Chemical Genomics: Reviews and Protocols (Methods in Molecular Biology)

METHODS IN MOLECULAR BIOLOGY ™

310

Chemical Genomics Reviews and Protocols Edited by

Edward D. Zanders

Chemical Genomics

M E T H O D S I N M O L E C U L A R B I O L O G Y™

John M. Walker, SERIES EDITOR 327. 327 Epidermal Growth Factor: Methods and Protocols, edited by Tarun B. Patel and Paul J. Bertics, 2006 326 In Situ Hybridization Protocols, ThirdEdition, 326. edited by Ian A. Darby and Tim D. Hewitson, 2006 325. 325 Nuclear Reprogramming: Methods and Protocols, edited by Steve Pells, 2006 324. 324 Hormone Assays in Biological Fluids, edited by Michael J. Wheeler and J. S. Morley Hutchinson, 2006 323. 323 Arabidopsis Protocols, Second Edition, edited by Julio Salinas and Jose J. Sanchez-Serrano, 2006 322 Xenopus Protocols: Cell Biology and Signal 322. Transduction, edited by X. Johné Liu, 2006 321 Microfluidic Techniques: Reviews and 321. Protocols, edited by Shelley D. Minteer, 2006 320. 320 Cytochrome P450 Protocols, Second Edition, edited by Ian R. Phillips and Elizabeth A. Shephard, 2006 319 Cell Imaging Techniques, Methods and 319. Protocols, edited by Douglas J. Taatjes and Brooke T. Mossman, 2006 318 Plant Cell Culture Protocols, Second Edition, 318. edited by Victor M. Loyola-Vargas and Felipe Vázquez-Flota, 2005 317 Differential Display Methods and Protocols, 317. Second Edition, edited by Peng Liang, Jonathan Meade, and Arthur B. Pardee, 2005 316. 316 Bioinformatics and Drug Discovery, edited by Richard S. Larson, 2005 315. 315 Mast Cells: Methods and Protocols, edited by Guha Krishnaswamy and David S. Chi, 2005 314 DNA Repair Protocols: Mammalian Systems, 314. Second Edition, edited by Daryl S. Henderson, 2005 313. 313 Yeast Protocols: Second Edition, edited by Wei Xiao, 2005 312. 312 Calcium Signaling Protocols: Second Edition, edited by David G. Lambert, 2005 311 Pharmacogenomics: Methods and 311. Applications, edited by Federico Innocenti, 2005 310 Chemical Genomics: Reviews and Protocols, 310. edited by Edward D. Zanders, 2005 309. 309 RNA Silencing: Methods and Protocols, edited by Gordon Carmichael, 2005

308 Therapeutic Proteins: Methods and Protocols, 308. edited by C. Mark Smales and David C. James, 2005 307. 307 Phosphodiesterase Methods and Protocols, edited by Claire Lugnier, 2005 306 Receptor Binding Techniques: Second 306. Edition, edited by Anthony P. Davenport, 2005 305. 305 Protein–Ligand Interactions: Methods and Applications, edited by G. Ulrich Nienhaus, 2005 304. 304 Human Retrovirus Protocols: Virology and Molecular Biology, edited by Tuofu Zhu, 2005 303. 303 NanoBiotechnology Protocols, edited by Sandra J. Rosenthal and David W. Wright, 2005 302. 302 Handbook of ELISPOT: Methods and Protocols, edited by Alexander E. Kalyuzhny, 2005 301 Ubiquitin–Proteasome Protocols, edited by 301. Cam Patterson and Douglas M. Cyr, 2005 300. 300 Protein Nanotechnology: Protocols, Instrumentation, and Applications, edited by Tuan Vo-Dinh, 2005 299. 299 Amyloid Proteins: Methods and Protocols, edited by Einar M. Sigurdsson, 2005 298 Peptide Synthesis and Application, edited by 298. John Howl, 2005 297. 297 Forensic DNA Typing Protocols, edited by Angel Carracedo, 2005 296. 296 Cell Cycle Control: Mechanisms and Protocols, edited by Tim Humphrey and Gavin Brooks, 2005 295. 295 Immunochemical Protocols, Third Edition, edited by Robert Burns, 2005 294. 294 Cell Migration: Developmental Methods and Protocols, edited by Jun-Lin Guan, 2005 293. 293 Laser Capture Microdissection: Methods and Protocols, edited by Graeme I. Murray and Stephanie Curran, 2005 292. 292 DNA Viruses: Methods and Protocols, edited by Paul M. Lieberman, 2005 291. 291 Molecular Toxicology Protocols, edited by Phouthone Keohavong and Stephen G. Grant, 2005 290. 290 Basic Cell Culture Protocols, Third Edition, edited by Cheryl D. Helgason and Cindy L. Miller, 2005 289. Epidermal Cells, Methods and Applications, edited by Kursad Turksen, 2005 288. Oligonucleotide Synthesis, Methods and Applications, edited by Piet Herdewijn, 2005

M ET H O D S I N M O L E C U L A R B I O L O GY™

Chemical Genomics Reviews and Protocols

Edited by

Edward D. Zanders, CamBP Ltd., Cambridge, UK

P hD

© 2005 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular BiologyTM is a trademark of The Humana Press Inc. All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover illustration: Fig. 1A from Chapter 11, "Human Solid Tumors," by Pietro Polito, Paola Dal Cin, Maria Debiec-Rychter, and Anne Hagemeijer. Production Editor: Tracy Catanese Cover design by Patricia F. Cleary For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected]; or visit our Website: www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $30.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [1-58829-399-8/05 $30.00 ]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 e-ISBN: 1-59259-148-6 Library of Congress Cataloging in Publication Data Chemical genomics : reviews and protocols / edited by Edward D. Zanders. p. ; cm. -- (Methods in molecular biology ; v. 310) Includes bibliographical references and index. ISBN 1-58829-399-8 (alk. paper) 1. Genomics. 2. Proteomics. 3. Pharmacogenetics. 4. Biochemical genetics. 5. DNA microarrays. 6. Combinatorial chemistry. [DNLM: 1. Molecular Probes--chemical synthesis--Laboratory Manuals. 2. Protein Array Analysis-methods--Laboratory Manuals. 3. Combinatorial Chemistry Techniques--Laboratory Manuals. 4. Genomics--methods--Laboratory Manuals. QU 25 C517 2005] I. Zanders, Edward D. II. Series. QH431.C45197 2005 572.8'6--dc22 2005013118

Preface Chemical genomics is an exciting new field that aims to transform biological chemistry into a high-throughput industrialized process, much in the same way that molecular biology has been transformed by genomics. The interaction of small organic molecules with biological systems (mostly proteins) underpins drug discovery in the pharmaceutical and biotechnology industries, and therefore a volume of laboratory protocols that covers the key aspects of chemical genomics would be of use to biologists and chemists in these organizations. Academic scientists have been exploring the functions of proteins using small molecules as probes for many years and therefore would also benefit from sharing ideas and laboratory procedures. Whatever the organizational backgrounds of the scientists involved, the challenges of extracting the maximum human benefit from genome sequencing projects remains considerable, and one where it is increasingly recognized that chemical genomics will play an important part. Chemical Genomics: Reviews and Protocols is divided into two sections, the first being a series of reviews to describe what chemical genomics is about and to set the scene for the protocol chapters. The subject is introduced by Paul Caron, who explains the various “flavors” of chemical genomics. This is followed by Lutz Weber and Philip Dean who cover the interaction between organic molecules and protein targets from the different perspectives of laboratory experimentation and in silico design. The protocols begin with the methods developed in Christopher Lowes’ laboratory (Roque et al.) for what could be described as a classical example of chemical genomics, namely the design of small molecules as affinity ligands for specific protein families. The theme is continued with detailed protocols for in silico docking by Jongejan et al. that highlights the importance of computational approaches to protein–small molecule interactions. The remaining protocols are directed towards the aim of producing highly diverse collections of proteins, carbohydrates, and small molecules for use in arrays containing large numbers of molecules. This highthroughput approach to screening for interaction between small and large biological molecules is the essence of chemical genomics. The chapters by Ryu, Doyle, Murphy, Sawasaki, Endo, Kohno, and Hoyt cover methods for the production of proteins and carbohydrates using different expression systems. Webster and Oxley give a protocol for analyzing the proteins using mass spectrometry. The techniques for arraying these proteins and carbohydrates on solid supports are detailed in the chapters by Blackburn, Marik, and Wang. Finally

v

vi

Preface

an in vivo method for identifying small molecule–protein interactions is described by Khazak et al. using the yeast two-hybrid system. Although we recognize that no single book on chemical genomics can be totally comprehensive in its coverage, we hope that the protocols here, in covering the key elements of the subject, will be of genuine use to the wide variety of scientists in this rapidly expanding field. Edward D. Zanders

Contents Preface .............................................................................................................. v Contributors ..................................................................................................... ix

PART I REVIEWS 1 Introduction to Chemical Genomics Paul R. Caron ........................................................................................ 3 2 Chemistry for Chemical Genomics Lutz Weber ......................................................................................... 11 3 Computer-Aided Design of Small Molecules for Chemical Genomics Philip M. Dean .................................................................................... 25

PART II PROTOCOLS 4 Design, Synthesis, and Screening of Biomimetic Ligands for Affinity Chromatography Ana Cecília A. Roque, Geeta Gupta, and Christopher R. Lowe ......... 43 5 The Role and Application of In Silico Docking in Chemical Genomics Research Aldo Jongejan, Chris de Graaf, Nico P. E. Vermeulen, Rob Leurs, and Iwan J. P. de Esch .................................................. 63 6 Synthesis of Complex Carbohydrates and Glyconjugates: Enzymatic Synthesis of Globotetraose Using β-1,3-NAcetylgalactosaminyltransferase LgtD From Haemophilus infuenzae Strain Rd Kang Ryu, Steven Lin, Jun Shao, Jing Song, Min Chen, Wei Wang, Hanfen Li, Wen Yi, and Peng George Wang ............... 93 7 High-Throughput Cloning for Proteomics Research Sharon A. Doyle ................................................................................ 107 8 Screening for the Expression of Soluble Recombinant Protein in Escherichia coli Sharon A. Doyle ................................................................................ 115 9 High-Throughput Purification of Hexahistidine-Tagged Proteins Expressed in E. coli Michael B. Murphy and Sharon A. Doyle ......................................... 123

vii

viii

Contents

10 The Wheat Germ Cell-Free Expression System: Methods for High-Throughput Materialization of Genetic Information Tatsuya Sawasaki, Mudeppa D. Gouda, Takayasu Kawasaki, Takafumi Tsuboi, Yuzuru Tozawa, Kazuyuki Takai, and Yaeta Endo ............................................................................. 11 Advances in Genome-Wide Protein Expression Using the Wheat Germ Cell-Free System Yaeta Endo and Tatsuya Sawasaki .................................................... 12 Production of Proteins for NMR Studies Using the Wheat Germ Cell-Free System Toshiyuki Kohno ............................................................................... 13 Adenoviral Expression of Reporter Proteins for High-Throughput Cell-Based Screening Jon Hoyt and Randall W. King .......................................................... 14 Fabrication of Protein Function Microarrays for Systems-Oriented Proteomic Analysis Jonathan M. Blackburn and Darren J. Hart ...................................... 15 Peptide and Small-Molecule Microarrays Jan Marik and Kit S. Lam .................................................................. 16 Peptide Mass Fingerprinting: Protein Identification Using MALDI-TOF Mass Spectrometry Judith Webster and David Oxley ...................................................... 17 A Practical Protocol for Carbohydrate Microarrays Ruobing Wang, Shaoyi Liu, Dhaval Shah, and Denong Wang ......... 18 Development of a Yeast Two-Hybrid Screen for Selection of Human Ras–Raf Protein Interaction Inhibitors Vladimir Khazak, Erica A. Golemis, and Lutz Weber ....................... Index ............................................................................................................

131

145

169

187

197 217

227 241

253 273

Contributors JONATHAN M. BLACKBURN • Department of Biotechnology, University of the Western Cape, Cape Town, South Africa; Procognia Ltd, Maidenhead, UK PAUL R. CARON • Head of Informatics, Vertex Pharmaceuticals, Cambridge MA MIN CHEN • The State Key of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, People’s Republic of China PHILIP M. DEAN • Chief Scientific Officer, De Novo Pharmaceuticals Ltd, Cambridge, UK SHARON A. DOYLE • Proteomics Group, DOE Joint Genome Institute, Walnut Creek, CA YAETA ENDO • Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan IWAN J. P. DE ESCH• Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research (LACDR), Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands ERICA A. GOLEMIS • Division of Basic Sciences, Fox Chase Cancer Center, Philadelphia, PA MUDEPPA D. GOUDA • Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan CHRIS DE GRAAF • Division of Molecular Toxicology, Leiden/Amsterdam Center for Drug Research (LACDR), Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands GEETA GUPTA • Institute of Biotechnology, University of Cambridge, Cambridge, UK DARREN J. HART • High Throughput Group, Grenoble Outstation, European Molecular Biology Laboratory, Grenoble, France JON HOYT • Department of Cell Biology, Institute of Chemistry and Cell Biology, Boston, MA ALDO JONGEJAN • Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research (LACDR), Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands TAKAYASU KAWASAKI • Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan VLADIMIR KHAZAK • Director of Biology, NexusPharma, Langhorne, PA RANDALL W. KING • Department of Cell Biology, Institute of Chemistry and Cell Biology, Boston, MA

ix

x

Contributors

TOSHIYUKI KOHNO • Laboratory of Structural Biology, Mitsubishi Kagaku Institute of Life Sciences (MITILS), Tokyo, Japan KIT S. LAM • Division of Hematology & Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California, Davis, CA ROB LEURS • Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research (LACDR), Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands HANFEN LI • Department of Biochemistry, The Ohio State University, Columbus, OH STEVEN LIN • Department of Biochemistry, The Ohio State University, Columbus, OH SHAOYI LIU • Columbia Genome Center, Columbia University College of Physicians & Surgeons, New York, NY CHRISTOPHER R. LOWE • Institute of Biotechnology, University of Cambridge, Cambridge, UK JAN MARIK • Division of Hematology & Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California, Davis, CA MICHAEL B. MURPHY • Proteomics Group, DOE Joint Genome Institute, Walnut Creek, CA DAVID OXLEY • Proteomics Research Group, Babraham Institute, Cambridge, UK ANA CECÍLIA A. ROQUE • Institute of Biotechnology, University of Cambridge, Cambridge, UK KANG RYU • Department of Biochemistry, The Ohio State University, Columbus, OH TATSUYA SAWASAKI • Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan DHAVAL SHAH • Columbia Genome Center, Columbia University College of Physicians & Surgeons, New York, NY JUN SHAO • Department of Biochemistry, The Ohio State University, Columbus, OH JING SONG • The State Key of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, People’s Republic of China KAZUYUKI TAKAI • Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan YUZURU TOZAWA • Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan TAKAFUMI TSUBOI • Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan

Contributors

xi

NICO P. E. VERMEULEN • Division of Molecular Toxicology, Leiden/ Amsterdam Center for Drug Research (LACDR), Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands DENONG WANG • Carbohydrate Microarray Laboratory, Departments of Genetics, Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA PENG GEORGE WANG • Department of Biochemistry, The Ohio State University, Columbus, OH RUOBING WANG • Carbohydrate Microarray Laboratory, Departments of Genetics, Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA WEI WANG • The State Key of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, People’s Republic of China LUTZ WEBER • President, NexusPharma, Langhorne, PA JUDITH WEBSTER • Proteomics Research Group, Babraham Institute, Cambridge, UK WEN YI • Department of Biochemistry, The Ohio State University, Columbus, OH EDWARD D. ZANDERS • CamBP Ltd, Cambridge, UK

Introduction to Chemical Genomics

I REVIEWS

1

2

Caron

Introduction to Chemical Genomics

3

1 Introduction to Chemical Genomics Paul R. Caron

1. Introduction Small-molecule drugs are a cost-effective way to treat and prevent disease. A study by the Slone Institute published in 2002 estimated that over 50% of the adult population in the United States used at least one pharmaceutical drug during the preceding week. The positive impact of small-molecule drugs on health care has been well documented (1,2). The discovery of novel drugs has traditionally been a combination of clever science, brute force, and good fortune. With the advent of high-throughput screening technology, combinatorial chemistry, and the completion of the human genome sequence in the late 1990s, the hope was that technology could address the brute-force aspect and the genome sequence would provide insights into the underlying science, and good fortune would continue. Although there are some exceptions, productivity in the industry overall has gone down. Some of this is owing to higher regulatory standards and more difficult therapeutic areas, but a significant portion is the result of the lack of well-validated targets to apply the technology to. The industry portfolio of pharmaceutical targets of approx 500 in 1996 (3) has not been significantly expanded. The availability of the human genome sequence and novel biological tools, such as siRNA, antisense, knockouts, and transgenics, suggests that over time, the physiological function of many of the genes in the genome may be deciphered. However, the time frame for this may be much greater than most people anticipated. For comparison, the first bacterial genome sequence was completed in 1995 (4), and although we may be able to now classify the majority of the genes by biochemical function, we don’t know most of their physiological roles. One

From: Methods in Molecular Biology, vol. 310: Chemical Genomics: Reviews and Protocols Edited by: E. D. Zanders © Humana Press Inc., Totowa, NJ

3

4

Caron

conceivable way to speed up the molecular dissection of the biology underlying various disease states is to use small-molecule compounds that specifically inhibit individual targets. There are several key factors required to be successful when using small molecules to explore biology. • The relative selectivity of the chemical probes that will be used must be known. • The correlations between the cellular readout(s) used and the pathway or phenotype that is being assessed must be independently validated. • All data must be fully integrated, allowing the user to navigate through biological pathways and supporting literature, assay results, and detailed information on compounds.

2. Different Flavors of Chemical Genomics As with any emerging field, there are often differences of opinion on terminology among researchers, sometimes subtle, sometimes not. I will attempt to capture and describe the major variants and apologize if I inadvertently leave out any major themes, or end up misrepresenting some differences in trying to summarize the field.

2.1. Chemical Genetics Chemical genetics, as described by Schreiber et al. in 1999 (5), refers to the use of small molecules to induce alterations in gene products in mammalian systems, in a manner similar to using mutations. This approach became feasible through the combination of high-throughput cellular assays and diverse libraries of compounds. The ability to perform genetic screens in cellular assays vastly increases the throughput—traditionally a key limitation when studying higher organisms. It also allows the separation of effects in somatic cells from those in development. It is critical to this chemical genetic approach to have a library of compounds that have a high probability of being relatively selective; otherwise, the ability to interpret the results becomes at least as complex as deciphering highly polygenetic phenotypes. To address this, diversity-oriented synthesis has been proposed to provide arrays of complex small molecules that are easily synthesized. The natural-product basis for many of the molecules and their complexity are believed to contribute to their cellular potency and selectivity (6). This chemical genetic approach has been applied to identify novel inhibitors of alphatubulin and histone deactylation (7). As in classical genetics, a chemical genetic approach involves screening with probes that potentially could interact with any target in the genome, while trying to identify specific phenotypes. An alternate approach, termed reverse chemical

Introduction to Chemical Genomics

5

genetics, is analogous to introducing specific gene disruptions. Here, compounds that are known to specifically interact with a given target are used in broad phenotype screens to help identify the physiological role of that target.

2.2. Reverse Chemical Genetics The key to a reverse chemical genetic approach is to have a one-to-one link between the small-molecule compound and the target of interest. This can be achieved by optimization of chemical reagents by thorough profiling against other potential targets, or alternatively by altering the target itself, to introduce changes that can be exploited for specificity. This approach has been most broadly applied to members of the protein kinase family, where specific changes can be made to the residues surrounding the active site that don’t significantly alter the affinities or kinetics for natural substrates, but now allow the binding of specific inhibitor analogs (8). Replacement of the wild-type copies of a given gene with these engineered mutants allows these compounds to be used to inhibit the function of the gene in cellular assays as well as in adult animals. The in vivo assay, complete with the complications of pharmacokinetics and pharmacodynamics, closely mimics the effect that would be expected from dosing an animal with a selective inhibitor against the wild-type target. Additionally, introducing mutations into the adenosine triphosphate (ATP)binding sites of targets allows the binding of labeled ATP analogs; these can then be used to trace biochemical pathways at the molecular level by looking directly at phosphorylated substrates, thus furthering the link between the target and the observed phenotype. A comparison between standard genetics and chemical genetics is shown in Fig. 1 (see also Table 1).

2.3. Screening Screening of large sets of compounds, often assembled to be quite diverse, is often one of the first steps in a drug discovery project. The assay used for the initial screen will both help define the likelihood of getting potent hits, as well as form the foundation for the follow-up path. Assays that are more physiological require that active molecules pass through additional filters depending on the assay, such as transversing the cell wall, serum binding, bioavailability, and metabolic stability. These factors tend to decrease the hit rate, but result in molecules with better overall properties. The downside to this approach is that further compound optimization may be hindered by a lack of knowledge about the molecular target(s) of the initial hits. Biochemical-based target screening is likely to yield hits that have a clearer path to optimization, but are at higher overall risk because the link between the target and the desired physiological

6

Caron

Fig. 1. Comparison of genetic and chemical genetics. In the traditional genetics approach genes are mutated resulting in missing or altered protein products. In a chemical genetic approach, compounds bind to specific proteins modulating their normal physiological functions. The throughput at steps A, B, C is used in Table 1 to help define the different chemical genetic approaches.

changes may not be strong and these compounds may be far from having the desired physical properties. Smaller compound sets and focused compound libraries can be used to screen more broadly for physiological phenotypes. In the typical high-content screening experiment, the effects of compounds on cellular assays are captured

Approach

7

Genetics

AcB

Reverse Genetics

baC

Screening

bAc

Genomics

ABC

Profiling

AC

ChemoBaC genomics

Randomly perturbate the system looking for changes in a specific phenotype Broad search for phenotype associated with perturbations of specific genes Search for compounds which modulate a target with a known phenotype Screen all targets for specific modulators, then try to identify phenotypes for each target Broadly screen for phenotypes associated with compounds, regardless of the target Use information on targets to identify specific modulators, then look for phenotypes

Goal Target/pathway identification New role for target

Step 1 Assemble large diverse chemical library Select target

Step 2 Assay for specific phenotype Find specific inhibitor

Step 3 Identify targets of compounds that induce the phenotype Broadly assay for phenotypes

Compounds/drugs Select target Assemble Screen for for validated with presumed large diverse modulators targets physiological chemical of target role library Target/pathway Assemble Screen for Broadly assay identification large diverse modulators for phenotypes chemical of any targets library Link between Assemble compounds and large diverse phenotype— chemical efficacy or library toxicity Compounds/drugs Select targets for novel targets

Introduction to Chemical Genomics

Table 1 Approaches to Chemical Genetics

Broadly assay for phenotypes

Identify inhibitor

Broadly assay for phenotypes

7

The steps in the second column refer to Fig. 1A–C. An uppercase letter indicates that the number of compounds, targets, or assays in the approach represents a large set. A lowercase letter is use to denote a small, focused set.

8

Caron

by multiple parameters. These range from cellular and subcellular morphology, intracellular translocation events, changes in state, such as phosphorylation, and proteomic and genomic profiling. The advances in recent years of highthroughput screening, miniaturization, and imaging have made it technically practical to assemble vast databases detailing the effects of specific compounds on a genome-wide scale. The current challenge in the field is to learn how to interpret this information to expand our knowledge of the underlying biological pathways. Whereas the primary goal of the high-content screening in this chemical genomic approach may be elucidation of the link between targets and the underlying physiology, the data collected on specific compounds can be used to drive drug programs.

2.4. Chemogenomics Chemogenomics refers to the generation of specific sets of compounds with drug-like properties, which are specific for a given set of targets. The distinction between this and chemical genomics is that the knowledge extracted from sequences, 3D structures, assay results, and known chemical properties are used in the compound-design phase so that the compounds have a high likelihood of being selective and the biological consequences of inhibiting the target are known. In a typical chemogenomic approach, there is a very focused set of assays used to drive potency and selectivity, and a broader set of assays used to optimize chemical properties. The different chemical genetic approaches described in the above subheadings are summarized in Table 1. An example of a chemogenomic approach is shown in Fig. 2, where the protein sequences of members of a gene family have been overlayed on the 3D structure of an inhibitor-binding site from a representative of one of the targets in this family. The colored surface suggests areas of the active site where specificity is likely to be obtained, and the superimposed ligand suggests locations where changes to the core scaffold should be directed. The overall similarity in sequence and protein structure allows information obtained from one member of the gene family to be transferred to other members. Using this common frame of reference allows knowledge gleaned from the optimization of one chemical scaffold to be extrapolated to guide the tuning of the potency and specificity of other scaffolds that bind to the same site. 3. Goal The goal of all of these approaches is to use chemical compounds to interrogate biological systems and ultimately provide insights that lead to improved health. A key limitation in the field is the ability to extrapolate from a molecular

Introduction to Chemical Genomics

9

Fig. 2. Mapping of conserved features onto three-dimensional structure. The sequence differences between chymotrypsin and its closest homologs are mapped onto a surface representation of the structure of chymotrypsin (PDB: 1ab9). A peptide ligand (TPGVY) is show in stick format.

phenotype, to cellular response, to animal pharmacology, to efficacy in the clinic. In a traditional target or cell-based screen, there are a limited number of readouts that are used to determine the efficacy of a compound on a system— certainly not enough data to safely or accurately predict any effects in the clinic. Broad screening of specific compounds using technologies such as gene profiling or proteomics can provide vast amounts of data at the cellular detail level, but it is still difficult to translate this into known biological phenotypes. Whereas a bottom-up approach, where events at the molecular level can be used to rationally explain physiology at a macroscopic, is an objective of some in systems biology, insights can be made by more empirical approaches. Correlations discovered between molecular readouts and more phenotypically relevant measurements, such as toxicity and efficacy, are proving to be quite powerful. These relationships will become more robust as more data are gathered and integrated into systems allowing cross-discipline statistical analyses. Although there is still no substitute for well-controlled clinical trials, the use of these technologies should improve the quality of the molecules entering the clinic and hopefully reduce failure rates, allowing treatments for more difficult diseases to be considered.

10

Caron

References 1. Lichtenberg, F. R. (2003) The economic and human impact of new drugs. J. Clin. Psychiatry 64(Suppl 17), 15–18. 2. Hansen, R. W. (1986) New pharmaceuticals reduce cost of illness. Can. Pharm. J. 119(6), 318–325. 3. Goodman, L. S., Hardman, J., and Limbird, L. (eds.). Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill, New York. 4. Fleischmann, R. D., Adams, M. D., White, O., et al. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269(5223), 496–512. 5. Stockwell, B. R., Haggarty, S. J., and Schreiber, S. L. (1999) High-throughput screening of small molecules in miniaturized mammalian cell–based assays involving post-translational modifications. Chem. Biol. 6(2), 71–83. 6. Spring, D. R., Krishnan, S., Blackwell, H. E., and Schreiber, S. L. (2002) Diversity-oriented synthesis of biaryl-containing medium rings using a one bead/one stock solution platform. J. Am. Chem. Soc. 124(7), 1354–1363. 7. Haggarty, S. J., Koeller, K. M., Wong, J. C., Butcher, R. A., and Schreiber, S. L. (2003) Multidimensional chemical genetic analysis of diversity-oriented synthesis-derived deacetylase inhibitors using cell-based assays. Chem. Biol. 10(5), 383–396. 8. Shogren-Knaak, M. A., Alaimo, P. J., and Shokat, K. M. (2001) Recent advances in chemical approaches to the study of biological systems. Annu. Rev. Cell Dev. Biol. 17, 405–433.

Chemistry for Chemical Genomics

11

2 Chemistry for Chemical Genomics Lutz Weber Summary New methods and strategies have been developed to design and use small molecules that allow the functional dissection of molecular pathways, cells, and organisms by selective small-molecule ligands or modulators. In this overview, we are focusing on diversity aspects, design methods, and chemical synthesis strategies for the application of small molecules as tools for chemical genomics. Examples for different successful chemicalgenomics strategies include the selection of diverse drug-like molecules, target family– focused compound libraries, natural-product chemistry, and diversity-oriented synthesis. Key Words: Chemical diversity; compound design; diversity-oriented synthesis; druglike compounds; molecular properties; natural products; rule of five; structure–activity relationship; target-focused compound libraries.

1. Introduction Organic chemistry is the science of the synthesis and properties of molecules that are constructed from only a few atom types, such as carbon, hydrogen, nitrogen, oxygen, and sulfur, with carbon atoms constituting the majority of the core of these chemicals. As these atoms are also the building blocks of naturally occurring peptides or oligonucleotides, this chemistry was termed organic, as opposed to other disciplines of chemistry. Such chemicals, commonly referred to as small molecules, are valuable as medicines to treat diseases ranging from headache to cancer. Small molecules have recently proven to be extremely useful tools to explore the functions of the cell at the genome level, giving rise to the new paradigm of chemical genomics. The functional dissection of molecular pathways, cells, and organisms by having a small-molecule ligand or modulator for every gene product, was the vision of Stuart L. Schreiber, one of the pioneers of the chemical genomics field (1). From: Methods in Molecular Biology, vol. 310: Chemical Genomics: Reviews and Protocols Edited by: E. D. Zanders © Humana Press Inc., Totowa, NJ

11

12

Weber

Several recent reviews and books deal with the implication of chemical genomics towards drug discovery (2–5). The chemical genomics paradigm is seen as a logical follow-up of the Human Genome Project, and several public initiatives on chemical genomics have been started on national levels. Thus, in the United States, the National Human Genome Research Institute has set up a chemical libraries plan that moves the National Institutes of Health into highthroughput screening and small-molecule development to “determine function and therapeutic potential of genes and to define molecular networks.” In Germany, the Nationale Genomforschungsnetz initiated a chemical genomics platform by assembling synthetic compound libraries as probes for protein function. These compounds will help to validate new targets for novel therapies more rapidly, and will enable researchers in the public and private sectors to take these targets and move them through the drug-development pipeline. In this overview we would like to focus on some chemical aspects, problems, and solutions to the application of small molecules as tools for chemical genomics. 2. Diversity of Small Molecules What are small molecules? For the sake of simplicity, let us consider only those organic chemicals as small molecules that have a molecular weight of less than 1000 Dalton. Such a definition is by its nature arbitrary, but allows separating out other classes of organic molecules such as molecules that are oligomers of smaller building blocks such as, for example, proteins, oligonucleotides, or oligosaccharides. The number of all possible and different small molecules with a molecular weight below 1000 is assumed to exceed 1060. To be a specific modulator of a target protein, a small molecule has to act as a ligand, binding to its target. As opposed to to large molecules, small molecules have an average interaction area of up to 400 Å2 with their target—which is, for example, the size of a typical enzyme substrate site. This relatively small interaction area poses a serious problem to the whole concept of chemical genomics—is it at all possible to find a specific small molecule for each gene product? Indeed, it appears more likely that small molecules are promiscuous and may interact rather with a range of targets that have similar binding sites. Many of these “unwanted” interactions might not result in undesired effects, giving way to selective drugs in vivo. In odd cases, such secondary interactions may cause toxicities or other side effects; in lucky cases, these secondary interactions might be the real reason why a particular small molecule is an effective drug. Therefore, the rational design of such dual- or triple-action compounds has emerged as a new paradigm (6). The functional, biological diversity of small molecules appears to be inherently more limited than that of large molecules. Whereas the diversity of oligomers

Chemistry for Chemical Genomics

13

can be described by metrics like sequence and secondary, tertiary, and quaternary structure, the diversity of small molecules is harder to capture. The development of qualitative and quantitative measures for the chemical diversity of small molecules has only started to evolve with the advent of combinatorial chemistry, which enabled the synthesis of a large number of small molecules in one experiment. As opposed to large molecules, the required high functional diversity of small molecules has to be packed into a rather small volume made up of only 20–30 non-hydrogen atoms. To bind efficiently at a protein-ligand site, most of the small-molecule ligand’s binding energy must come from sites not exploited by the natural protein ligand. The limited molecular volume has to interact with the target protein with a maximum binding energy per atom to achieve both the required affinity and specificity. This binding energy has been calculated for maximal free-energy contributions per non-hydrogen atom with approx 1.5 kcal/ mol across a wide variety of macromolecule–small-molecule interactions. The empirical data also revealed a significant trend to smaller contributions per atom as the relative molecular mass of the ligand increases (6). Thus, smallmolecule ligands are binding to their targets with approximately three times more binding energy per unit area than protein ligands (8). As subnanomolar binding can be achieved with ligands containing as few as 10–20 atoms, the remaining 10–20 atoms could potentially be used to obtain selectivity. The likelihood of obtaining the desired functional diversity for small molecules is also correlated to the nature of the biological targets of interest. Thus, it is more likely to find enzyme inhibitors than small molecules that block protein–protein interactions (8). Functional diversity of small molecules for chemical genomics experiments can therefore be defined in terms of obtaining maximum affinity to a given target protein by a minimum of molecular volume in order to minimize unwanted binding to other proteins. The realization of this Max–Min concept in a library of small-molecule compounds poses a serious challenge to organic chemists. As the molecular volume of small molecules is rather similar and does not leave enough room for major variations, the only way to obtain both affinity and selectivity is a maximum diverse distribution of atoms in this small volume. This can not be achieved by varying a small number of building blocks like the 20 amino acids for proteins, but requires the whole repertoire of organic chemistry to assemble novel chemical scaffolds. This recent understanding has led not only to a reappreciation of natural-product chemistry but also to a series of initiatives that aim at the development of novel chemistries to obtain chemical diversity.

2.1. Drug-Like Compound Libraries A large range of small-molecule physico-chemical properties can be computed and used for diversity selection and drug likeliness. Thus, drug-like mole-

14

Weber

cules should follow the “rules of five” that requires a logP 1 y. By printing, processing, and assaying arrays using these protocols, we have been able to achieve spot-to-spot CVs of 4–5% (Fig. 8). Fluorophores other than Cy3—for example, the Alexa dyes (Molecular Probes) or Oyster dyes (Denovo Biolabels)—also work well in these applications. However, when using any amine-reactive dye to label the protein, buffers containing primary amino groups such as Tris and glycine should be avoided, because they will inhibit the conjugation reaction. The presence of low concentrations (