324

S. Chiacchio et a L / Pharmaceutica Acta Heluetiae 74 (2000) 315-326

mouse

mouse

anti-c-myc z .!

E

Z

~

I

anti-HA

!

~a

z! Z

206000 117000 79000

483(X)

.'M7(K) 29300 21300

Fig. 5. Immunoblotting of lysates from COS-7 cells expressing epitope-tagged c-myc-M~ and haemagglutinin (HA)-M 3 muscarinic receptors. Crude membrane preparation were immunoblotted following S D S / P A G E with the anti c-myc or the anti-HA policlonal antibodies. The blots reveal immunoreactive bands corresponding to the expected monomeric forms of M 2 and M 3. The two proteins migrated as a doublet under the 79 kDa marker. Presumably, the doublet reflects glycosylated (the larger form) and nonglycosylated (the lower form) receptors.

form) and nonglycosylated (the lower form) receptors (Debburman et al., 1995).

8. Conclusions There is currently much debate on the role of (hetero/homo)dimers in G protein-coupled receptor, but four recent works, three of which on GABA B receptors (Kaupmann et al., 1998; Jones et al., 1998; White et al., 1998) and one on ~ and K opioid-receptors (Jordan and Devi, 1999) have shed light on the functional significance

of this phenomenon. In the three GAB A B receptor papers, neither GABA BR 1 nor GABA BR2, when expressed individually, activates inwardly rectifying K § channels, however, the combination of GABABR1 and GABABR2 confers robust stimulation of K + channel activity. This indicates that heteromeric assembly of the two GABA B receptor subunits is essential to confer function. In the opioid-receptor work, heterodimerization of two fully functional opioid receptors, g and K, results in a new receptor that exhibits ligand binding and functional properties that are distinct from those of either receptor. The molecular mechanism that caused dimerization is not known, but

325


~N

muscarinic M 3 receptor

.~~

.~ ~ ~ ~ .....~~ .

~

N

muscarinic M 2 receptor ~-..:~.. ~.-".S'.-:-

-

~ ~ i i : . ,

~~ ......... ~

~

~

~

,

...'::.. . . . . . . . . . i ! i ~ j ~ ! .............

.

.

,

~-~,:~ - ~-;.'~-i

,, . e0

i~

- i~$A~~~"

~-:

COOH

chimeric muscarinic M2-truncAVI3-tailreceptor Fig. 6. Model depicting the hypothetical interaction between the wild type M 2 and M 3 receptors. The length of the third cytoplasmic loop is thought to promote the exchange of the Mz-trunc and M3-tail portions between the M 2 and M 3 receptors. The elliptic background highlights the formation of the hybrid M 2-trunc/M 3-tail receptor.

recently Gouldson et al. (1997; 1998), using computer simulations, have proposed domain swapping in G protein-coupled receptor dimerization. Domain swapping is a very efficient method of forming dimers since the interaction within the monomer are reused in the dimer. It is likely that domain swapping is the mechanism by which M 2 and M 3 muscarinic receptors interact (as depicted in Fig. 6). At the moment, we do not know the functional implications that muscarinic receptor interaction may have and further experiments will be necessary to reveal the biological significance of this phenomenon. As GTP~/S experiments indicate that the split chimeric M z-trunc/M3-tail receptor binds to G proteins, it is possible that the interaction between M 2 and M 3 receptors could result in the acquisition of a novel function, similar to what has been shown for the GAB A B and the ~ and K opioid receptors. As co-expression of different subtypes of muscarinic receptors in the same cells occurs in several tissues (Weiner et al., 1990; Fukamauchi et al., 1993; Eglen et al., 1994), it is likely that chimeric receptors could be formed in vivo and represent new pharmacological entities. In this respect it is interesting to note that recently, several articles pre-

dicted a M 2/M3 interaction to explain functional results in smooth muscle (Zholos and Bolton, 1997; Boxall et al., 1998; Sawyer and Ehlert, 1999).

Acknowledgements

The enantiomer (1S,2S)S-1 of rociverine was from Laboratory Guidotti (Pisa, Italy). [SS 62, CH3I] and [SS 63, CHgI] were a gift of Prof. Fulvio Gualtieri. Tripitramine and all its derivatives were synthesized in the laboratory of Prof. Carlo Melchiorre (Department of Pharmaceutical Sciences, University of Bologna, Italy). Tagged c-myc-M 2 and HAM 3 muscarinic receptors were kindly provided by Dr. Tatsuya Haga and Dr. Jurgen Wess, respectively.

References

Angeli, P., Gualtieri, F., Maggio, R., Paparelli, F., Scapecchi, S., 1993. Affinity profiles at five cloned human muscarinic receptors (ml-m5) of a new series of antimuscarinic drugs. Pharm. Pharmacol. Lett. 3, 84-87.

326


Barbier, P., Colelli, A., Bolognesi, M.L., Minarini, A., Tumiatti, V., Corsini, G.U., Melchiorre, C., Maggio, R., 1998. Antagonist binding profile of the split chimeric muscarinic m2-trunc/m3-tail receptor. Eur. J. Pharmacol. 355, 267-274. Barbier, P., Renzetti, A.R., Turbanti, L., Di Bugno, C., Fornai, F., Vaglini, F., Maggio, R., Corsini, G.U., 1995. Stereoselective inhibition of muscarinic receptor subtypes by the eight stereoisomers related to rociverine. Eur. J. Pharmacol. 290, 125-132. Barbier, P., Maggio, R., Colelli, A., Vaglini, F., Fornai, F., Corsini, G.U., 1996. Reconstitution of functional dopamine receptors by coexpression of N- and C-terminal receptor domains. Soc. Neuroci. Abstracts 26th Annual Meeting, 329, 1. Bliiml, K., Mutschler, E., Wess, J., 1994. Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic acetylcholine receptors. J. Biol. Chem. 269, 18870-18876. Bonner, T.I., Buckley, N.J., Young, A.C., Brann, M.R., 1987. Identification of a family of muscarinic acetylcholine receptor genes. Science 237, 527-532. Bonner, T.I., Young, A.C., Brann, M.R., Buckley, N.J., 1988. Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes. Neuron 1,403-410. Boxall, D.K., Ford, A.P.D.W., Choppin, A., Nahorski, S.R., Challiss, R.A.J., Eglen, R.M., 1998. Characterization of an atypical muscarinic cholinoceptor mediating contraction of the guinea-pig isolated uterus. Br. J. Pharmacol. 124, 1615-1622. Debburman, S.K., Kunapuli, P., Benovic, J.L., Hosey, M.M., 1995. Agonist-dependent phosphorylation of human muscarinic receptors in Spodoptera frugiperda insect cell membrane by G protein-coupled receptor kinases. Mol. Pharmacol. 47, 224-233. Eglen, R.M., Reddy, H., Watson, N., Challiss, R.A.J., 1994. Muscarinic acetylcholine receptor subtypes in smooth muscle. Trends Pharmacol. Sci. 15, 114-119. Fukamauchi, F., Saunders, P.L., Hough, C., Chuang, D.M., 1993. Agonist-induced down-regulation and antagonist-induced up-regulation of m2- and m3-muscarinic acetylcholine receptor mRNA and protein in cultured cerebellar granule cells. Mol. Pharmacol. 44, 940-949. Gouldson, P.R., Snell, C.R., Reynolds, C.A., 1997. A new approach to docking in the [32-adrenergic receptor which exploits the domain structure of G-protein coupled receptors. J. Med. Chem. 40, 38713886. Gouldson, P.R., Snell, C.R., Bywater, R.P., Higgs, C., Reynolds, C.A., 1998. Domain swapping in G-protein coupled receptor dimers. Protein Eng. 11, 1181-1193. Jakubik, J., Wess, J., 1999. Use of a sandwich enzyme-linked immunosorbent assay strategy to study mechanism of G protein-coupled receptor assembly. J. Biol. Chem. 274, 1349-1358. Jones, K.A., Borowsky, B., Tamm, J.A., Craig, D.A., Durkin, M.M., Dai, M., Yao, W.-J., Johnson, M., Gunwaldsen, C., Huang, L.-Y., Tang, C., Shen, Q., Salon, J.A., Morse, K., Laz, T., Smith, K.E., Nagarathnam, D., Noble, S.A., Branchek, T.A., Gerald, C., 1998. GABA B receptors function as a heteromeric assembly of the subunit GABABR1 and GABABR2. Nature 396, 674-679. Jordan, B.A., Devi, L.A., 1999. G-protein-coupled receptor heterodimerization modulates receptor function. Nature 399, 697-700. Hulme, E.C., Birdsall, N.J.M., Buckley, N.J., 1990. Muscarinic receptor subtypes. Annu. Rev. Pharmacol. Toxicol. 30, 633-673. Kahn, T.W., Engelman, D.M., 1992. Bacteriorhodopsin can be refolded from two independently stable transmembrane helices and the complementary five-helix fragment. Biochemistry 31, 6144-6151.

Kaupmann, K., Malitschek, B., Schuler, V., Heid, J., Froestl, W., Beck, B., Mosbacher, J., Bischoff, S., Kulik, A., Shigemoto, R., Karschin, A., Bettler, B., 1998. GABAB-receptor subtypes assemble into functional heteromeric complexes. Nature 396, 683-687. Kobilka, B.K., Kobilka, T.S., Daniel, K., Regan, J.W., Caron, M.G., Lefkowitz, R.J., 1988. Chimeric Otz-,[3z-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity. Science 240, 1310-1316. Kubo, T., Fukuda, K., Mikami, A., Maeda, A., Takahashi, H., Mishina, M., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., Kojima, K., Matsuo, H., Hirose, T., Numa, S., 1986. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature 323, 411-416. Liao, M.J., London, E., Khorana, H.G., 1983. Regeneration of the native bacteriorhodopsin structure from two chymotryptic fragments. J. Biol. Chem. 258, 9949-9955. Maggio, R., Vogel, Z., Wess, J., 1993a. Reconstitution of functional muscarinic receptors by co-expression of amino- and carboxyl-terminal receptor fragments. FEBS Lett. 319, 195-200. Maggio, R., Vogel, Z., Wess, J., 1993b. Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular "cross-talk" between G-protein linked receptors. Proc. Natl. Acad. Sci. U.S.A. 90, 3103-3107. Maggio, R., Barbier, P., Bolognesi, M.L., Minarini, A., Tedeschi, D., Melchiorre, C., 1994. Binding profile of selective muscarinic receptor antagonist tripitramine. Eur. J. Pharm. 268, 459-462. Maggio, R., Barbier, P., Fornai, F., Corsini, G.U., 1996. Functional role of the third cytoplasmic loop in muscarinic receptor dimerization. J. Biol. Chem. 49, 31055-31060. Maggio, R., Barbier, P., Colelli, A., Salvadori, F., De Montis, G., Corsini, G.U., 1999. G protein-linked receptors: pharmacological evidence for the formation of heterodimers. J. Pharmacol. Exp. Ther. 291, 251-257. Peralta, E.G., Ashkenazi, A., Winslow, J.W., Smith, D.H., Ramachandran, J., Capon, D.J., 1987. Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. EMBO J. 6, 3923-3929. Popot, J.L., Gerchman, S.E., Engelman, D.M., 1987. Membrane folding and oligomerization: the two-stage model. J. Mol. Biol. 198, 655-676. SchiSneberg, T., Liu, J., Wess, J., 1995. Plasma membrane localization and functional rescue of truncated forms of G protein-coupled receptor. J. Biol. Chem. 270, 18000-18006. Sch~Sneberg, T., Yun, J., Wenkert, D., Wess, J., 1996. Functional rescue of mutant V2 vasopressin receptors causing nephrogenic diabetes insipidus by a co-expressed receptor polypeptide. EMBO J. 15, 1283-1291. Sawyer, G.W., Ehlert, F.J., 1999. Muscarinic M 3 receptor inactivation a pertussis toxin-sensitive contractile response in the guinea pig colon: evidence for M 2 / M 3 receptor interactions. J. Pharmacol. Exp. Ther. 289, 464-476. Weiner, D.M., Levey, A.I., Brann, M.R., 1990. Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia. Proc. Natl. Acad. Sci. U.S.A. 87, 7050-7054. White, J.H., Wise, A., Main, M.J., Green, A., Fraser, N.J., Disney, G.H., Barnes, A.A., Emson, P., Foord, S.M., Marshal, F.H., 1998. Heterodimerization is required for the formation of a functional GAB A B receptor. Nature 396, 679-682. Zholos, A.V., Bolton, T.B., 1997. Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle. Br. J. Pharmacol. 122, 885-893.



Constitutive activity of G protein coupled receptors and drug action Rob Leurs

*,

Maria Sol Rodriguez Pena, Remko A. Bakker, Astrid E. Alewijnse, Henk Timmerman

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, Netherlands

Keywords: G protein coupled receptor (GPCR); Constitutive activity; Inverse agonist; Neutral antagonist; Drug design; Disease; Autoantibodies

1. Introduction

2. Constitutive activity and the two-state model

An interesting and new concept in G protein coupled receptor (GPCR)-mediated drug action is the occurrence of constitutive activity of GPCRs in the absence of agonist stimulation. Originally reported for the ~-opioid receptor in NG108-15 cells (Costa and Herz, 1989), this GPCR activity has now been described for a variety of GPCRs (Milligan et al., 1995). Concomitant with the notion of agonist-independent signalling it has been realised that the constitutive GPCR activity can be inhibited by some antagonists (inverse agonists), but not by all antagonists (neutral antagonists) introducing the need for a pharmacological reclassification of GPCR antagonists (Milligan et al., 1995). Moreover, as previously described for agonists also for inverse agonist a full spectrum of (negative) intrinsic activity from - 1 to 0 can be observed (Milligan et al., 1995; Leurs et al., 1998). Neutral antagonists, i.e., compounds with actually no intrinsic activity, are quite rare. Nevertheless, for some GPCRs, ligands with virtually no (negative) intrinsic activity are found. For example, the H z receptor (the first described H 2 antagonists burimamide) has been identified as a ligand with very low (Alewijnse et al., 1998) or no intrinsic activity (Smit et al., 1996). Burimamide is able to block the effects of both an agonist (histamine) and an inverse agonist (cimetidine) on the cAMP levels in CHO cells expressing the H 2 receptor (Fig. 1), with apparent p K B values that match its p K~ value (Alewijnse et al., 1998).

To explain the above described findings the GPCR protein is thought to isomerise between (at least) two different states, an inactive (R) and active (R*) conformation. In this oversimplified scheme the R* state is considered to be responsible for effective G protein activation and the equilibrium between R and R* determines the level of basal, constitutive GPCR signalling (Milligan et al., 1995; Kenakin, 1996; Leurs et al., 1998). Agonist binding is thought to shift the equilibrium towards R*, leading to an increased GPCR activity. Such a scheme also implicates that, by favouring the inactive R state, inverse agonists can inhibit constitutive GPCR signalling. In this scheme neutral antagonists do not distinguish between the R and R* state and are thus also not able to shift the equilibrium between R and R*. Consequently, neutral antagonists do not modulate constitutive GPCR signalling (Milligan et al., 1995; Kenakin, 1996; Leurs et al., 1998).

* Corresponding author. Tel.: +31-204447579; fax: +31-204447610; e-mail: [email protected]

3. Constitutive activity in a cellular context

It is accepted for some time that agonists can have different intrinsic activities in different cell systems. Similarly, the degree of constitutive GPCR activity and the concomitant inverse agonistic behaviour of GPCR ligands are also highly dependent upon the cellular context. As for GPCR agonism, one of the main determinants for constitutive GPCR signalling is the expression level of the respective GPCR. As can be seen in Fig. 2A, the transient expression of the histamine H~ receptor in COS-7 cells leads to a expression-dependent increase in luciferase ac-

0031-6865/00/$- see front matter 9 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 1 - 6 8 6 5 ( 9 9 ) 0 0 0 4 2 - 4

328

R. Leurs et al. / Pharmaceutica Acta Helvetiae 74 (2000) 327-331

125

A

125]

B

a,

=:o,

=-

Receptor Chemistry Towards the Third Millennium

Plant Genetic Engineering: Towards the Third Millennium

Anion receptor chemistry

Heroes Of The Third Millennium

Heroes Of The Third Millennium

Calvin for the Third Millennium

Fluorine Chemistry at the Millennium

Towards A Third Theatre

HyperReality: Paradigm for the Third Millennium

Intravenous Immunoglobulins in the Third Millennium

A CREED FOR THE THIRD MILLENNIUM

Intravenous Immunoglobulins: In The Third Millennium

A Creed For The Third Millennium

A Global Strategy for Housing in the Third Millennium (Technology in the Third Millennium, Vol 2)

Organic Chemistry, Third Edition

Soil Chemistry, Third Edition

The Delta Receptor

The Third Millennium: Living in the Posthistoric World

The Evolving Self: A Psychology for the Third Millennium

The Delta Receptor

The Nuclear Receptor FactsBook

Millennium

Millennium

The New Reformation (Ministry for the Third Millennium)

Welfare State Change: Towards a Third Way?

ACTH Receptor

b Receptor

Millennium

Millennium

C5a Receptor

Receptor Chemistry Towards the Third Millennium

Plant Genetic Engineering: Towards the Third Millennium

Anion receptor chemistry

Heroes Of The Third Millennium

Heroes Of The Third Millennium

Calvin for the Third Millennium

Fluorine Chemistry at the Millennium

Towards A Third Theatre

HyperReality: Paradigm for the Third Millennium

Intravenous Immunoglobulins in the Third Millennium

A CREED FOR THE THIRD MILLENNIUM

Intravenous Immunoglobulins: In The Third Millennium

A Creed For The Third Millennium

A Global Strategy for Housing in the Third Millennium (Technology in the Third Millennium, Vol 2)

Organic Chemistry, Third Edition

Soil Chemistry, Third Edition

The Delta Receptor

The Third Millennium: Living in the Posthistoric World

The Evolving Self: A Psychology for the Third Millennium

The Delta Receptor

The Nuclear Receptor FactsBook

Millennium

Millennium

The New Reformation (Ministry for the Third Millennium)

Welfare State Change: Towards a Third Way?

ACTH Receptor

b Receptor

Millennium

Millennium

C5a Receptor

Recommend Documents