Medicinal Chemistry of Nucleic Acids (Wiley Series in Drug Discovery and Development)

MEDICINAL CHEMISTRY OF NUCLEIC ACIDS MEDICINAL CHEMISTRY OF NUCLEIC ACIDS Edited by LI-HE ZHANG Peking University, B...

Author: Li-He Zhang | Zhen Xi | Jyoti Chattopadhyaya | Sidney M. Hecht

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MEDICINAL CHEMISTRY OF NUCLEIC ACIDS

MEDICINAL CHEMISTRY OF NUCLEIC ACIDS Edited by

LI-HE ZHANG Peking University, Beijing, China

ZHEN XI Nankai University, Tianjing, China

JYOTI CHATTOPADHYAYA Uppsala University, Uppsala, Sweden

A JOHN WILEY & SONS, INC., PUBLICATION

Cover image credits: Shi, H., Moore, P.B. The crystal structure of yeast phenylalanine tRNA at 1.93 A resolution: a classic structure revisited. RNA, 6, pp. 1091–1105, 2000. Pley, H.W., Flaherty, K.M., McKay, D.B. Three-dimensional structure of a hammerhead ribozyme. Nature, 372, pp. 68–74, 1994. Juneau, K., Podell, E., Harrington, D.J., Cech, T.R. Structural basis of the enhanced stability of a mutant ribozyme domain and a detailed view of RNA—solvent interactions. Structure, 9, pp. 221–231, 2001. Vicens, Q., Westhof, E. Crystal Structure of a Complex between the Aminoglycoside Tobramycin and an Oligonucleotide Containing the Ribosomal Decoding A Site. Chem. Biol., 9, pp. 747– 755, 2002. Copyright © 2011 John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: Medicinal chemistry of nucleic acids / edited by Li He Zhang, Zhen Xi, Jyoti Chattopadhyaya. p. ; cm. Includes index. ISBN 978-0-470-59668-5 (cloth) 1. Pharmaceutical chemistry. 2. Nucleic acids–Therapeutic use. I. Zhang, Li He. II. Xi, Zhen. III. Chattopadhyaya, Jyoti. [DNLM: 1. Nucleic Acids–pharmacology. 2. Nucleic Acids–therapeutic use. QV 185] RS400.M438 2011 615 .19—dc22 2011008300 Printed in the United States of America oBook ISBN: 978-1-118-09280-4 ePDF ISBN: 978-1-118-09283-5 ePub ISBN: 978-1-118-09281-1 10 9 8 7 6 5 4 3 2 1

CONTENTS

FOREWORD

vii

CONTRIBUTORS

xi

INTRODUCTION

xv

1

RECENT ADVANCES IN CARBOCYCLIC NUCLEOSIDES: SYNTHESIS AND BIOLOGICAL ACTIVITY

1

Jianing Wang, Ravindra K. Rawal, and Chung K. Chu

2

STRUCTURES AND FUNCTIONS OF NUCLEIC ACIDS MODIFIED WITH S, Se, AND Te AND COMPLEXED WITH SMALL MOLECULES

101

Wen Zhang, Jia Sheng, and Zhen Huang

3

UNRAVELING OF THE NAD CYCLIZING AND CALCIUM SIGNALING FUNCTIONS OF HUMAN CD38

142

Hon Cheung Lee

4

DNA AND RNA BINDING SMALL MOLECULES

164

Shibo Li and Zhen Xi

v

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5

CONTENTS

G-QUADRUPLEX DNA AND ITS LIGANDS IN ANTICANCER THERAPY

206

Zhi-Shu Huang, Jia-Heng Tan, Tian-Miao Ou, Ding Li, and Lian-Quan Gu

6

MOLECULAR MODELING IN NUCLEIC ACID-TARGETED DRUG DESIGN

258

Lidan Sun, Hongwei Jin, Liangren Zhang, and Lihe Zhang

7

STRUCTURE OF 10–23 DNAzyme IN COMPLEX WITH THE TARGET RNA IN SILICO—A PROGRESS REPORT ON THE MECHANISM OF RNA CLEAVAGE BY DNA ENZYME

272

Oleksandr Plashkevych and Jyoti Chattopadhyaya

8

LABELING OLIGONUCLEOTIDES TOWARD THE BIOMEDICAL PROBE

292

Il Joon Lee and Byeang Hyean Kim

9

LOCKED NUCLEIC ACID OLIGONUCLEOTIDES TOWARD CLINICAL APPLICATIONS

335

Rakesh N. Veedu and Jesper Wengel

10 THE PHARMACOKINETICS RESEARCH OF NUCLEIC ACID DRUGS

349

Shengqi Wang, Dandan Lu, Qingqing Wang, Haifeng Song, Chan Gao, Caihong Liu, and Ying Wang

11 INDUCIBLE RNAi AND DRUG TARGET VALIDATION

390

Wei Xiong, Jing Zhao, Yufan Zhang, Jinxi Wang, Yong-Xiang Zheng, Qiu-Chen He, Li-He Zhang, and De-Min Zhou

12 siRNA: THE SPECIFICITY AND OFF-TARGET EFFECTS

405

Quan Du, Huang Huang, and Zicai Liang

INDEX

423

FOREWORD

Nucleic acids have now been of interest to the research communities of chemists and biochemists for a number of decades. Although the synthesis of DNA, and even RNA, is now considered “routine,” the current level of sophistication was achieved only after meeting the challenges associated with the preparation of the nucleoside building blocks of DNA and RNA. The synthesis of DNA and RNA oligonucleotides is actually quite complex, requiring the identification and use of suitable protecting groups for the nucleobases and sugar moieties of RNA and DNA, not to mention efficient methods for creating phosphate ester linkages between individual nucleosides. Because nucleic acids are polyanions, the newly synthesized DNAs and RNAs also required novel methods for purification, which took cognizance of their ready solubility only in aqueous and other polar solvents. Another major challenge has been the analysis of the primary, secondary, and tertiary structures of DNA and RNA, as well as their interactions with macromolecular and low-molecular-weight ligands. The discovery and development of biophysical and biochemical techniques has enabled this challenge to be met with increasing facility and sophistication. The chemical and biochemical communities have worked together productively for many years to drive new discoveries involving nucleic acids. These discoveries have created new opportunities for both communities. The finding that nucleic acids participated in the decoding of genetic information as well as its storage provided numerous opportunities for chemical intervention in the mechanisms of RNA synthesis and splicing, in addition to that of protein synthesis. The resulting probe molecules and their analogues, such as puromycin, actinomycin D, and chloramphenicol, in turn facilitated the mechanistic analyses of biochemical function. The remarkable discovery that certain RNA molecules are vii

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responsible for their own biosynthetic processing, reflecting the existence of an early world driven by RNAs as the primary catalysts, has enabled the identification of RNAs and DNAs capable of effecting highly selective transformations not represented in nature. The techniques used to identify novel processes in recognition and catalysis employ iterative cycles of molecular interactions/selections and amplifications and have involved increasingly sophisticated and complex biological systems. The recent findings that gene expression can be regulated by gene transpositions, G-quadruplex structures associated with specific genes, RNA interference, and riboswitches have further enriched our understanding of nucleic acid function. Perhaps equally importantly, they provide new opportunities for intervention leading to further mechanistic understanding and therapeutic gain. The structural integrity of DNA is essential to enable its role as the repository of genetic information. Agents that alter DNA structure are mutagenic and potentially carcinogenic. Accordingly, organisms have evolved elaborate systems to recognize and repair DNA damage. Because these systems protect cancerous as well as normal cells, using DNA as a target for therapeutic intervention does not intrinsically provide a source of tumor cell selectivity. Efforts in antitumor therapy have led to numerous clinically used agents that function at the level of nucleic acids, but the poor therapeutic indices for such agents have often limited their utility. Ongoing efforts to better understand the mechanisms that control gene expression have progressed impressively in recent years, providing targets (e.g., telomeric assemblies, micro RNAs, and G-quadruplex structures associated with individual genes) that seem likely to lead to more selective therapy of cancer and other diseases. New chemistries used for the elaboration of therapeutic oligonucleotides, as well as new strategies for their more effective delivery, have significantly enhanced the therapeutic potential of these agents; successes in advanced clinical trials make it seem likely that agents of this type will appear with some frequency as newly marketed drugs. The discovery of multiple natural mechanisms for the regulation of gene expression with oligonucleotides will undoubtedly extend the therapeutic reach of such drugs. This book provides an excellent overview of a number of current research activities relevant to the medicinal chemistry of nucleic acids. This includes Chapters 1 and 4, which deal with carbocyclic nucleosides and small molecules that bind to DNA or RNA. These areas have attracted significant attention over a period of years due to the novel chemistry involved and the biochemical/biological activities associated with many of these compounds. Studies of this type have led to numerous important clinically used agents, whose mechanisms involve disruption of nucleic acid synthesis and function, especially in viruses and cancers. Additional drugs of this type will undoubtedly be found. Strongly enabling future studies in this area are modeling studies that permit the nature of small molecule–nucleic acid interaction to be better understood at the levels of affinity and selectivity, and thereby enhance our capacity for the design of improved agents. These are ably summarized in Chapter 6. Structural studies of nucleic acids play a critical role in monitoring interactions of nucleic acids with both large and small substrates and in providing

FOREWORD

ix

high-resolution information pertinent to such binding events. Such studies are represented in Chapter 8, dealing with the labeling of oligonucleotides with suitable reporter groups, and in Chapter 2 from Professor Zhen Huang’s laboratory, which summarizes strategies for the preparation and x-ray crystallographic characterization of nucleic acid analogues containing S, Se, and Te. More recently addressed opportunities in medicinal chemistry include a focus on nucleoside-containing cofactors, such as cyclic ADP-ribose, a substrate for the multifunctional enzyme CD38 (Chapter 3), which is involved in intracellular calcium signaling. The critical functions of G-quadruplex structures makes them a logical focus for medicinal chemistry studies, and their structural variety promises potentially enhanced selectivity of action, as evidenced by the data summarized in Chapter 5. Increasing sophistication in the preparation and characterization of nucleic acids has brought research on oligonucleotides firmly within the realm of medicinal chemistry. Tools required for success in this area at a therapeutic level include optimization of the chemistries employed to facilitate the delivery and stability of oligonucleotide probes of interest. Chapter 10, dealing with the pharmacokinetic issues involved, provides a summary of current studies in this area. Better chemistries are required to realize improved potency and selectivity of oligonucleotide targeting, and Chapter 9 by Veedu and Wengel describes locked nucleic acid oligonucleotides, which achieve important increases in potency through conformational constraint of the nucleoside building blocks. The efficiency of nucleic acid targeting could be improved dramatically if the therapeutic agents functioned catalytically; Chapter 7 from the Chattopadhyaya laboratory provides an insightful account of a DNAzyme, which cleaves an RNA target. Finally, Chapters 11 and 12 deal with different aspects of RNA interference (RNAi). In addition to its importance in genomic studies, and as a tool for drug target validation, the study of the mechanism RNAi can provide mechanistic information of potential utility in improving the delivery and efficacy of therapeutic oligonucleotides. The range of topics in this volume accurately reflects the vigor of current investigations over a range of topics in the area of nucleic acids and underscores the expanding opportunities for medicinal chemistry in this central discipline. Sidney M. Hecht Arizona State University

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CONTRIBUTORS

Jyoti Chattopadhyaya, Bioorganic Chemistry Program, Department of Cell & Molecular Biology, Box 581, Biomedical Center, Uppsala University, SE75123 Uppsala, Sweden Chung K. Chu, College of Pharmacy, The University of Georgia, 30602 Athens, GA Quan Du, Institute of Molecular Medicine, Peking University, Beijing 100871, China Chan Gao, Beijing Institute of Radiation Medicine, Taiping Road 27, Haidian District, Beijing 100850, People’s Republic of China Lian-Quan Gu, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China Qiu-Chen He, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Sidney M. Hecht, Arizona State University, Department of Chemistry and Biochemistry, Box 871604, 85287-1604 Tempe, AZ Huang Huang, Institute of Molecular Medicine, Peking University, Beijing 100871, China Zhen Huang, Department of Chemistry, Georgia State University, 30303 Atlanta, GA Zhi-Shu Huang, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China xi

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Hongwei Jin, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Byeang Hyean Kim, Laboratory for Modified Nucleic Acid Systems, Department of Chemistry, BK School of Molecular Science, Pohang University of Science and Technology (POSTECH), Pohang 790-784, South Korea Hon Cheung Lee, Department of Physiology, University of Hong Kong, 4/F Lab Block, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong Il Joon Lee, Laboratory for Modified Nucleic Acid Systems, Department of Chemistry, BK School of Molecular Science, Pohang University of Science and Technology (POSTECH), Pohang 790-784, South Korea Ding Li, School of Pharmaceutical Guangzhou 510006, China

Sciences,

Sun

Yat-sen

University,

Shibo Li, Department of Chemical Biology and State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China Zicai Liang, Institute of Molecular Medicine, Peking University, Beijing, China Caihong Liu, Beijing Institute of Radiation Medicine, Taiping Road 27, Haidian District, Beijing 100850, People’s Republic of China Dandan Lu, Beijing Institute of Radiation Medicine, Taiping Road 27, Haidian District, Beijing 100850, People’s Republic of China Tian-Miao Ou, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China Oleksandr Plashkevych, Bioorganic Chemistry Program, Department of Cell & Molecular Biology, Box 581, Biomedical Center, Uppsala University, SE75123 Uppsala, Sweden Ravindra K. Rawal, College of Pharmacy, The University of Georgia, 30602 Athens, GA Jia Sheng, Department of Chemistry, Georgia State University, 30303 Atlanta, GA Haifeng Song, Beijing Institute of Radiation Medicine, Taiping Road 27, Haidian District, Beijing 100850, People’s Republic of China Lidan Sun, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Jia-Heng Tan, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China Rakesh N. Veedu, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072, Australia

CONTRIBUTORS

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Jianing Wang, College of Pharmacy, The University of Georgia, 30602 Athens, GA Jinxi Wang, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Qingqing Wang, Beijing Institute of Radiation Medicine, Taiping Road 27, Haidian District, Beijing 100850, People’s Republic of China Shengqi Wang, Beijing Institute of Radiation Medicine, Taiping Road 27, Haidian District, Beijing 100850, People’s Republic of China Ying Wang, Beijing Institute of Radiation Medicine, Taiping Road 27, Haidian District, Beijing 100850, People’s Republic of China Jesper Wengel, Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, Odense M-5230, Denmark Zhen Xi, Department of Chemical Biology and State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China Wei Xiong, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Liangren Zhang, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Li-He Zhang, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Yufan Zhang, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Wen Zhang, Department of Chemistry, Georgia State University, 30303 Atlanta, GA Jing Zhao, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Yong-Xiang Zheng, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China De-Min Zhou, State Key Laboratory of Natural and Biomimetic Drugs, 38 Xueyuan Road, Beijing 100191, China

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INTRODUCTION

L. H. ZHANG Peking University, Beijing, China

Z. XI NanKai University, Tianjing, China

J. CHATTAPADHYAYA Uppsala university, Uppsala, Sweden

Medicinal chemistry is an extremely significant and challenging area, and the development of drug discovery truly brings fruitful benefits to science and humans. The process of drug discovery has become completely transformed. Rapid advances in genomics, proteomics, bioinformatics and automation, combinatorial chemistry, and high-content screening appear to be principally responsible for driving such a rapidly evolving discovery process. However, data indicate that the number of new drugs has slipped in recently years. Medicinal scientists face increased barriers, including how to predict the efficacy, pharmacokinetic problems, toxicity and clinical safety of drug candidates during the discovery process. Researchers are seeking ways to make drug discovery and development more productive from target identification and validation to lead discovery and optimization and clinic testing.

xv

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INTRODUCTION

NUCLEOSIDE AND NUCLEOTIDE DRUGS

In the area of nucleoside and nucleotide drugs, small molecule nucleosides and nucleotides have attracted much attention due to a remarkable increase for the treatment of cancer and viral diseases. The nucleoside and nucleotide analogs can be regarded as prodrugs, as they need activation for their anticancer or antiviral efficacy through a phosphorylation process to their nucleoside diphosphates or nucleoside triphosphates that functions as the inhibitor for the DNA polymerase. During the past two decades, antiviral nucleosides have become reliable in the treatment of several viral infections such as HSV, HBV, HCV, HIV, and so on. The antiviral nucleosides are first phosphorylated by the cellular nucleoside kinases to nucleoside monophosphates, which are subsequently further phosphorylated by corresponding enzymes to the diphosphates and the triphosphates. The nucleoside triphosphate is then incorporated into the nascent viral DNA chain and blocks viral DNA synthesis. Virus doesn’t encode its own viral kinase, and the phosphorylation of nucleoside analogs is entirely dependent on the cellular kinase. The intial phosphorylation catalyzed by the host nucleoside kinase like TK1, TK2, dCK, dGK, UCK1, UCK2, and ADK are often the rate-limiting step in the activation process. The further phosphorylation to triphosphate is effected by the cellular nucleotide kinases [1,2]. Extensive research efforts have been directed toward the development of nucleoside therapeutics; structural modified nucleoside inhibitors can vary in either (or both) the ribose or base portion of the molecule. The continuing research work for novel nucleosides and nucleotides is successfully publishing many nucleoside analogs in market. For example, Entecavir (Figure 1) is a cyclopentyl guanosine analog launched in 2005 for the once-daily oral treatment of chronic hepatitis B virus infection, and it is the third nucleoside or nucleotide analog to be marketed for this indication. In mammalian cells, Entecavir is efficiently phosphorylated to the active triphosphate form, which competes with the natural substrate deoxyguanosine triphosphate and functionally inhibits all three activities of HBV polymerase: (1) base priming; (2) reverse transcription of the negative strand from the pregenomic messager NH2

O N

N N

H2C

N

NH2

N

O HO

HO

N

NH N

Cl

F OH

OH Entecavir

Clofarabine

Figure 1 Chemical structures of nucleic acids based drugs Entecavir (antiviral drug against hepatitis B infection) and Clofarabine (anti-leukemia drug against relapsed/refractory acute lymphoblastic leukemia in children).

INTRODUCTION

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RNA; and (3) synthesis of the the positive strand of HBV DNA [3a]. The absolute configuration of the glycosyl moiety in most of modified nucleoside drugs is D. During the past 10 years, the interesting biological activities of several “unnatural” l-nucleosides have been discovered [3b]. Another anti-HBV nucleoside drug, clevudine (L-FMAU; Figure 2), is already approved in some Asian countries and launched in 2007 [3c]. However, this drug was discontinued in its Phase III QUASH studies for the treatment of chronic hepatitis B (HBV) infection in 2009 due to safety concerns, in particular myopathy (muscle damage). Now this drug is being investigated for focus on developing therapies for hepatitis C virus (HCV) [3d]. Adenosine-related antimetabolites, such as Cladribine and Fludarabine, have proven successful in treating low-grade lymphomas, chronic lymphocytic leukemia, and hairy-cell leukemia. Clofarabine (Figure 2) is a second-genenation purine nucleoside analog launched in 2005 for the treatment of pediatric patients with relapsed or refractory acute lymphoblastic leukemia. A key differentiator for Clarabine is the presence of a fluorine in the C−2 position, which renders it less susceptible to phosphorolytic cleavage of the glycosydic bond and inactivation by purine nucleoside phosphorylases. In addition, the C−2 fluoro group improves the acid stability relative to its predecessors. As seen with other purine nucleoside analogs, the mechanism of action of Clofarabine involves intracellular phosphotylation to active triphosphate by 2 -deoxycytidine kinase, and subsequent inhibition of RNA reductase and DNA polymerase α [3e]. Nelarabine (Figure 2) is a pro-drug of 9-β-D-arabinofuranosylguanine (ara-G), which was launched in 2006 as an intravenous infusion for treating relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) after at least two prior chemotherapy regimens [3f]. Despite a large increase on the studies of nucleosides and nucleotides, the number of new drugs known as new chemical entities (NCEs) has risen only slightly. It seems that simply making more compounds does not translate into finding more drugs, and adverse events associated these nucleoside drugs were

OCH3

O N

N

NH N O O

N

OH HO

NH2

O

F OH Clevudine (L-FMAU)

N

OH

OH

Nelarabine

Figure 2 Chemical structures of nucleic acids analogs based drugs Clevudine (L-FMAU) for treatment of hepatitis B and Nelarabine lymphoblastic leukemia.

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INTRODUCTION

similar to other chemotherapy agents, including vomiting, nausea, febrile neutropenia, and diarrhea. Now most studies on nucleoside and nucleotide drugs have become more selective, targeting compounds on biomolecules likely to be effective and tolerated by the body. The evolution of drug discovery into a knowledge-based predictive science lies in the assembly and integration of all pharmacologically relevant information, at both the molecular and phenotype level. To deal with these challenges and to fatten their production pipelines, many new approaches have been used for the development of nucleoside and nucleotide drugs.

TARGETING RNA WITH SMALL MOLECULES

A wealth of biological information has been discovered in the past 20 years, which has fundamentally changed our perspective of the biological role of RNA. In medicinal chemistry and pharmaceuticals, the traditional targets are proteins, most commonly active sites of enzymes [4]. A lot of molecular scaffolds and ideas on how to target protein active sites have been widely discussed in literature. The increasing awareness of the central role of RNA has led to realization that RNA is a potential drug target [5] that has remained largely unexplored. Keeping with the growing trend of recent years, targeting RNA with small molecules has appeared as an attractive strategy for the new drug discovery. Many efforts have been made on taking 3-D structure of RNA to design small molecules that selectively target RNA sites and might be therapeutically useful. In February of 2001, the initial draft of the human genome was published [6]. Many genes have been correlated with disease. In all cells the genetic information in DNA is first translated into messenger RNA and then converted by ribosomes into proteins. The human genome sequence contains noncoding RNA genes, regulatory sequences, and structural motifs. Because RNAs are able to achieve intricate tertiary structures [7,8], many interesting and important functions are conferred. Now RNA is becoming increasingly amenable to small molecule therapy [9,10], as more structural and functional information accumulates with regard to important RNA functional domains. Ribosomes are the major player in biology’s central dogma. To make that happen, dozens of different proteins and strands of RNA form a complicated machine divided into two principal components. The smaller component, known as the 30S subunit, works mainly to decode the genetic code in messenger RNA. The larger 50S subunit then takes this information and uses it to stitch together amino acids in the proper sequence to make up the final protein. Three scientists, Ada Yonath of the Weizmann Institute of Science in Rehovot, Israel, Thomas Steitz of Yale University; and Venkatraman Ramakrishnan of the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK revealed the atomic structure and inner workings of the ribosome, and they shared the 2009 Nobel Prize in Chemistry [11]. The exploration of 3-D RNA structure may open a new way for the drug discovery. Different from RNA folding from an unfolded polynucleotide chain, many ribonucleic acids

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(RNAs) can adopt more than a single three-dimensional structure. RNA structures are stabilized by a variety of interactions, including nonelectrostatic interactions (hydrophobic, van der Waals, and base–pair interactions), as well as translational, rotational, vibrational, and configurational entropy. Due to the highly charged nature of the RNA, electrostatic interactions with surrounding solvent and with different ions or other charged molecules in solution are of particular importance. Researchers are now investigating a new generation of small molecules aimed at RNA structures, both in pathogens and in human cells. How to create small molecules with the right hydrophobicity, aromaticity, and other properties to make them selective for specific RNAs is a big challenge for medicinal chemists. For example, the bacterial ribosome in particular is the site of action for antibiotics such as aminoglycosides and tetracyclines and a key target for the design of new antibiotics as well. Now, drug discoverers are increasingly testing the idea that non-nucleotide small molecules that selectively target RNA sites might be therapeutically useful. The effort to discover such molecules with druglike properties is intensifying. Many efforts have been made on taking the rational drug design route to small molecule-RNA interaction. The molecular biology community has ensured public access to all gene and protein sequences and 3-D protein structures. A database of RNA-binding ligands and the RNA structures or motifs to which the ligands bind is available. The short RNA sequences (24–48 bps), which were identified from sequence-conserved and functionally important regions of several disease-related bacterial, viral, or human RNAs, such as the bacterial ribosomal 16S A-site, E. coli transglycosidase mRNA, hepatitis C virus (HCV) internal ribosome entry site (IRES) RNAs [12,13], HIV frameshift signal [14], HIV protease mRNA, human oncogenic Bcr-Abl mRNA [15], and human tyrosine sulfotransferase mRNA, have been used as a target for the study of the binding affinity and specificity of small molecules. Using a structure-guided approach, Mobashery and coworkers took into account steric and electronic contributions to interactions between RNA and aminoglycosides to make a random search of 273,000 compounds from the Cambridge structural database and the National Cancer Institute 3-D database of ribosomal aminoglycoside-binding pocket [16]. Because of the good affinity with RNA, the study of aminoglycoside antibiotics and their binding to RNA has been a paradigm for understanding the way in which small molecules can be developed to affect the function of RNA. Aminoglycoside antibiotics are a group of clinically important antibacterial drugs. However, their widespread use over the last decades has been significantly compromised by otoand nephrotoxicity and the rapid emergence of bacterial resistance. To overcome the undesirable properties of parent structures, it is highly desirable to synthesize modified aminoglycosides that will possess higher RNA binding affinity, better selectivity, better antibacterial activity, and stronger resistance against the aminoglycoside-modifying enzymes compared to their parent structures [17]. Another viral RNA genome being targeted by small molecules is that of human immunodeficiency virus (HIV), the cause of AIDS. The replication

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of human immunodeficiency virus type 1 (HIV-1) can be activated by two RNA-protein interactions [18,19]. One of them is the transactivator protein (Tat) and its responsive RNA element (TAR), and the other is the regulator of virion expression (Rev) and its responsive RNA (RRE). Host-cell translation of HIV’s genome is boosted greatly when the HIV protein Tat binds to TAR, which has a hairpin shape. It is known that the binding site of HIV-1 RRE RNA and TAR RNA is a relatively small fragment composed of 47 and 31 nucleotides, respectively. It is also known that the binding domains of the Rev and Tat proteins are small fragments of the peptide composed of 17 and 9 amino acids, which are called Rev peptide (Rev34–50) and Tat peptide (Tat49–57), respectively [20]. Intensive research over the past decade has enriched the structural and biological knowledge of the transactivation mechanism involving a Tat–TAR interaction [21]. Therefore, blocking Tat–TAR complex formation seems to be a promising target for inhibiting the multiplication of the HIV-1 virus [22]. The interaction of small molecule to RNA target is usually governed by the mutual electrostatic properties and the p–p stacking between aromatic rings, and hydrogen bonding between the nucleobases is a naturally existing specific interaction in the recognition of DNA or RNA. Many small molecules, such as aminoglycosides and their derivatives [23] 2,4-diaminoquinozaline or quinoxaline-2,3-diones [24], aminoalkyl-linked acridine-based compounds [25], beta-carboline [26] and isoquinoline [27] derivatives, have been developed through high-throughput screening or rational drug design. One interesting RNA target in a noncoding region is the rCUG triplet repeat expansion in the 3 UTR of the dystrophia myotonica protein kinase (DMPK) gene. The rCUG triplet repeat expansion in the 3 UTR of the dystrophia myotonica protein kinase (DMPK) gene results in a gain-of-function for the RNA and causes myotonic muscular dystrophy type 1 (DM1). The toxic rCUG repeat folds into a hairpin that contains regularly repeating UU mismatches flanked by GC pairs (5 CUG/3 GUC) within the stem. These regularly repeating 5 CUG/3 GUC internal loop motifs bind to the alternative splicing regulator muscleblind-like 1 protein (MBNL1). Formation of the DM1 RNA-MBNL1 complex compromises function of MBNL1, which leads to the misregulation of alternative splicing for a specific set of pre-mRNAs. A bisbenzimidazole pentamer designed by this route inhibits with low nanomolar potency; perhaps this approach can be applied toward targeting other toxic repeating RNAs [28,29]. Recently, naturally occurring RNA switches (riboswitches) have significantly attracted attention due to their important functions in gene regulation. RNA switches belong to the noncoding part of the mRNA and are mostly found in the 5 -untranslated regions (5 -UTR) of messenger RNA (mRNA). RNA switches consist of an aptamer domain or sensor region and the so-called expression platform. The aptamer domain could bind to small molecule ligands as diverse as coenzymes and vitamins, amino acids, glucosamine-6-phosphate, and the purine bases guanine and adenine. The expression platform transmits the ligand-binding state of the aptamer domain through a conformational change and thereby modulates gene expression either at the level of transcription or translation. Structural analysis of many of the aptamer–ligand complexes have already been described

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in review articles [30–33]. Thus, new knowledge space could be created by analyzing the relationship between the RNA conformational change and the modulation of gene expression; mapping RNA switches data in its entirety enables the development of methods for the rational design of therapeutic agents. DNA and RNA G-quadruplexes are another interesting targets for drug design. It is well known that at physiological concentration of monovalent ions, G-rich oligonucleotides can form four-stranded structures called G-quadruplexes. G-quadruplex structures comprise stacked tetrads in which four guanines are arranged in a square-planar array, and each guanine serves as both hydrogen bond acceptor and donor in a reverse Hoogsteen base-pair. Several types of G-quadruplex structures can be classified based on their strand orientation, strand stoichiometry, and glycosidic conformation. Several biologically important genomic regions such as telomeres, the immunoglobulin switch regions, the promoter regions of genes, and recombination sites were found to have the propensity to form G-quadruplex structures. Three scientists, Elizabeth Blackburn of the University of California, San Francisco; Carol Greider of Johns Hopkins University School of Medicine in Baltimore, Maryland; and Jack Szostak of Harvard Medical School in Boston, discovered a key mechanism that cells use to protect their genetic information and received the 2009 Nobel Prize in Physiology. They demonstrated that chromosome ends, called telomeres, and the enzyme that makes them, known as telomerase, protect chromosomes and ensure that they’re faithfully copied each time a cell divides. The discovery has launched major research efforts in areas where cell division takes center stage, including aging and cancer [34]. Many small molecules can stabilize the G-quadruplex structure and inhibit telomerase, making the G-quadruplex DNA a promising drug target for cancer therapy and aging research. In addition to inhibiting telomerase, quadruplexes may have a range of other important biological functions. And quadruplexes may be present in thousands of gene promoters and thus may affect gene expression, suggesting they could exert broad influence over a wide range of processes in the body. To better understand the biological function of G-quadruplexes and guide the design of drugs that interact with them, scientists have been characterizing quadruplexes with X-ray crystallography [35] and nuclear magnetic resonance spectroscopy (NMR). Phan, Neidle, Patel, and coworkers recently reported the NMR structure of a quadruplex that forms in c-Kit, a gene involved in gastrointestinal tumors [36]. Although RNA quadruplexes have been less commonly studied than DNA quadruplexes, these RNA structures may also have important functional and clinical significance. Balasubramanian and coworkers recently reported that an RNA quadruplex in the transcript of a human oncogene inhibits expression of that gene as well [37]. Now researchers suspect that hundreds of thousands of DNA sequences sprinkled throughout the human genome are potential quadruplexforming sites. And directing drugs to these sites might be a way of artificially regulating gene expression and thus providing medicinal benefits such as anticancer activity. RNA G-quadruplex with specificity has obvious potential as a molecular target for small-molecule therapeutic agents.

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INTRODUCTION

SHORT RNA SEQUENCES THAT INTERFERE WITH TRANSLATION OF MESSENGER RNA

A total of 20,000 to 30,000 protein-coding genes are thought to reside within the human genome, for example, but interestingly only an estimated 1–3% of total genomic DNA actually codes for protein. Moreover, of the total transcriptional output identified in human cells, it is believed that approximately 98% consists of non-protein-coding RNA (ncRNA) [38]. RNA interference (RNAi) is a new field to describe the use of small inhibitory double-stranded RNA (siRNA) to target for degradation sequence-specific cellular mRNAs and, as a result, to silencing gene expression [39]. RNA interference (RNAi) is a system within living cells that helps to control which genes are active and how active they are. Two types of small RNA molecules—microRNA (miRNA) and small interfering RNA (siRNA)—are central to RNA interference. Endogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer, which binds and cleaves double-stranded RNAs (dsRNAs) to produce double-stranded fragments of 21–25 base pairs with a few unpaired overhang bases on each end [40–42]. These short double-stranded fragments are called small interfering RNAs (siRNAs). Exogenous dsRNA is detected and bound by an effector protein, known as RDE-4 in C. elegans and R2D2 in Drosophila, that stimulates Dicer activity [43]. These RNA-binding proteins then facilitate transfer of cleaved siRNAs to the RNA-induced silencing complex (RISC) [44]. Part of the RISC complex components are discovered, and more proteins that are taking part in the RNAi process are still yet to be characterized in details. The active components of RISC are endonucleases called argonaute proteins, and the structural basis for binding of RNA to the argonaute protein was examined by x-ray crystallography of the binding domain of an RNA-bound argonaute protein [45]. With the more recent development of RNAi in mammalian systems, investigators are not only dissecting gene function but also attempting the development of new therapeutic approaches in human genetics and/or infectious diseases. Although it is difficult to introduce long dsRNA strands into mammalian cells due to the interferon response, the use of short interfering RNA mimics has been more successful [46]. The first studies on the therapeutic effects of siRNA show that this new “drug” class holds great promise for therapeutic intervention [47]. The main challenge for translating the experimental success of siRNA into clinical applications is how to solve the problems of the stability of siRNA in blood and the delivery to target. Especially, all an academic lab or biotech firm needs to do is to figure out how to deliver siRNA, the key double-stranded molecule in this gene-silencing pathway, to cell. Scientists are working hard to transition their research from the bench top to mice, primates, and humans [48]. Another challenge for the clinical application of siRNA is a lack of specificity. A computational genomics study estimated that the error rate of off-target interactions is about 10% [49]. Off-target activity can complicate the interpretation of phenotypic effects following gene-silencing experiments and can potentially lead to unwanted or unexpected toxicities [50].

INTRODUCTION

xxiii

Chemically synthesized siRNA has great advantages in accommodating chemical modifications, delivery methods, and dosing changes. Indeed, synthetic siRNA was chosen in all currently ongoing clinical trials. Among ncRNAs are microRNAs (miRNA)s that represent a class of small, processed RNAs that are able to silence gene expression through interactions with specific target messenger RNAs (mRNAs) via either translational inhibition or target RNA cleavage (depending on their homology to the target mRNA) [51–53]. siRNA and miRNA actually share pretty much of the same RNA interference machinery. The recent development on the regulation of microRNA and noncoding RNA also open a new approach for the drug design, it may help to understand the complex network of the interaction between drug/DNA, RNA, and proteins. As many modified antisense oligos are being tested in clinical trials, up to now only Fomivirsen (Vitravene; ISIS) was approved by the U.S. Food and Drug Administration as the first oligo drug for the treatment of eye cytomegalovirus infection. The development of short RNA sequences-based therapeutics is obstructed by its intrinsic qualities, such as poor intracellular uptake, limited blood stability, off-target effect, nonspecific immune stimulation, and so forth. In this book, modified nucleosides, cyclonucleotides, and sequence-based oligonucleotides therapeutics will be described in detail and a general discussion on the interaction between RNA and small molecules will also be provided. A review describes a potential drug target CD38, which is a novel multifunctional enzyme catalyzing the metabolism of two messenger molecules, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate; both are central in intracellular Ca2+ signaling. Despite the intrinsic challenges (e.g., potential toxicity of nucleoside drugs, complexity of delivery and pharmacokinetic profiling of sequence-based oligonucleotides, and variability of 3-D RNA structure), nucleoside and nucleotide drug research will continue to provide the increasing opportunities for drug design.

ACKNOWLEDGMENTS

We sincerely thank the authors for their great contributions and John Wiley & Sons for publishing this book, which allows us to share these very interesting topics with our readers. IntroductionREFERENCES

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17. Zhou, J., Wang, G. N., Zhang, L. H., Ye, X. S. (2007). Modifications of aminoglycoside antibiotics targeting RNA. Medicanl Research Reviews, 27 , 279–316. 18. Jain, C., Belasco, J. G. (1996). A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy. Cell , 87 , 115–125. 19. Tan, R., Chen, L., Buettner, J. A., Hudson, D., Frankel, A. D. (1993). RNA recognition by an isolated a helix. Cell , 73 , 1031–1040. 20. Faber, C., Sticht, H., Schweimer, K., Rosch, P. (2000). Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B. Journal of Biological Chemistry, 275 , 20660–20666. 21. Karn, J. (1999). Tackling tat. Journal of Molecular Biology, 293 , 235–254. 22. Froeyen, M., Herdewijn, P. (2002). RNA as a target for drug design, the example of Tat–TAR interaction. Current Topics in Medicinal Chemistry, 2 , 1123–1145. 23. Wang, S., Huber, P. W., Cui, M., Czarnik, A. W., Mei, H. Y. (1998). Binding of neomycin to the TAR element of HIV-1 RNA induces dissociation of Tat protein by an allosteric mechanism. Biochemistry, 37 , 5549–5557. 24. Mei, H. Y., Cui, M., Heldsinger, A., Lemrow, S. M., Loo, J. A., Sannes-Lowery, K. A.; et al . (1998). Inhibitors of protein-RNA complexation that target the RNA: Specific recognition of human immunodeficiency virus type 1 TAR RNA by small organic molecules. Biochemistry, 37 , 14204–14212. 25. Gelus, N., Hamy F., Bailly, C. (1999). Molecular basis of HIV-1 TAR RNA specific recognition by an acridine tat-antagonist. Bioorganic & Medicinal Chemistry, 7 , 1075–1079. 26. Yu, X. L., Lin, W., Li, J. Y., Yang, M. (2004). Synthesis and biological evaluation of novel beta-carboline derivatives as Tat-TAR interaction inhibitors. Bioorganic & Medicinal Chemistry Letters, 14 , 3127–3130. 27. He, M.Z., Yuan, D. K., Lin, W., Pang, R. F., Yu, X. L., Yang, M. (2005). Synthesis and assay of isoquinoline derivatives as HIV-1 Tat-TAR interaction inhibitors. Bioorganic & Medicinal Chemistry Letters, 15 , 3978–3981. 28. Pushechnikov, A., Lee, M. M., Childs-Disney, J. L., Sobczak, K., French, J. M., Thornton, C. A.; et al . (2009). Rational Design of Ligands Targeting Triplet Repeating Transcripts That Cause RNA Dominant Disease: Application to Myotonic Muscular Dystrophy Type 1 and Spinocerebellar Ataxia Type 3. Journal of the American Chemical Society, 131 , 9767–9779. 29. Lee, M. M., Pushechnikov, A., Disney, M. D. (2009). Rational and modular design of potent ligands targeting the RNA that causes myotonic dystrophy 2. ACS Chemical Biology, 4 , 345–355. 30. Schwalbe, H., Buck, J., Furtig, B., Noeske, J., Wohnert, J. (2007). Structures of RNA switches: Insight into molecular recognition and tertiary structure. Angewandte Chemie International Edition, 46 , 1212–1219. 31. Reichow, S., Varani, G. (2006). Structural biology: RNA switches function. Nature, 441 , 1054–1055. 32. Nudler, E. (2006). Flipping riboswitches. Cell , 126 , 19–22. 33. Shapiro, P. (2006). Discovering new MAP kinase inhibitors. Chemistry & Biology, 13 , 805–807. 34. Vogel, G., Pennisi, E.U.S. (2009). Researchers recognized for work on telomeres. Science, 326 , 212–213.

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35. Parkinson, G. N., Lee, M. P. H., Neidle, S. (2002). Crystal structure of parallel quadruplexes from human telomeric DNA. Nature, 417 , 876–880. 36. Phan, A. T., Kuryavyi, V., Burge, S., Neidle, S., Patel, D. J. (2007). Structure of an unprecedented G-quadruplex scaffold in the human c-kit promoter. Journal of the American Chemical Society, 129 , 4386–4392. 37. Kumari, S., Bugaut, A., Huppert, J. L. (2007) Balasubramanian, S. An RNA Gquadruplex in the 5 UTR of the NRAS proto-oncogene modulates translation. Nature Chemical Biology, 3 , 218–221. 38. Sontheimer, E.J., Carthew, R.W. (2005). Silence from within. Endogenous siRNAs and miRNAs. Cell , 122 , 9–12. 39. Fire, A., Xu, S., Montgomery, M. K., Kostas, S.A., Driver, S.E., Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391 , 806–811. 40. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411 , 494–498. 41. McManus, M.T., Sharp, P.A. (2002 ). Gene silencing in mammals by small interfering RNAs. Nature Reviews Genetics, 3 , 737–747. 42. Hannon, G. J. (2002). RNA interference. Nature, 418 , 244–251. 43. Parker, G., Eckert, D., Bass, B. (2006). RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. RNA, 12 , 807–818. 44. Liu, Q. H., Rand, T. A., Kalidas, S., Du, F. H. Kim, H. E., Smith, D. P.; et al . (2003). R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science, 301 , 1921–1925. 45. Ma, J. B., Yuan, Y. R., Meister, G., Pei, Y., Tuschl, T., Patel, D. J. (2005). Structural basis for 5 -end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature, 434 , 666–670. 46. Tong, A. W., Zhang, Y.A., Nemunaitis, J. (2005) Small interfering RNA for experimental cancer therapy. Current Opinion in Molecular Therapeutics, 7 , 114–124. 47. Huttenhofer, A., Schattner, P., Polacek, N. (2005). Non-coding RNAs: hope or hype? Trends in Genetics, 21 , 289–297. 48. Whitehead, K. A., Langer, R., Anderson, D. G. (2009). Knocking down barriers: advances in siRNA delivery. Nature Review: Drug. Discovery, 8 , 129–138. 49. Qiu, S., Adema, C., Lane, T. (2005). A computational study of off-target effects of RNA interference. Nucleic Acids. Res., 33 , 1834–1847. 50. Jackson, A. L., Linsley, P. S. (2010). Recognizing and avoiding siRNA offtarget effects for target identification and therapeutic application. Nature Review: Drug Discovery, 9 , 57–67. 51. Wang, Q. L., Li, Z. H. (2007). The functions of microRNAs in plants. Frontiers in Bioscience, 12 , 3975–3982. 52. Zhao, Y., Srivastava, D. (2007). A developmental view of microRNA function. Trends in Biochemical Sciences, 32 , 189–197. 53. Lu, J., Getz, G., Miska, E.A., Alvarez-Saavedra, E., Lamb, J., Peck, D.; et al . (2005). MicroRNA expression profiles classify human cancers. Nature, 435 , 834–838.

a)

b)

3′

3′

Gse

A C

C

T

A

Gse

G

T

C U

A 3′

5′

c) 3.48

3.16

2.59

Figure 2.3 See page 113 for full caption. 2.87 Å

2.77 Å (b) 2.37 (a) 2.10

(c)

Figure 2.6 See page 118 for full caption.




(a)

(b)


(a)

(b)

(c)


DNAzyme with Mg2+(active)

(a)

DNAzyme with K+ (not active)

(b)

DNAzyme T8 C8 with Mg2+ (not active) (c)

DNAzyme G2 A2 with Mg2+ (not active)

(d)


(e)

DNAzyme G14 A14 with Mg2+ (not active) (f)


Cleavage products H N

O

O

N O

H2O H

N

N

rG10

H2O

O O O

Mg1

O

O

5′

Target RNA

NH

N

O

O

O

O

NH2

O

3′

3′

O

O

O

O P

O

5′

O

O

N

O

DNA enzyme

3′ –

O

C13

O

O

O

N

N

O OH

O

P

NH2

O

N

O

HO

O –

O P

O

N

–

O

+

5′

N N

N

C13

N NH2

H2N

G14 N H

O

Distances: Mg1 to O atom of the scissile phosphate: 2.0 Å Mg2 to O atom of the scissile phosphate: 4.3 Å Mg2 to O5′ of rU11: 5.4 Å Mg3 to O atom of the scissile phosphate: 6.0 Å Mg3 to 2′-O of rG10: 5.9 Å Mg3 to O5′ of rU11: 4.9 Å


10-23 DNAzyme + Mg2+

O NH

N O

O

P O

N

N

O Mg2

O

NH2

N

3' O

rU11

NH

G14

OH

Mg3

NH2

rG10

5′ O

N

HN

O

rU11

DNAzyme pro-S sulfur DNAzyme pro-Rpsulfur substituted at P5 + Mg2+ substituted at P5 + Mg2+

B

D


(a)

(b)


(a)

(b)


(a)

(b)





CHAPTER 1

RECENT ADVANCES IN CARBOCYCLIC NUCLEOSIDES: SYNTHESIS AND BIOLOGICAL ACTIVITY JIANING WANG, RAVINDRA K. RAWAL, and CHUNG K. CHU The University of Georgia, College of Pharmacy, Athens, GA

1.1. INTRODUCTION

As fundamental building blocks of nucleic acids, nucleosides are essential to the process of replication and transcription of genetic information in living organisms [1]. Therefore, a nucleoside analog is able to interfere with the replication of pathogenic agents or with the proliferation of cancer cells by competing with their natural counterparts, and this conception has attracted considerable attention in the field of chemotherapy. Indeed, the past decades have witnessed the emergence of numerous therapeutically important nucleosides. In antiviral chemotherapy, eight nucleosides/nucleotides are currently licensed for the treatment of human immunodeficiency virus (HIV) infection, and five nucleosides/nucleotides have been approved for anti-hepatitis B virus (HBV) therapy. A number of other nucleoside analogs are widely used against herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), influenza virus, respiratory syncytial virus (RSV), and hepatitis C virus (HCV) [2]. In cancer chemotherapy, several nucleoside analogs have also demonstrated their clinical application [3]. Carbocyclic nucleosides are analogs of natural nucleosides in which a methylene group replaces the oxygen atom in the carbohydrate ring. This modification results in the loss of the labile glycosidic bond and thus increases their metabolic stability toward phosphorylase and/or hydrolase [4]. Although carbocyclic nucleosides were first conceived and synthesized by medicinal chemists Medicinal Chemistry of Nucleic Acids, First Edition. Edited by L.H. Zhang, Z. Xi and J. Chattopadhyaya. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

1

2


[5], aristeromycin 1 and neplanocin A 3, two naturally occurring antibiotics [6,7], prompted extensive investigations in carbocyclic nucleosides. Although synthesis of carbocyclic nucleosides have been mainly focused on the five-membered ring system, three-, four-, and six-membered carbocyclic nucleosides have also been synthesized and discussed in this review as well. Thus, a large number of novel carbocyclic nucleosides have been prepared, and many of these compounds are endowed with interesting biological activities. Particularly, this strategy has successfully led to the discoveries of abacavir (a pro-drug of carbovir) and entecavir as clinically useful anti-HIV and anti-HBV agents, respectively. Despite the substantial progress that has been achieved [8–13], the effort to discover novel chemotherapeutic agents with enhanced biological activity and reduced toxicity continues, in order to treat emerging infectious organisms. This chapter covers the most recent advances in carbocylic nucleosides up to September 2010.

1.2. FIVE-MEMBERED CARBOCYCLIC NUCLEOSIDES 1.2.1. Aristeromycin and neplanocin analogs

Although the majority of carbocyclic nucleosides are of synthetic origin, nature has provided two of the most interesting compounds, aristeromycin (1) and neplanocin A (3) (Figure 1.1). The d-(-)-aristeromycin was first isolated from Streptomyces citricolor in 1968 [7], although the racemic form was chemically synthesized in 1966 [5]. The discovery of neplanocin A, another carbocylic nucleoside, was achieved later in 1981 [6]. These two carbocyclic furanose nucleosides exhibit significant antitumor as well as antiviral activity. In particular, the broad-spectrum antiviral activity of these agents has been correlated with potent inhibitory effect of S -adenosyl-L-homocysteine (SAH) hydrolase [14–19]. It is well known that SAH hydrolase is one of the key enzymes in regulating the methylation reactions, which are essential to a number of important biological processes [20–22]. For instance, methylation of mRNA (i.e., 5 -capping) is required for mRNA maturation in many viruses. As shown in Figure 1.2, after the methyltransfer reaction, S -adenosyl-L-methionine (SAM) is converted to S -adenosyl-L-homocysteine (SAH), which is a powerful feedback inhibitor of

Figure 1.1 Aristeromycin and neplanocin A and their 3-deaza analogs.

FIVE-MEMBERED CARBOCYCLIC NUCLEOSIDES

3

Figure 1.2 Inhibition of SAH hydrolase by aristeromycin and neplanocin A [13].

this cycle. SAH hydrolase efficiently removes SAH by cleaving it to adenosine and homocysteine, which maintains the balance of SAM and SAH. Inhibition of SAH hydrolase (tight binding) results in increased level of SAH and consequent inhibition of viral mRNA methylation [13]. There are two types of mechanisms by which SAH hydrolase acts. The first one was elucidated by Palmer and Abeles [20]. The reaction proceeds with oxidation, deprotonation, elimination, Michael-type addition, and reduction, with all steps reversible. Methionine-bound aristeromycin and neplanocin A appear to act as analogs of SAH and are oxidized by SAH hydrolase at the 3 -position. This process leads to a depletion of enzyme-bound NAD, and SAH hydrolase can no longer initiate the catalysis cycle [19,23,25]. Such inhibition of SAH hydrolase can be reversed after incubation with NAD+ or dialysis. On the other hand, the second type of inhibition is caused not only by the NAD+ depletion, but also by the covalent binding of the inhibitor to SAH hydrolase, which cannot be rescued by the addition of NAD+ [26,30]. The inhibition of SAH by aristeromycin and neplanocin A, as well as their 3deaza analogs (2 and 4), has been investigated (Table 1.1). All these compounds, especially 3-deazaneplanocin A (4) (Ki 0.05 nM), are potent SAH inhibitors [31]. De Clercq and coworkers have also demonstrated that vaccinia virus (VV) and vesicular stomatitis virus (VSV) are markedly senstitive to these carbocyclic TABLE 1.1 Inhibition of SAH and antiviral activity of compounds 1–4 [18,31] Inhibitory constants against SAH

Antiviral potency EC50 (μg/ml)

Compounds

Source

Aristeromycin (1) Beef liver 3-Deazaaristeromycin (2) Beef liver Neplanocin A (3) Beef liver 3-Deazaneplanocin A (4) Hamster liver

Ki (nM) Cell VV 5 4–13 2–8.4 0.05

PRK PRK PRK PRK

N/A 0.8 0.03 0.07

VSV

CC50 (μg/ml)

N/A 0.2 0.01 0.07

N/A >400 40 >400

4


nucleosides. Presumably, the mRNA, which needs to be heavily methylated, is indispensable to the life cycle of VV and VSV. Shuting down the methylation pathway of mRNA, therefore, results in the inhibition of viral replication [31]. In addition to VV (belonging to poxvirus) and VSV (belonging to rhabdovirus), a number of species of viruses, encompassing paramyxoviruses, herpesviruses, reoviruses, arenavirus, and retroviruses, have been shown to be susceptible to the SAH inhibitors [31]. In view of the interesting mechanism of action, as well as the significant biological activity, syntheses of SAH hydrolase inhibitors, particularly aristeromycin and neplanocin A, as well as their analogs, has been the subject of a number of investigations. 1.2.1.1. Aristeromycin (1) A number of approaches have been used to synthesize aristeromycin since Shealy’s original work [5]. Several published methods took advantage of aminotriol 5 as an important intermediate for building up the target nucleosides, whereas others focused on utilizing alcohol 6 (or appropriate derivatives therefrom) as a pseudosugar source to couple with the base moiety (Figure 1.3). In general, the aminotriol routes always started from achiral materials and generated racemic compounds [5,32–37]. Although in other instances, the chirality can be resolved by using enzymatic resolutions [38], asymmetric cycloaddition [39–42], or palladium-catalyzed reactions [43–45], the synthesis of optically pure aristeromycin 1 is unsatisfactory due to lengthy sequences and low yields, as well as scale-up difficulties. On the contrary, the pathway via alcohol 6 has been more fruitful. Borchardt and coworkers [46,47] developed a more direct method starting from d-ribonic acid γ-lactone 7, via an enone intermediate 8, which was treated with lithium di-(tert-butoxymethylene)cuprate followed by DIBAL-H to provide the desired key intermediate 10. Condensation of triflate 11 with adenine salt followed by deprotection afforded the chiral aristeromycin (Scheme 1.1). Chu and coworkers made modifications of this scheme in which d-ribose was converted to the key intermediate 14 as well as its enantiomer 16 in eight steps in large scale (>40 g) in good yield (d-series >54%; l-series >45%, Scheme 1.2) [48,49]. A series of l-aristeromycin analogs have been prepared using 16 as a key intermediate [50,51].

Figure 1.3 General approaches of the synthesis of aristeromycin.


5

Scheme 1.1 Synthesis of aristeromycin by Borchardt and coworkers [46]. Reagents and conditions: (a) (i) cyclohexanoe, FeCl3 , NaIO4 , NaOH; (ii) 2-propanol, PPTS; (iii) CH3 PO(OMe)2 , nBuLi, THF; (b) (t BuOCH2 )2 CuLi; (c) DIBAL-H; (d) Tf2 O/Py; and (e) (i) adenine, NaH, (ii) TFA/H2 O.

Scheme 1.2 Modified sequences of the synthesis of aristeromycin by Chu and coworkers [48,49]. Reagents and conditions: (a) (i) 2,2-dimethoxypropane, p-TSA; (ii) TBDMSCl, Im; (b) (i) vinylmagnesium bromide; (ii) TBAF; (iii) NaIO4 ; (iv) NaH, DMSO, Ph3 PMeBr; (c) (i) NaH, DMSO, Ph3 PMeBr; (ii) DCC, DMSO, Py, TFA; (iii) vinylmagnesium bromide; (iv) TBAF; (v) NaIO4 ; (vi) NaBH4 , CeCl3 · 7H2 O; (d) (i) Grubbs’ catalyst; (ii) ˚ MS, AcOH. PDC, 4 A

Recently, Schneller and coworkers also described a hybrid sequence of the previously reported methods to produce aristeromycin (Scheme 1.3) [52]. The key step of this scheme is the 1,4-addition using a vinyl anion instead of tertbutoxymethylene. By this modification, the deprotection step of tert-butyl group is avoided, which requires very harsh conditions, resulting in incompatibility of some protecting groups. 1.2.1.2. Neplanocin (3) An endocyclic double bond in the carbocyclic ring distinguishes neplanocin A from aristeromycin. Therefore, the allylic alcohol 19, which is more reactive than the saturated alcohol 6, has been employed frequently as a precursor for the synthesis of neplanocin A (Figure 1.4). A representative sequence for synthesis was developed by Lim and Marquez (Scheme 1.4) [53], in which neplanocin A was synthesized from ribonolactone 20, which in turn was available in two steps from d-ribonic acid γ-lactone 7 [54]. Treatment of lactone 20 with lithium dimethyl methylphosphonate followed by

6


Scheme 1.3 Synthesis of aristeromycin by Schneller and coworkers [52]. Reagents and conditions: (a) (i) (MeO)2 CMe2 , MeOH; (ii) Ph3 P, I2 , Im; (iii) Zn; (iv) vinylmagnesium bromide; (v) Grubbs’ catalyst; (vi) PCC; (b) (i) vinylmagnesium bromide, TMSCl, HMPA, CuBr • Me2 S; (ii) LiAlH4 ; (c) Ph3 P, DIAD, 6-chloropurine; (d) (i) NaIO4 , OsO4 ; (ii) NaBH4 ; (iii) NH3 /MeOH; (iv) HCl/MeOH.

Figure 1.4 General synthesis approach via allylic alcohol 19.

Scheme 1.4 Synthesis of neplanocin A by Lim and Marquez [53]. Reagents and conditions: (a) (i) LiCH2 P(O)(OCH3 )2 ; (ii) NaOMe; (b) CrO3 , Py; (c) K2 CO3 , 18-crown-6; (d) NaBH4 , CeCl3 ; (e) (i) p-CH3 PhSO2 Cl; (ii) 6-chloropurine, NaH; (iii) NH3 /MeOH; (iv) BCl3 .

sodium methoxide in methanol afforded keto phosphonate 21. Oxidation of 21 with modified Collins reagent produced ketone 22, which underwent intramolecular cyclization under basic condition to generate key intermediate 23. Regioselective reduction of 23 to allylic alcohol 24, followed by condensation condensation with 6-chloropurine, eventually afforded the chiral neplanocin A.


7

Optimization of this sequence was carried out by Johnson and coworkers [55], who utilized the enone 16 as a precursor, which was converted to acetate 26 via a sequence of 1,2-addition/acetylation/1,3-σ rearrangement (Scheme 1.5). Deprotection of 26 provided known compound 24, which was converted to the neplanocin A 3 by the known chemistry. In view of the interesting biological activity as well as the unique structure of neplanocin A, Chu and coworkers conducted the SAR study of d- and l-neplanocin analogs and observed interesting antiviral activity (vida infra, Table 1.11). It is noteworthy that the Mitsunobu reaction was performed to construct the nucleosides instead of classic SN2 coupling reaction (Scheme 1.6) [56]. More recently, Strazewski and Michel reported a short pathway to neplanocin A in good overall yield (Scheme 1.7) [57]. This approach used allylic alcohol 36 as the key intermediate, which was prepared from d-ribose in eight steps. Mitsunobu reaction coupled the carbocyclic moiety with di -Boc protected adenine moiety to give the desired nucleoside 37, which was deblocked to provide neplanocin A (3).

Scheme 1.5 Synthesis of neplanocin A by Johnson and coworkers [55]. Reagents and conditions: (a) (i) n-Bu3 SnCH2 OBn, n-BuLi; (ii) Ac2 O, Et3 N, DMAP; (b) PdCl2 (CH3 CN)2 , benzoquinone; (c) (i) K2 CO3 ; (ii) MsCl, Et3 N; (iii) adenine, K2 CO3 , 18-crown-6; (iv) Pd(OH)2 , cyclohexene; (d) HCl/MeOH.

Scheme 1.6 Synthesis of d- and l-neplanocin analogs by Chu and coworkers [56]. Reagents and conditions: (a) (i) (CH3 )3 COCH3 ,t BuOK, sec-BuLi; (ii) Ac2 O, Et3 N, DMAP; (b) (i) PdCl2 (CH3 CN)2 , benzoquinone; (ii) K2 CO3 ; (c) Mitsunobu conditions, proper base moieties.

8


Scheme 1.7 Synthesis of neplanocin A by Strazewski and Michel [57]. Reagents and conditions: (a) (i) acetone, H+ ; (ii) TBDPSCl, Et3 N, DMAP; (b) (i) Ph3 PMeBr,t BuOK; (ii) (COCl)2 , DMSO, Et3 N; (c) vinylmagnesium bromide; (d) Neolyst dichloride; (e) (i) ˚ MS; (ii) NaBH4 , CeCl3 ; (f) (i) PPh3 , DIAD, base; (ii) TBAF; (g) TFA. PDC, 4A

1.2.2. Aristeromycin and neplanocin A analogs

Although aristeromycin and neplanocin A are potent SAH inhibitors, their therapeutic utility has been limited due to their significant toxicity, which was shown to be mediated through phosphorylation by adenosine kinase and subsequent conversion to the corresponding cytotoxic nucleotides (Figure 1.5) [58–61]. Therefore, modifications based on the prototypes of these natural products have generated some of the carbocyclic analogs that retain the inhibitory activity toward SAH hydrolase, but are devoid of toxicity (Figure 1.6). 1.2.2.1. Aristeromycin analogs 5 -Fluoro-5 -deoxyaristeromycin 38 has been prepared via a Mitsunobu coupling of (1S,2S,3R,4S)-2,3-(cyclopentylid-

Figure 1.5 Metabolites of aristeromycin and neplanocin A.


9

Figure 1.6 Biologically active aristeromycin analogs.

enedioxy)-4-fluoromethylcyclopentan-1-ol (78) with N6 -bis-boc protected adenine (Scheme 1.8) [62]. This procedure is adaptable to preparing a number of 5 -fluoro-5 -deoxycarbocyclic nucleoside analogs with diversity in the heterocyclic base. To gain a glimpse into the biological potential of 38, it was subjected to antiviral evaluation against herpes simplex-1, herpes simplex-2,

10


Scheme 1.8 Synthesis of 5 -fluoro-5 -deoxyaristeromycin by Schneller and coworkers [62]. Reagents and conditions: (a) NaIO4 , OsO4 , MeOH/H2 O, then NaBH4 , MeOH, 53%; (b) DAST, Pyridine, CH2 Cl2 /H2 O, 60%; (c) DDQ, CH2 Cl2 /H2 O, 60%; (d) DIAD,TPP, Ad(Boc)2 ; (e) 3N HCl/MeOH, 58% for two steps.

herpes simplex-1 (TK− ), vaccinia, cowpox, vesicular stomatitis, coxsackie B4, respiratory syncytial, parainfluenza 3, reovirus-1, Sindbis, Punta Toro, rhinovirus, adenovirus, hepatitis C, virus, WestNile virus, and feline coronavirus. No activity was found against these viruses. However, antiviral test found compound 38 (Figure 1.6) showed activity against measles (MO6) with EC50 s 2.8 μM in neutral red assay and 13 μM in visual assay. No cytotoxicity was observed in the cell lines. 2-Modified aristeromycin derivatives 39 and the related analog 40 were synthesized to investigate their inhibitory activity against human and Plasmodium falciparum S -adenosyl-L-homocysteine hydrolase (PfSAHH) as shown in Scheme 1.9 [63]. The 2-fluorinated compound 39 and 2-amino compound 40 showed strong inhibitory activities against PfSAHH with IC50 value of 1.98 and 4.51 and selective index 24 and 20, respectively (Table 1.2), and a complete resistance to adenosine deaminase. Given the fact that the cytotoxicity of aristeromycin is attributed to the metabolism to its 5 -phosphates, Schneller and coworkers addressed this situation by preparing (±) 5 -noraristeromycin 41 (Figure 1.6) to avoid the

Scheme 1.9 Synthesis of 2-modified aristeromycins and their analogs [63]. Reagents and conditions: (a) 2-fluoroadenine or 2-amino-6-chloropurine, NaH, DMSO, then (Ph3 P)4 Pd, Ph3 P, THF, 55◦ C, 73% (for 81) or 46% (for 82); (b) NH3 /MeOH, 0◦ C, 99% (for 83) or 120◦ C, 71% (for 84); (c) OsO4 , NMO, THF–H2 O, rt, 45%, 52%, 35%, and 40% (for 39, 40, and 2 ,3 -epimer).

11


TABLE 1.2 Inhibitory activities of aristeromycin and noraristeromycin carbocyclic nucleosides analogs against human and P. falciparum SAH hydrolases [63–65] IC50 (μM) Compound 1 39 40 42 43 44 45 46 47 48 50

HsSAHH

PfSAHH

SIa

4.85 47.2 90.7 1.1 200 NDb NDb NDb 63 79 9

57 1.98 4.51 3.1 NDb NDb NDb 220 13 7.6 18

0.085 24 20 0.35 4.5 4.8 9.6 0.5

a SI: b

mean of IC50 values for HsSAH/mean of IC50 value for PfSAHH. No inhibitory activity showed at 500 μM.

phosphorylation step by displacing the 5 -phosphate-accepting hydroxyl group from its original place [66]. Surprisingly, nucleoside 41 was found to be nontoxic to host cells but still active against a variety of viruses (Table 1.3). Subsequently, the enantiomerically pure d-5 -noraristeromycin and its l-isomer were synthesized using chiral precursors 85 and 88 as starting materials. The Pd-catalyzed reaction was conducted to couple the base and sugar to provide the target nucleosides (Scheme 1.10) [67]. As shown in Table 1.3, d-form 42 was, on the average, 10-fold more potent than its l-enantiomer 91 in inhibiting virus replication. The 4 -epimer of 5 TABLE 1.3 Antiviral Activity of (–)- and (+)-5 -noraristeromycin (41 and 42) EC50 (μg/mL) Virus

Cell

Compd 41 Compd 42 Compd 91 (racemic) (d-form) (l-form)

Vaccinia virus E6 SM Vesicular stomatitis virus E6 SM Parainfluenza-3 HeLa Reovirus-1 Vero Cytomegalovirus HEL Measles Vero Respiratory syncytial virus Hela Tacaribe Vero a b

Positive control for compound 41. Positive control for compounds 42 and 91.

0.3 0.07 0.4 0.07 0.4 0.4 2.0 1.0

0.04 0.1 0.07 0.7 0.01–0.05 / / 8

0.7 2.0 0.2 7.0 5–20 / / 50

Neplanocin A 0.2a/0.2b 0.2a/2.0b 0.4a/0.2b 0.4a/0.7b 0.4a/0.2–0.5b 0.4a 0.2a 0.4a/0.4b

12


Scheme 1.10 Synthesis of d and l-noraristeromycin by Siddiqi and coworkers [67]. Reagents and conditions: (a) (EtO)2 P(=O)Cl, Py; (b) (i) NH3 /MeOH; (ii) Pd(PPh3 )4 , Ph3 P, Base; (c) (i) OsO4 , NMO; (ii) NH4 OH/MeOH.

noraristeromycin 48 [68] (Figure 1.6) and 5 -homoaristeromycin 56 [69] (Figure 1.6) were also prepared. Compound 48 inhibited the replication of various DNA and RNA viruses at concentrations similar to those for neplanocin A, but were significantly less cytotoxic. Interestingly, an extension of 5 -hydroxylmethyl chain (56) also retained antiviral activity against vaccinia (EC50 1.2 μg/mL), cowpox (EC50 0.12 μg/mL), and moneypox (EC50 0.12 μg/mL) viruses without cytotoxicity up to 100 μg/mL. Kitade and coworkers [64] synthesized 4 -fluorinated analogue of 9 [(1 R,2 S,3 R)-2 ,3 -dihydroxy-cyclopentan-1 -yl]adenine (DHCaA) and their related analogues under the Mitsunobu and palladium(0) coupling conditions followed by fluorination with inversion of the configuration by using diethylaminosulfur trifluoride as shown in Scheme 1.11. The 4 -β-Fluoro DHCaA (43 and 44) and 2-amino-4 -α-fluoro DHCaA (45 and 46) demonstrated slight inhibitory activity against human and P. falciparum S -adenosyl-L-homocysteine hydrolase (Table 1.2), respectively. A favorable antiviral activity of compound 43 was observed toward measles (IC50 of 1.2 μg/mL by the neutral red assay and 14 μg/mL by the visual assay), but this was accompanied by the cytotoxicity in the CV-1 host cells (21–36 μg/mL) [70]. The same group also synthesized 4 -modified noraristeromycin (NAM) analogs [65], 2-fluoronoraristeromycin 47, 5 -α-noraristeromycin 48, 4 -sulfo-, 4 -sulfamoy, 4 -azido, and 4 -amino-NAM. The inhibitory activities of these analogs and related compounds against P. falciparum and human S -adenosylL-homocysteine hydrolase were investigated (Table 1.2), but none of them demonstrated good activity. Borchardt and coworkers also described several other aristeromycin analogs without a 5 -OH [71,72]. Among these compounds, nucleoside 50 (Figure 1.6) was one of the most potent SAH hydrolase inhibitors. A number of other aristeromycin analogs with modifications at the 5 -position have been reported [73–77]. However, no significant antiviral activity was observed for those


13

Scheme 1.11 Synthesis of 4 -fluorinated carbocyclic nucleoside [64]. Reagents and conditions: (i) adenine (for 93) or 2-amino-6-chloropurine (for 94), Ph3 P, DEAD, THF, rt; (ii) NH3 , MeOH, rt (for 95), 55◦ C (for 100), 100◦ C (for 96 and 102); (iii) DAST, CH2 Cl2 , 0◦ C; (iv) OsO4 , NMO, THF–H2 O, rt; (v) N6 -benzoyladenine (for 99) or 2-amino-6chloropurine (for 100), NaH, (Ph3 P)4 Pd, Ph3 P, DMSO, THF, 55◦ C.

molecules with one exception of nucleoside 57 (Figure 1.6), which showed potent antiviral activity against yellow fever (EC50 0.32 μg/mL). Seley-Radtke and coworkers synthesized 5 -deoxy pyrimidine analogues 52 and 53 [78] and evaluated against SAH, but they were inactive. The replacement of the 2 -hydrogen of natural ribonucleosides with a methyl group yields compounds with excellent RNA chain-terminating properties as antiHCV agents. Among them, 2 -C -methyladenosine [79] and 2 -C -methylcytidine [80] were discovered as potent anti-HCV agents and have undergone clinical trials. To explorer the effect of substitution in the carbocyclic moiety of the aforementioned antiviral agents, Hong and coworkers decided to synthesize the nucleoside analogues 54 and 55 containing a novel 2 ,3 -dimethylcarbasugar [81]. These compounds were evaluated as inhibitors of HCV in Huh-7 cell in vitro. But these nucleosides failed to inhibit HCV RNA replication in the cell-based replication assay. Modification of aristeromycin was carried out on its 6 -position as well. Nucleoside 58 (Figure 1.6) is a racemic compound with a fluorine atom on the 6 -β face [82]. The synthesis of 58 was accomplished, starting from the epoxide precursor 105, which was subjected to nucleophilic attack by an azide anion to open up the epoxide ring. Fluorination and azide reduction provided the amine intermediate 107, which was used to construct the target nucleoside 58 (Scheme 1.12). Although 58 and its α-epimer 59 are good SAH hydrolase inhibitors (IC50 8 and 80 nM for β and α epimers, respectively), its analog 60 is not active (IC50 28,000 nM). As shown in Scheme 1.13, the mechanism of this type of SAH hydrolase inhibitors has been proposed. The intermediate 115, generated from 6 -fluoroaristeromycin, is the same one produced by the action of SAH hydrolase

14


Scheme 1.12 Synthesis of 6 -modified aristeromycin analogs [82]. Reagents and conditions: (a) (i) NaN3 ; (ii) 2,2-dimethoxypropane, H+ ; (b) (i) Tf2 O, Py; (ii) TASF; (iii) H2 /Lindlar catalyst; (c) (i) 5-amino-4,6-dichloropyrimidine, Et3 N; (ii) diethoxymethyl acetate; (iii) NH3 /MeOH; (iv) cyclohexene/Pd(OH)2 /C; (v) HCl; (d) p-anisylchlorodiphenylmethane, Py; (e) Tf2 O, 2,6-di-tert-butyl-4-methylpyridine; (iii) lithium benzonate; (iv) NH3 /MeOH; (f) i) Tf2 O, 2,6-di-tert-butyl-4-methylpyridine; (ii) TBAF; (g) adenine, K2 CO3 ; (h) (i) formic acid; (ii) cyclohexene/Pd(OH)2 /C.

Scheme 1.13 Proposed mechanism of 6 -F-neplanocin analogs as SAH hydrolase inhibitors [82].


15

on neplanocin A and can irreversibly bind with the enzyme and consequently inhibits the SAH hydrolase. Therefore, the poor activity of 60 may be explained by the fact that the 6 -hydroxyl group would be more difficult to eliminate than 6 fluorine to generate active intermediate 115. On the basis of this information, the d-enantiomer of 6 -β-fluoroaristeromycin (61) was synthesized [83]. However, no biological data have been reported. Installing exocyclic double bond on the 6 -position of aristeromycin produced novel nucleoside 436 (Figure 1.15) which will be discussed in detail in the next section (vide infra). One of important discoveries of selective SAH hydrolase nucleoside inhibitors is the replacement of adenine moiety with a 3-deazaadenine moiety. This modification provides the reduced metabolic susceptibility of the nucleoside to the adenosine deaminase as well as adenosine kinase [84,85], which may result in increased antiviral potency and/or reduced toxicity profile (Figure 1.7). For instance, 3-deazaaristeromycin 2 exhibited potent antiviral activity against VV and VSV in vitro (vida supra Table 1.1). In vivo, it decreased mortality rate in newborn mice infected with VSV at doses of 20, 100, and 500 μg/day [85]. The 3-Deazanoraristermycin analog 49 (Figure 1.6) showed inhibition of VV and VSV with EC50s of 0.4 and 0.7 μg/mL, respectively, and was nontoxic in E6 SM, Hela, Vero, and MDCK cells at concentrations up to 200 μg/mL [86]. The other interesting 3-deaza analog is compound 51 (Figure 1.6), which does not have 5 -substitution. This nucleoside inhibited L929 cell SAH with IC50 values of 14.3 nM and was not a substrate or inhibitor of cellular adenosine deaminase [71]. Miller and coworkers reported a diastereoselective synthesis of spironoraristeromycin 62-(±) using an acylnitroso Diels–Alder reaction [87,88]. (See Figure 1.6.) The affinity-labeling probes were prepared for the elucidation of the molecular mechanism of SAHHs. Kitade and coworkers reported novel noraristeromycin analogs possessing epoxy functional groups at the 3 ,4 -positions (63–65 in Figure 1.6) [89] as potential affinity-labeling probes, for the elucidation of the catalytic site of SAHH and their affinities with both HsSAHH and PfSAHH, as shown in Schemes 1.14 and 1.15. The values of Ki and Kinact , which are useful for evaluating the affinity and reactivity of an affinity-labeling reagent, are summarized in Table 1.4. The Ki and Kinact values of epoxy compound 64 against HsSAHH were weaker than those of previously reported FDPHA. In addition, compound 64 did not show any Kinact against PfSAHH. It is noteworthy that 3 ,4 -epoxy-2-fluoronoraristeromycin 65 had moderate Kinact against HsSAHH and PfSAHH.

Figure 1.7 Favorable metabolic profile of carbocyclic 3-deaza nucleoside.

16


Scheme 1.14 Synthesis of 2 ,3 -β-epoxynoraristeromycins [89]. Reagents and conditions: (a) TBDMSCl, imidazole, DMF, rt 5 h, 33%; (b) methanesulfonyl chloride, DMAP, Et3 N, CH2 Cl2 , 0◦ C, 0.5 h, 86%; (c) Bu4 NF, THF, rt, 0.5 h, 86%; (d) t-BuOK, DMF, rt 0.5 h, 42%.

Scheme 1.15 Synthesis of 2 ,3 -α-epoxynoraristeromycins [89]. Reagents and conditions: (a) HC(OEt)3 , p-toluenesulfonic acid monohydrate, acetone, 1 h, rt 88–97%; (b) methanesulfonyl chloride, DMAP, Et3 N, CH2 Cl2 , 0◦ C, 0.5 h, 79–97%; (c) TFA/H2 O (1:1), rt, 2 h, 95–99%; (d) t-BuOK, DMF, rt 0.5 h, 42–79%. TABLE 1.4 Ki values and Kinact values of compounds (64–66) against HsSAHH and PfSAHH [89,90] HsSAHH

PfSAHH

Compound

Ki (μM)

Kinact (min)

Ki (μM)

Kinact (min)

FDPHA 64 65 66

8.8 12.4 20.3 5 ± 0.9

0.09 0.552 0.133 —

— ND 14.3 —

— ND 0.099 —


17

The blending of key structural features from the purine and pyrimidine nucleobase scaffolds gives rise to a new class of hybrid nucleosides. The purine–pyrimidine hybrid nucleosides can be viewed as either N-3 ribosylated purines or 5,6-disubstituted pyrimidines, thus recognition by both purine- and pyrimidine-metabolizing enzymes is possible. Given the increasing reports of the development of resistance in many enzymatic systems, a drug that could be recognized by more than one enzyme, could prove highly advantageous in overcoming resistance mechanisms related to binding site mutations. In this regard, Seley-Radtke and coworkers designed and synthesized carbocyclic uracil derivatives with either a fused imidazole or thiazole ring compound 66 and 67 (Figure 1.6) [90]. The targets were screened for their ability to inhibit SAHase and DNA methyltransferase (DNA MeTase). Weak to no activity was observed against DNA MeTase (data not shown) for either compound; however, 66 exhibited good activities against SAHase, whereas 67 showed no appreciable activity. The Ki value for compound 66 was found to be 5.0±0.9 μM against SAHase in the hydrolysis direction based on a Km value of 7.9 μM for the SAH substrate. Modification in the vicinity of the 2 -hydroxy of the ribose in natural ribonucleosides can produce effective RNA chain terminator. For example, 2 -C -methylcytidine [80] and 2 -C -methyladenosine [79] were discovered as potent anti-HCV agents, and 2 -C -methylcytidine had studied in phase II clinical trials. The 4 -homologated stavudine and thiostavudine analogues are molecules of considerable interest as antiviral and antitumor agents. Modeling studies demonstrated the presence of a narrow, relatively hydrophobic 4 -pocket that can accommodate these substitutions, contributing to the observed enhancement in potency. On the basis of these findings, that the branched nucleoside analogues exhibited excellent anti-HCV activities, Hong and coworkers have synthesize 4 (α)-ethyl-2 (β)-methyl carbodine analogues (68 and 69) [91]. The synthesized nucleoside analogues 68 and 69 were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line (LucNeo#2). However, the synthesized nucleosides did not show either any significant antiviral activity or toxicity up to 50 μM. Chu and coworkers synthesized enantiomerically pure cyclopentyl cytosine [(-)-carbodine (72); Scheme 1.16] [92] from d-ribose and evaluated for its antiinfluenza activity in vitro in comparison to the (+)-carbodine (71), (±)-carbodine (70), and ribavirin. The (-)-Carbodine (72) exhibited potent antiviral activity against various strains of influenza A and B viruses as shown in Tables 1.5 and 1.6. The same group also synthesized carbocyclic-6-benzylthioinosine analogues as part of their continuing efforts to develop potent and selective antitoxoplasmic agents (Scheme 1.17) [93]. Various substituents on the aromatic ring of the 6benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compounds. The nature of the substituent at the para position seems to have a substantial impact on binding to the enzyme. A cyano substitution at the para position decreased binding, whereas the methyl substitution

18


Scheme 1.16 Synthesis of (-)-carbodine [92]. Reagents and conditions: (a) MeSO2 Cl, TEA, DCM, rt, quantitative; (b) NaN3 , DMF, 140◦ C, 4 h, 89%; (c) 10% Pd/C, EtOH, rt, 30 psi, 2 h, quantitative; (d) β-methoxyacryloyl isocyanate, DMF,—20◦ C to rt, 10 h, 72%; (e) 30% NH4 OH, EtOH/dioxane (1:1), 100◦ C, 18 h, 85%; (f) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et3 N, 30% NH4 OH, rt, 17 h, 78%; (g) TFA/H2 O (2:1), 60◦ C, 3 h, 82%.

was the best substrate in this series. Modeling studies showed carbocyclic 6benzylthioinosine and 6-benzylthioinosine have similar sugar ring puckering with C2 -endo and anti -base conformation, which led to their binding into the active site of T. gondii adenosine kinase. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, compounds 73 and 74 demonstrated potent antitoxoplasma activity IC50 11.9 and 14.5 μM in cell culture, respectively, without any apparent host toxicity (Table 1.7). 1.2.2.2. Neplanocin A Analogs Neplanocin A analogs are specifically designed to improve the selectivity and reduce the toxicity of this class of nucleosides. Chemical modifications mainly focused on the 5 - and/or 6 -positions and base moiety (Figure 1.8). As mentioned earlier (Table 1.1), 3-deazaneplanocin A (4) exhibited potent inhibitory effect toward SAH hydrolase (Ki 0.05 nM, vida supera, Table 1.1) without toxicity after 24-h exposure up to 100 μM [86]. In newborn mice, it showed marked protective effect against a lethal infection with VSV at a dose of 0.5 mg/kg/day. This compound is one of the most potent SAH inhibitors known so far and has received particular attention from researchers. Chu and coworkers rescreened the antiviral activity of 3-deazaneplanocin A (4) and noticed interesting antiviral activity against HCV and HBV (Table 1.8). It has been recognized that the introduction of a halogen atom at the 2-position of adenine nucleosides allowed resistance to the adenosine deaminase; for instance, arabinosyl-2-fluoroadenine and 2-chloro-2 -deoxyadenosine are

19

(−)-carbodine 72 (+)-carbodine 71 (±)-carbodine 70 Ribavirin (−)-carbodine 72 (+)-carbodine 71 ( ± )-carbodine 70 Ribavirin (−)-carbodine 72 (+)-carbodine 71 ( ± )-carbodine 70 Ribavirin (−)-carbodine 72 (+)-carbodine 71 ( ± )-carbodine 70 Ribavirin

60 ± 46

53 ± 40

84 ± 91

139 ± 156

Duck H5N1

Gull H5N1

Hong Kong H5N1

Vietnam H5N1

0.8 ± 0.2 >41 1.0 ± 0.4 8.4 ± 5.9

0.7 ± 0.1 >41 0.7 ± 0.2 4.3 ± 0.9

0.8 ± 0.3 >41 1.1 ± 0.6 23.6 ± 1.7

1.8 ± 0.5 >414 2.4 ± 0.9 35.4 ± 11.0

Visual CPE

1.5 ± 0.9 >41 2.9 ± 2.3 13.9 ± 10.0

0.8 ± 0.04 >41 1.8 ± 1.0 9.4 ± 0.6

1.0 ± 0.3 >41 1.4 ± 0.5 27.4 ± 6.8

b

5.7 ± 5.4 >41 12.1 ± 9.3 11.6 ± 9.8

2.1 ± 0.4 >41 2.0 ± 0.06 16.8 ± 14.3

1.6 ± 1.0 >41 1.6 ± 0.5 20.3 ± 4.7

10.5 ± 10.1 >414 17.6 ± 13.8 43.6 ± 15.0

Virus yield reduction

EC90 d (μM)

>414 >414 >414 >1310

>414 >414 >414 >1310

>414 >414 >414 >1310

>414 >414 >414 >1310

IC50 e (μM)

>518 4 14 >156

>591 591 >305

>518 376 >55

>230 1 >173 >37

Visual CPE

Anti-influenza activity (H5N1)a

2.0 ± 1.4 >414 5.0 ± 1.9 32.5 ± 8.6

Neutral red

of triplicate experimental data (duplicate for Hong Kong). Approximate CCID50 inoculated per well, average ± SD from triplicate assays. c Effective concentration required to reduce influenza virus-induced cytopathic effect by 50%. d Concentration required to reduce infectious virus titer by 90%. e The 50% cell inhibitory concentration of drug without virus. f In vitro selectivity index (IC50 /EC50 ), and for virus yield reduction (IC50 /EC90 ).

a Average

Compound

Inoculumb

Virus type

EC50 (μM)

c

>276 143 >94

>518 230 >139

>414 296 >48

>207 1 >83 >40

Neutral red

SIf

>73 34 >113

>197 207 >78

>259 259 >65

>39 1 >24 >30

Virus yield reduction

TABLE 1.5 Anti-influenza in vitro activity of (−)-carbodine (72), (+)-carbodine (71), (±)-carbodine (70), and ribavirin against various H5N1 strains [92]

20

21

10

27

A/Solomon Island/03/ 2006/H1N1

A/Wisconsin/ 67/2005/ H3N2

B/Malaysia/ 2506/2004

(−)-carbodine 72 (+)-carbodine 71 ( ± )-carbodine 70 Ribavirin




Compounds

Neutral Red 1.6 ± 0.3 >414 1.9 ± 1.0 19.4 ± 1.8 0.6 ± 0.3 >41 2.0 ± 0.4 25.1 ± 3.6 2.0 ± 0.9 >41 4.6 ± 3.9 20.2 ± 0.6 0.8 ± 0.1 >41 1.3 ± 0.3 19.5 ± 5.6

Visual CPE 1.3 ± 0.20 >414 1.5 ± 1.0 11.2 ± 6.3 0.7 ± 0.4 >41 1.9 ± 0.5 23.3 ± 0.7 1.6 ± 0.7 >41 2.8 ± 0.7 16.4 ± 7.2 0.6 ± 0.2 >41 0.8 ± 0.0 10.2 ± 5.6

b Approximate

Average of triplicate experimental data. CCID50 inoculated per well; same inoculum used for all replicate tests. c Effective concentration required to reduce influenza virus-induced cytopathic effect by 50%. d Concentration required to reduce infectious virus titer by 90%. e The 50% cell inhibitory concentration of drug without virus. f In vitro selectivity index (IC50 /EC50 ) and for virus yield reduction (IC50 /EC90 ).

a

100

Inoculumb

A/California/ 07/2009/ H1N1(swine flu strain)

Virus type

EC50 c (μM)

1.5 ± 1.0 >41 3.8 ± 0.9 22.2 ± 1.9

SIf

>414 >591 >414 414 >218 >1310 >56

>414 >690 >414 414 >518 >1310 >128

>518 318 >67

>207 90 >65

>690 207 >52

>259 1 >218 >68

>276 109 >59

>166 118 >60

>318 180 >49

>109 1 >94 >101

Visual Neutral Virus Yield CPE Red Reduction

>414 >318 >414 1 >414 >276 >1310 >117

IC50 (μM)

e

2.5 ± 0.4 >414 >259 >41 >414 414 >148 21.7 ± 11.9 >1310 >80

1.3 ± 0.9 >41 2.3 ± 0.4 27.0 ± 6.4

3.8 ± 0.3 >414 4.4 ± 2.3 13.0 ± 4.2

Virus Yield Reduction

EC90 d (μM)

Anti-influenza Activity (H1N1, H3N2 and FluB)a

TABLE 1.6 Anti-influenza in vitro activity of (-)-carbodine (72), (+)-carbodine (71), (±)-carbodine (70), and ribavirin against various H1N1, H3N2, and influenza type B strains [92]


21

Scheme 1.17 Synthesis of 6-mercaptopurine derivatives [93]. Reagents and Conditions: (a) NaBH4 , MeOH, 0◦ C, 1 h, 96%; (b) (i) MsCl, Et3 N, CH2 Cl2 , 0◦ C, 1 h, 92%; (ii) NaN3 , DMF, 150◦ C, 6 h, 85%; (iii) H2 , 10% Pd/C, MeOH, 30 psi, 2 h; (iv) 5-amino-4,6dichloropyrimidine, Et3 N, n-PrOH, reflux, 24 h, 68% (two steps); (c) CH(OEt)3 , p-TsOH, rt, 14 h, 65%; (d) thiourea, EtOH, reflux, 3 h; (e) TFA/H2 O (2:1, v/v), 50◦ C, 3 h, 52% (two steps); (f) appropriate benzylhalide, NH4 OH/H2 O, rt, 11h.

TABLE 1.7 The effect of carbocyclic 6-(p-methylbenzylthio)inosine (73), carbocyclic 6-(p-chlorolbenzylthio)inosine (74), and therapeutic compounds on host-toxicitya and percent survivalb of wild-type (RH) and adenosine kinase deficient (TgAK−3 ) strains of Toxoplasma gondii grown in human fibroblasts in culture [93] Concentration (μM) Compound 73 (p-CH3 )

74 (p-Cl)

pyrimethaminec sulfadiazinec

Infection wild type TgAK−3 none wild type TgAK−3 none wild type none wild type none

(RH)

(RH)

(RH) (RH)

0

5

10

25

50

IC50 (μM)

100 100 100 100 100 100 100 100 100 100

43 99 100 73 100 100 99 101 93 98

2.5 100 100 12 100 100 55 100 58 100

0 100 100 0 100 100 25 108 53 100

0 100 100 0 100 100 23 108 46 102

11.9 ± 0.4 14.5 ± 1.3 16.1 ± 2.5 27.3 ± 3.3

a

Host toxicity of uninfected cells was measured by MTT method in at least two independent experiments each of three replica as previously described [15–21]. b Percent survival of parasites was measured by incorporation of [5, 6-13 H]uracil in at least two independent experiments of three replica each as previously described. c Therapeutic compounds.

potent antitumor agents resistant to adenosine deaminase. The 2-Halo derivatives of adenosine have also been known to inhibit AdoHcy hydrolase. On the basis of these results, Matsuda and coworkers designed and synthesized 2-fluoroNPAs (135) as adenosine deaminase–resistant equivalents of NPA (Scheme 1.18) [94,95]. The 2-Fluoroneplanocin A (135) showed an antiviral potency and a

22


Figure 1.8 Biologically active neplanocin A analogs.

Scheme 1.18 Synthesis of 2-fluoroneplanocine [94]. Reagents: (a) K2 CO3 , 18-crown-6, 2-fluoroadenine, DMF; (b) BCl3 , CH2 Cl2 ; (c) HCl, MeOH.

23


TABLE 1.8 Antiviral activity of 3-deazaneplanocin A (4) Entry

Virus

Assay

Cell line

EC50 (μM)

CC50 (μM)

SI

1

Measles

2 3 4 5 6

Vaccinia HCMV HCV HBV Tacaribe

NR Visual CPE CPE HRR VIR NR Visual

CV-1 CV-1 HFF HFF Huh7 ET Huh7 ET Vero 76

0.4 0.9 2.9 0.36 1.44 0.59 103 508 0.62 32 >1 >220

NR, neutral red; CPE, cytopathic effect; HRR, HCV RNA replicon; SI, selective index (EC50 /CC50 ).

spectrum that was comparable to that of neplanocin A (3) (Table 1.9). It was particularly active against vaccinia virus, vesicular stomatitis virus, parainfluenza virus, reovirus, arenaviruses, and human cytomegalovirus (i.e., those viruses that fall within the scope of the S -adenosyl-L-homocysteine hydrolase inhibitors). In addition to these analogs, a series of 6 -position modified neplanocin A analogs were prepared by the same group [96–98]. Compound (6 R)-6 methylneplanocin A (136, RMNPA) demonstrated excellent antiviral potency and selectivity superior to that of the neplanocin A (Table 1.9). In mice study, 136 (EC50 1.0 mg/kg/day) [99] was found to be superior to chloroquine (EC50 1.8 mg/kg/day). Interestingly, its diastereomer, (6 S)-6 -methylneplanocin A (137, SMNPA), was completely biologically inactive. The synthesis of RMNPA 136 was accomplished starting from neplanocin A, as depicted in Scheme 1.19. Protection of 2 - and 3 -positions as well as the amino group on the base moiety left a free 6 -OH, which was oxidized, alkylated, and then separated by HPLC to provide optically pure 136 and 137. A recent report described an improved asymmetric synthesis of 136 via a chelation-controlled stereoselective addition reaction [99].

TABLE 1.9 Antiviral activity and cytotoxicity of compounds neplanocin A 3, 2-F-neplanocin (135) and 6 -(R)-(RMNPA) in vero cells Antiviral activity, IC50 (μg/mL)a

Cytotoxicity, CC50 (μg/mL)b

Compounds

VSV

Measles

Mumps

Vero cells

Neplanocin 3 2-F-neplanocin 135 6 -(R)-(RMNPA) 136

0.25 0.25 0.25

0.10 0.03 0.09

0.11 1.2 0.19

155 >200 >200

a

Inhibitory concentration, required to reduce virus-induced cytopathicity (VSV) or virus plaque formation (measles and mumps) by 50%. b The 50% cytotoxic concentration, required to reduce the number of viable cells by 50%.

24


Scheme 1.19 Synthesis of RMNPA [98]. Reagents and conditions: (a) (i) TMSCl, Py, (ii) BzCl, (iii) NH4 OH; (b) HClO4 , acetone; (c) BaMnO4 ; (d) Me3 Al; (e) (i) 90% HCOOH; (ii) NH4 OH, Dioxane; (f) HPLC (C18 ) separation.

The 5 -nor derivative 138 or 139 [46], prepared via enone 8 through a convergent approach (Scheme 1.20), has been demonstrated to be approximately 10-fold better than the parent compound 3 in terms of antiviral activity (CC50 /IC50 ) [100]. A comparative study analyzed the antiviral activity and toxicity of neplanocin A (3), 5 -norneplanocin A (138) and 5 -nor-3-deazaneplanocin A (139) against a wide range of DNA and RNA viruses [100], in which 138 and 139 showed greater selectivity than neplanocin A against vesicular stomatitis virus and rotavirus (Table 1.10). Schneller and coworkers, in their pursuit of neplanocin A analogues with nontoxic, antiviral potential, found the 6 -isoneplanocin A analogues 140–141 (Figure 1.8) were synthesized [101]. Another interesting compound is 6 -homoneplanocin A (142), which displayed particular activity against human cytomegavirus (EC50 0.15–0.5 μg/mL), vaccinia virus (EC50 0.1 μg/mL), and vescular stomatitis virus (EC50 1.0 μg/mL)

Scheme 1.20 Synthesis of 5 -norneplanocin analogs [46]. Reagents and conditions: (a) NaBH4 , CeCl3 ; (b) (i) TsCl, Et3 N; (ii) base, NaH; (c) HCl.


25

TABLE 1.10 Antiviral activity against VV and cytotoxity of 138, 139, and NPA Compounds Compd 138 Compd 139 Neplanocin A (3)

IC50 (μM)

ID50 (μM)

Antiviral selectivity (ID50 /IC50 )

0.28 0.95 0.08

15 56 0.5

61 59 6

[102]. Starting from enone 16, the key intermediate 187 was obtained via an addition/reduction/rearrangement sequence. Treating mesylated alcohol 187 with adenine or 3-deazaadenine salt, followed by deprotection, afforded desired 6 homoneplanocin analogs (Scheme 1.21). Jeong and coworkers synthesized fluoroneplanocin A 144 (Scheme 1.22), which was believed to inhibit SAH hydrolase based on the type II mechanism [26]. The fluorosugar was prepared starting from eneone 23, which was converted to the iodo derivative 190 by treating with I2 /CCl4 /pyridine. Compound 190 was then reduced, protected, fluorinated, and deprotected to provide desired key intermediate 191. The preparation of target nucleoside 144 was accomplished after obtaining the compound 191 in hand, via SN2 type coupling reaction followed by deprotection steps. The 5 -Fluoroneplanocin A (144) has been found to exhibit

Scheme 1.21 Synthesis of 6 -homoneplanocin A [102]. Reagents and conditions: (a) (i) (TMS)2 NH, BuLi, EtOAc; (ii) LiBH4 ; (iii) TBSCl, Im; (iv) Ac2 O, DMAP, Et3 N; (v) PdCl2 (MeCN)2 , p-benzoquinone; (vi) K2 CO3 ; (b) (i) MsCl, DMAP; (ii) base, NaH, 15crown-5; (c) HCl.

23

190

191

144

Scheme 1.22 Synthesis of fluoroneplanocin A [26]. Reagents and conditions: (a) I2 , CCl4 , Py; (b) (i) NaBH4 , CeCl3 ; (ii) TBDPSCl, Im; (iii) n-BuLi, N -fluorobenzenesulfonimide; (v) TBAF; (c) (i) MsCl, Py; (ii) adenine, K2 CO3 , 18-crown-6; (iii) BBr3 ; (iv) Ac2 O, Py; (v) NH3 /MeOH.

26


better and irreversible SAHH inhibition and more potent antitumor activity than neplanocin A. According to their results, after conversion of 3 -hydroxy group to a keto group by NAD+ the fluorine atom acts as a leaving group during a Michael addition-elimination reaction induced by a nucleophile present in SAHH, as shown in Figure 1.9. Generally, an iodide is a better leaving group than a fluoride. Therefore, it was of great interest to synthesize 5 -iodoneplanocin A (145 in Figure 1.8) [103,104]. Moon and coworkers synthesized 5 -iodoneplanocin A (145), an isostere of 5 -fluoroneplanocin A, and its analogs, 146 and 147 [104], having different purine nucleobases and to evaluate their antiviral activity and cytotoxicity. But none of them showed antiviral activity. Considering that neplanocin A and its fluoro-substituted analogues showed good biological activities, the cyclopentene template of these nucleosides can be assumed to be a good template for phosphorylations by kinases. That’s why Moon and coworkers designed and synthesized 2 -β-C -methylneplanocin A (148) [105]. Disappointingly, compound 148 did not show significant activity against HCV. The antiviral and antitumor activity of NPA (3) seems to be interesting due to an efficient inhibition of S -adenosylhomocysteine (AdoHcy) hydrolase. However, the main drawback for the therapeutic utilization of NPA as an antiviral agent comes from its significant cytotoxicity. In contrast to NPA, ara-neplanocin A (149; ara-NPA) has shown less cytotoxicity while still retaining reasonable antiviral activity [106]. However, a thorough literature search indicated that little attention has been paid to the ara-neplanocin family of compounds. Apart

Figure 1.9 A plausible inhibitory mechanism of SAHH by fluoro-neplanocin A via Michael addition-elimination process (SAHH, S -adenosylhomocysteine hydrolase; Ade, Adenine) [104].


27

from the adenine derivative 149 (ara-NPA), only the cytosine derivative 150 (ara-NPC) has been reported so far, which was evaluated only for its antitumor activity. Whereas the ara-NPA was obtained from a divergent approach starting from NPA in four steps, the ara-NPC was obtained from a 2,2 -anhydro-uridine intermediate, which was the by-product of 2 -deoxygenation reaction. Considering the poor versatility of the reported approaches, Chu and coworkers decided to develop a convergent strategy for the synthesis of ara-neplanocin A analogues have been developed [106]. Microwave-assisted Mitsunobu reaction proved to be an essential tool both for the 2 -β-hydroxy inversion and for the coupling reaction with the heterocyclic bases, as shown in Scheme 1.23. The exploitation of the present approach allowed generating a family of ara-neplanocins of which biological potential is still unexplored. In comprehensive structure activity relationship studies of d- and l-neplanocin analogs (vide supra, Scheme 1.6), Chu and coworkers showed that the cytosine (155) and 5F-cytosine analog (156) exhibited potent anti-orthopoxvirus as well as anti-West Nile virus activities as shown in Table 1.11 [56,107]. On the basis of inhibitory activity of truncated cyclopentenyl cytosine against adenosylhomocysteine hydrolase (SAH), its fluorocyclopentenyl pyrimidine derivatives (157 and 158) were efficiently synthesized from d-ribose via electrophilic fluorination as a key step by Jeong and coworkers (Scheme 1.24) [108]. The final nucleosides were evaluated for SAH inhibitory activity, among which the uracil derivative 157 showed significant inhibitory activity (IC50 8.53

192

154

193

194

195

197

196

Scheme 1.23 Synthesis of ara-neplanocin A (3) analogues under subzero microwaveassisted conditions [106]. Reagents and conditions: (a) BzCl, pyridine, rt, 2 h; (b) Dowex, MeOH, MW, open vessel, 65◦ C, 30 min (two cycles); (c) t-Bu2 Si(OTf)2 , DMF, 0◦ C, 30 min; (d) p-NO2 PhCO2 H, Ph3 P, DIAD, benzene, MW, 180◦ C, sealed tube, 5 min; (e) THF/ MeOH/H2 O (3:2:1), LiOH, rt, 30 min; (f) Ph3 P, DIAD, THF, MW, −40◦ C, 100 W, 5 min; (g) Et3 N · 3HF, rt, 30 min; (h) Sat. NH3 in MeOH, rt, 1 h; (i) Sat. NH3 in MeOH, 90◦ C, 18 h; (j) 2N HCl/MeOH, 60◦ C 15 h; (k) (i) formic acid, 90◦ C, MW, 5 min; (ii) Sat. NH3 in MeOH, rt, overnight.

28


TABLE 1.11 Antiviral activity and toxicity of nepalnocin A (3), 155 and 156 [56,107] Activity (μg/mL) Compds

Toxicity (μg/mL)

Virus

MK2

Vero

MK2

Vero

Nepalnocin A (3)

Smallpox (7124) Smallpox (BSH) Cowpox Monkeypox Vaccinia virus West Nile virus

0.10 0.10 100 0.26 2.62 N/A

0.03 0.14 >100 0.21 >100 >51 (μM)

23 36 100 42 31 N/A

50 10 20 57 >100 3.5 (μM)

Cytosine analog 155

Smallpox (7124) Smallpox (BSH) Cowpox Monkeypox Vaccinia virus West Nile virus

0.08 0.03 0.06 0.1 0.12 N/A

100 0.05 100 >100 70 >100 100 N/A

39 100 >100 100 >100 27.4 (μM)

Scheme 1.24 Synthesis of truncated fluorocyclopentenyl pyrimidines (157 and 158) [108]. Reagents and conditions: (a) I2 , pyridine, CCl4 , rt, 1.5 h; (b) NaBH4 , CeCl3 -H2 O, MeOH, 0◦ C, 40 min; (c) TBDPSCl, imidazole, DMF, rt, overnight; (d) NFSI, n-BuLi, THF, −78◦ C, 10 min; (e) TBAF, THF, rt, 1 h. (f) N3 -benzoyl-uracil, PPh3 , DEAD, THF, rt, 2.5 h; (g) NH3 in MeOH, rt, overnight; (h) 50% aq. TFA, rt, 3 h; (i) 4chlorophenyldichlorophosphate, 1,2,4-triazole, pyridine, rt, overnight; (ii) 1,4-dioxane: 28% NH4 OH = 1:2, rt, 3 h.


29

μM). They were also evaluated for cytotoxic effects in several human cancer cell lines such as fibro sarcoma, stomach cancer, leukemia, and colon cancer, but they did not show any cytotoxic effects up to 100 μM, indicating that 4 -hydroxymethyl groups are essential for the anticancer activity. A number of apio nucleosides, in which 4 -hydroxymethyl group are shifted to C3 position have been synthesized to search for novel antiviral agents. Apio-ddA, mimicking the parent compound ddA, exhibited comparable anti-HIV activity to ddA. On the basis of these findings, apio-neplanocin A (159) and its analogues (160–162) [109] were asymmetrically synthesized, but unlike compounds 3 and 135, these compounds did not show inhibitory activity against AdoHcy hydrolase. Fluoroneplanocin A (135, F-NPA), which was synthesized by Moon’s group, exhibited more potent inhibition of AdoHcy hydrolase than NPA (3), as well as a significant antiviral activity, but also exhibited high cytotoxicity to the cells. Cytotoxicity of NPA (3) and F-NPA (135) seems to be derived from the inhibition of cellular polymerases by their 5 -triphosphate metabolites. On the other hand, it has been known that the inhibitory ability of AdoHcy hydrolase is derived from nucleosides by themselves, such as NPA (3) and F-NPA (135), not their triphosphates. Therefore, nucleosides, which show potent inhibitory ability against AdoHcy hydrolase but which cannot be phosphorylated by kinases, have been considered a promising target for the development of new antiviral agents. On the basis of these findings, apio fluoroneplanocin A (apio F-NPA, 163) [110] and its uracil analog 164 was synthesized as a potential AdoHcy hydrolase inhibitor. On the basis of chemical and biological properties of C-nucleosides as well as carbocyclic nucleosides, it was of interest to synthesize hybrid nucleosides, carbocyclic C-nucleosides. Although some carbocyclic nucleosides and C-nucleosides are naturally occurring, so far no natural carbocyclic C-nucleosides have been identified. Therefore, it was of great interest to synthesize optically active carbocyclic C-nucleosides, possessing a cyclopentenyl moiety as analogs of neplanocin. Chu and coworkers reported the enantiomeric synthesis of purine and pyrimidine cyclopentenyl C-nucleosides 165–168 (Scheme 1.25) [111] from the key intermediate, 2,3-(isopropylidenedioxy)-4-(trityloxymethyl)-4-cyclopenten-1-O-mesylate (204), which was prepared from d-ribose in eight steps and evaluated as potential antiviral agents against HIV, SARSCoV, Punta Toro, West Nile, and cowpox viruses. However, only 9-deazaneplanocin A (165) exhibited moderate anti-HIV activity (EC50 2.0 ± 1.1) [111]. Recently, Chu’s group also reported that the triazole analog 169 (Scheme 1.26) was a potent antiviral agent against vaccinia virus with an EC50 of 0.4 μM, whereas the positive control cidofovir had an EC50 of 6 μM [112]. Recently, the same group also described the synthesis and antiviral activity of 7-deazaneplanocin A (7-DNPA) against orthopoxviruses (vaccinia and cowpox virus) with EC50 values of 1.2 and 3.4 μM, respectively [113]. In addition, the further screening of the compound 170 revealed significant anti-HBV and anti-HCV activity with low cytotoxicity. In view of these interesting biological

30


Scheme 1.25 Enantioselective synthesis of purime cyclopentenyl C-nucleoside (165 and 166) [111]. Reagents and conditions: (a) NCCH2 CO2 Et, NaH, THF, rt then to 55◦ C, 40 h; (b) (i) DIBAL-H, Et2 O,−78◦ C, 30 min; (ii) H2 NCH2 CN.H2 SO4 , NaOAc.3H2 O, MeOH, rt, 24 h; (c) (i) ClCO2 Et, DBU, CH2 Cl2 0◦ C then to reflux, 6 h; (ii) K2 CO3 , MeOH, rt, 1 h; (d) HC(=NH)NH2 .AcOH, EtOH, reflux, 8 h; (e) (i) 12% HCl/MeOH, rt, 2 h; (ii) NaHCO3 /MeOH, rt, 2 h; (f) (i) DIBAL-H, Et2 O,−78◦ C, 30 min; (ii) H2 NCH2 CO2 Et.HCl, NaOAc.3H2 O, MeOH, rt, 24 h; (g) (i) BzN = C = S, CH2 Cl2 , 0◦ C, 1 h; (ii) MeI, DBN, CH2 Cl2 , rt, 2 h; (iii) NH3 /MeOH, 95◦ C, 16 h.

Scheme 1.26 Synthesis of triazol analog [112]. Reagents and conditions: (a) (i) MsCl, Et3 N, (ii) NaN3 ; (b) (i) methyl propiolate, CuI, Et3 N; (ii) NH3 /MeOH; (c) HCl.

results, it was of interest to explore the structure-activity relationships of 7substituted-7-DNPA analogues as potential antiviral agents. These 7-substitutions were introduced by using 7-substituted-7-deaza heterocyclic base precursors (F, Cl, Br, and I) or via substitution reactions after the synthesis of the carbocyclic nucleosides (Scheme 1.27) [114]. Among the synthesized compounds, 170–176 exhibited significant anti-HCV activity (EC50 ranged from 1.8 to 20.1 μM, Table 1.12) [114]. 7-DNPA 170 displayed anti-HCV activity with an EC50


31

Scheme 1.27 Synthesis of 7-deazaneplanocin analogues [114]. Reagent and conditions: (a) PPh3 , DIAD, THF, room temp; (b) NH3 , MeOH, 80◦ C; (c) HCl, MeOH, THF, 50◦ C; (d) (i) NaH, p-methoxybenzyl chloride, DMF; (ii) HCl/MeOH/THF; (iii) Ac2 O, pyridine; (iv) DDQ, CH2 Cl2 , H2 O; (e) Bu3 Sn(CHCH2 ), Pd(PPh3 )4 , Et3 N, CuI, DMF; (f) TMSCtCH, Pd(PPh3 )4 , Et3 N, CuI; (g) Bu3 SnCN, (PPh3 )4 Pd, ClCH2 CH2 Cl, reflux; (h) H2 O2 , NH4 OH.

32


value of 2.5 μM without any cytotoxicity at a concentration up to 100 μM (Table 1.12). In the additional studies of the anti-HCV activity of 7-DNPA, 170 is at least comparable to, if not better than, the positive controls, 2 -C Me-cytosine (2 -C -Me-C) and 2 -F-C -Me-cytosine (2 -F-C -Me-C; Table 1.13) [114]. Compounds 172–174 showed moderate to potent anti-HBV activity (EC50 0.3–3.3 μM). Among synthesized nucleosides, the 7-ethylnyl substituted compound 174 exhibited interesting anti-HBV activity against wild type as well as several lamivudine and adefovir-associated HBV mutants, including rtL180M, rtM204I, rtM204V and rtN236T as shown in Table 1.14 [114]. 1.2.3. Neplanocin F

Neplanocin F, a minor constituent of the family of neplanocin antibiotics, was synthesized as a racemate from racemic cyclopentenone 23, which in turn was available from d-ribonolactone by Marquez and co-workers as shown in Scheme 1,28 [115]. The carbocyclic ring of neplanocin F corresponds to the

TABLE 1.12 Anti-HCV activity of 7-DNPA analogues based on an HCV RNA replicon assay and cytotoxicity [114] Compounds 170 171a 172 173a 174 175a 176 2 -C -Me-adenine

Anti-HCV activity, EC50 (μM)

Cytotoxicity, CC50 (μM)

2.5 9.5% 2.1 49.2% 20.1 0.5% 1.8 0.15

>100 112.7% 15.0 99.7% 48.9 103.3% 11.8 >10.0

SI >40 7.1 2.4 6.6 >66.7

a

Compounds assessed at a single-concentration of 20 (μM) with percentage donating inhibition level compared to control cells.

TABLE 1.13 Additional studies of anti-HCV activity and cytoxicity of 7-DNPA 170 in the hcv rna replicon huh7 assay in comparison to the known agents (2 -F-C -Me-C and 2 -C -Me-C) [114] Compounds 170 2 -F-C -Me-cytosine 2 -C -Me-cytosine a b

Anti-HCV activity, EC50 (μM)a

Cytotoxicity, EC90 (μM)a

CC50 (μM)b

0.9 1.7 3.5

8.2 5.3 11

>50 >50 >50

Effective concentrations required for reducing HCV level by 50% and 90% in 5 days. Cytotoxicity concentration required for reducing the rRNA levels by 50% in 5 days.

33


TABLE 1.14 In vitro antiviral activity of compound 174 against HBV mutants based on the intracellular HBV DNA replication assay [114] EC50 (μM)a Strain WT rtL180M rtLM/rtMV rtM204I rtM204V rtN236T a

Anti-HBV activity 174

3TC

Adefovir

2.5 2.0 2.8 3.0 2.1 5.6

0.2 10.0 >100 >100 >100 0.3

1.3 1.6 1.2 1.8 1.5 7.7

rtLM/rtMV) rtL180M/rtM204V double mutant.

Scheme 1.28 Synthesis of (±)-neplanocin F (223) [115]. Reagents and conditions: (a) (i) 40% TFA; (ii) Ac2 O/Et3 N/DMAP; (b) MeSO2 C1/Et3 N; (c) (i) LiN3 /DMSO/110C; (ii) NaOMe/MeOH; (iii) NaH/DMF, BnBr; (d) [H2 ]/Lindlar catalyst/MeOH, rt; (e) 5-Amino-4,6-dichloropyrimidine/Et3 N/n-BuOH/145◦ C; (f) (i) (EtO)3 CH/HCl/rt; (ii) BCl3 /CH2 Cl2 /−78◦ C; (g) NH3 /MeOH/110◦ C.

allylic rearranged isomer of neplanocin A. Regiospecific reduction of the racemic cyclopentenone 23 and protection of the resulting α-alcohol as a benzyl ether 231 produced, after removal of the isopropylidene moiety, a compound (232) having allylic and homoallylic secondary alcohol functionalities. Differences in the reactivity of these two secondary alcohols were successfully manipulated to prepare the homoallylic substituted azide 234, which was then reduced and converted to the desired adenine ring by conventional methods. In contrast to its bioactive isomer, neoplanocin A, neplanocin F was devoid of cytotoxicity and in vitro antiviral activity (Figure 1.10). Rodr´ıguez and coworkers described an improved procedure for the synthesis of (+)-neplanocin F (224) as shown in Scheme 1.29 [116]. The cyclopentenol, which was achieved from d-ribono-1,4-lactone 7, reacted with benzyl bromide in DMF at 0◦ C to afford the corresponding benzyl ether 238 in good yield,

34


Figure 1.10 Neplanocin F and its analogues.

Scheme 1.29 Synthesis of (+)-neplanocin F [116]. Reagents and conditions: (a) ref [117]; (b) (i) 60% AcOH, 50◦ C; (ii) trimethyl orthoformate, CH2 Cl2 , CAN, rt, 2h; (iii) DIBAL,−78◦ C, 1h; (c) 6-chloropurine, DEAD, PPh3 , THF, rt, 16h; (d) (i) CF3 COOH, CH2 Cl2 , rt, 16h; (ii) MeOH/NH3 , 70◦ C, 5h; (iii) BCl3 , CH2 Cl2 ,−78◦ C; (iv) MeOH,−78◦ C.

which after treatment with acetic acid at 60◦ C followed by MOM protection at the allylic position exclusively, gave rise to compound 239. The 6-chloropurine was then coupled with 239 via the Mitsunobu reaction to produce solely N-9 alkylated product 240. The compound 240 was treated first with trifluoroacetic acid, NH3 /MeOH and then followed by BCl3 in methylene chloride gave rise to target molecule (+)-neplanocin F (224). To evaluate the biological properties of such a carbanucleoside against emerging pathogens, its enantioselective synthesis was reconsidered. In addition, (-)-neplanocin F (225) could be considered an attractive template giving an access, through appropriate chemical modifications, to new series of nucleoside derivatives displaying potential biological properties. Rodr´ıguez and coworkers synthesized (-)-neplanocin F (225) as shown in Scheme 1.30, in which the synthesis of (-)-neplanocin F (225) was stereospecificaly achieved from the known cyclopentenone 14, which was obtained from commercially available 2,3-O-isopropylidene-d-1,4-ribonolactone 7 according to literature protocols [118]. Briefly, treatment of 14 with [(benzyloxy)methyl](tributyl)stannane in the presence of n-BuLi in THF at−78◦ C yielded stereoselectively the 1,2-addition product, which on benzoylation provided compound 241. Palladium-catalyzed rearrangement of 241 gave the corresponding isomeric allylic benzoate 242


35

Scheme 1.30 Synthesis of (-)-neplanocin F (225) [118]. Reagents and conditions: (a) (i) BnOCH2 SnBu3 , n-BuLi/THF, −78◦ C; (ii) BzCl/pyridine, rt; (b) (i) PdCl2 [CH3 CN]2 , p-benzoquinone/THF, 85◦ C; (ii) NaOH 1%, MeOH, rt; (iii) BnBr/NaH/DMF, rt; (c) (i) 60% AcOH, 50◦ C; (ii) HC(OMe)3 Ce(NH4 )2 (NO3 )6 , CH2 Cl2 , rt; (iii) DIBAL,−78◦ C; (iv) Tf2 O/DMAP/CH2 Cl2 , 0◦ C; (d) adenine, K2 CO3 , 18-crown-6 ether, DMF, 60◦ C; (e) (i) 18% TFA in CH2 Cl2 , rt; (ii) BCl3 , CH2 Cl2 ,−78◦ C.

with good yield. Introduction of the heterocyclic base was achieved via the preparation of the triflate 243, which on reaction with adenine gave solely the N-9 alkylated product. Removal of the MOM group by treatment with TFA/CH2 Cl2 as well as the two benzyl ethers by treatment with BCl3 /CH2 Cl2 at −78◦ C provided the target molecule (-)-neplanocin F (225). Chu and coworkers describe the synthesis and antiviral activity of (±)-5 deoxyneplanocin F analogues 226–230 as shown in Scheme 1.31 [119]. Among the compounds 226–230, 5 -deoxyneplanocin F adenine (226) exhibited moderate anti-HIV activity (EC50 13.8 μM) in human lymphocytes without any marked cytotoxicity up to 100 μM. 1.2.4. Carbovir, Abacavir and Their Analogs

Vince and coworkers first reported (±)1.2.4.1. Carbovir and Abacavir Carbovir with potent anti-HIV activity and low cytotoxicity [120]. Although the (-)-d form is approximately 75-fold more potent than its enantiomer, triphosphates of (-)-d and (+)-l-carbovir are equally active against HIV reverse transcriptase [120,121]. Therefore, the reduced activity seen with (+)-l-carbovir in vitro could, in part, be attributed to low levels of conversion to its phosphates. Cytosolic 5 nucleotidase converts (-)-d-carbovir to its triphosphate, which can incorporate into viral DNA and disturb viral replication but have no interaction with host cell DNA polymerase α, β, and γ. Unfortunately, the low aqueous solubility and poor oral bioavailability, as well as inefficient central nervous system penetration, prevented it from further developing as anti-HIV agents [122–124]. To improve

36


Scheme 1.31 Synthesis of (±)-neplanocin F analogues (226–230). Reagents and conditions: Synthesis of adenine derivatives. Reagents and conditions: (a) (i) (Boc)2 O, DMAP, Et3 N, CH2 Cl2 , rt, 3 h; (ii) m-CPBA, CH2 Cl2 , refluxed, 2 h; (b) MeONa, MeOH, rt, overnight; (c) (i) CF3 COOH, CH2 Cl2 , 0◦ C, 2.5 h; (ii) Et3 N, EtOH, 4,6dichloro-5-nitropyrimidine, 0◦ C, 2 h; (d) (i) SnCl2 .2H2 O, EtOH; 80◦ C, 10 min; (ii) CH(OCH3 )3 , CH3 SO3 H, rt, 1 h; (e) (i) DIBAL-H, THF, CH2 Cl2 ,—78◦ C, 4 h; (ii) NH3 /CH3 OH, 110◦ C, 24 h; (f) p-Nitrobenzoic acid, DIAD, Ph3 P, THF, 0◦ C, 6 h; (g) (i) DIBAL-H, BF3 .Et2 O, CH2 Cl2 ,—78◦ C, 2.5 h; (ii) Ac2 O, pyridine, CH2 Cl2 , 0◦ C, 3 h; (h) (i) CF3 COOH, CH2 Cl2 , 0◦ C, 2.5 h; (ii) N -(4,6-dichloro-5-nitropyrimidin-2-yl)acetamide, Et3 N, EtOH, 3 h; (i) (i) SnCl2 .2H2 O, EtOH, 60◦ C, 30 min; (ii) CH(OCH3 )3 , CH3 SO3 H, rt, 3 h, in two steps; (iii) HSCH2 CH2 OH, CH3 ONa, CH3 OH, reflux, 7 h; (j) (i) CF3 COOH, CH2 Cl2 , 0◦ C, 2.5 h; (ii) Et3 N, methacryloyl isocyanate, rt, overnight; (k) (i) CF3 COOH, CH2 Cl2 , 0◦ C, 2.5 h; (ii) Et3 N, 3-methoxy-2-methylacryloyl isocyanate, toluene, 0◦ C, 3 h; (l) NH4 OH, CH3 OH, 85◦ C, 24 h.


37

Figure 1.11 Carbovir and abacavir.

its preclinical profiles, a number of prodrug of carbovir were prepared, including a 6-cyclopropylamino substituted analog, which was later known as the clinically useful drug abacavir (Figure 1.11) [123]. Abacavir exhibits significant anti-HIV activity with low cytotoxicity [123], and more important, it has excellent pharmacokinetic as well as toxicological profiles. The unique activation process of abacavir to its triphosphate is described in Figure 1.12: (1) adenosine phosphortransferases are responsible for the monophosphorylation; (2) deamination to carbovir monophosphate is performed by cytosolic deaminase; and (3) cytosolic enzymes are responsible for the conversion of carbovir monophosphate to triphosphate [125]. In December 1998, abcarvir was approved by the FDA for the treatment of HIV infection under the trade name of Ziagen™. It has been used in combinations with AZT and 3TC (Trizivir™) and later with 3TC (Epizcom™). Vince’s procedure [120,126] (Scheme 1.32) utilized the racemic compound 245 as the starting material, which underwent a sequence of hydrolysis/esterification/reduction/deprotection to generate the key intermediate 258.

Figure 1.12 Activation pathway of abacavir [125].

Scheme 1.32 Synthesis of racemic carbovir [120,126]. Reagents and conditions: (a) (i) H+ ; (ii) MeOH, H+ ; (iii) esterification; (b) (i) LiBH4 ; (ii) H+ ; (c) base construction.

38


The compound 258 was converted to the target nucleoside (±)- as well as other analogs by reported methodology [127]. Later, it was found that bicycle compound (±) 245 can be resolved by using Psedomomonas solanacearum [128,129]. Pig liver esterase (PLE) distinguishes the two enantiomers of (±) 257 [130], and adenosine deaminase recognizes either (-) or (+) 261 at different temperature. By using these enzymatic methods, optically pure (-) 255 could be prepared (Scheme 1.33) [126]. Enantioselective synthesis of (-)-carbovir was reported by Jones and coworkers from Glaxo [131]. Starting from the same chiral epoxide 262, two different routes were developed, in which the first one (route a) generated a double bond at the nucleoside level, whereas the second one (route b) produced a vinyl epoxide 268 at the very beginning of the sequence. However, both routes needed an extra step to remove the 6 -hydroxyl group by Barton-McCombie radical reaction (Scheme 1.34). Crimmins’s approach (Scheme 1.35) [132] to prepare (-)-carbovir relies on the Trost’s palladium-catalyzed nucleophilic coupling reaction [44]. The racemic homoallylic chloride 273 was converted to optically active endocyclic vinyl compound 277 in four steps by using a chemical resolution method. The compound 277 was then coupled with base moiety under Trost’s conditions (allylpalladium dichloride dimer/Ph3 P) to give nucleoside 278, which was further hydrolyzed to afford the target compound 255.

Scheme 1.33 Enzymatic resolution reactions in the synthesis of carbovir [126,128–130]. Reagents and conditions: (a) Psedomomonas solanacearum; (b) PLE; (c) adenosine deaminase.


39

Scheme 1.34 Synthesis of carbovir from epoxide 262 [131]. Reagents and conditions: (a) PMBCl, NaH; (b) Ph3 P, DIAD, base; (c) (i) PhOCSCl, DMAP, (ii) BuSnH, AIBN; (d) DDQ; (e) (i) MsCl, DMAP, (ii) NaOCH2 CH2 OMe; (f) base derivation and deprotection; (g) (i) MsCl, DMAP, (ii) TBAF; (h) BF3 • Et2 O, Ac2 O; (i) NaNO2 , AcOH.

Scheme 1.35 Synthesis of carbovir from chloride compound 273 [132]. Reagents and conditions: (a) Mg, CO2 , recrystallization as (-)-(α-phenylethyl)amine salt; (b) LAH; (c) BuLi, CO2 , I2 ; (d) DBU; (e) 2-amino-6-chloropurine, allylpalladium chloride dimmer, PPh3 ; (f) NaOH.

40


Scheme 1.36 Chiral auxiliary assisted asymmetric synthesis of carbovir [132]. Reagents and conditions: (a) n-BuLi, pentenoic pivalic mixed anhydride; (b) Bu2 BOTf, Et3 N, CH2 = CHCHO; (c) Grubbs’ catalyst; (d) LiBH4 ; (e) Ac2 O, Et3 N, DMAP; (f) 2-amino-6-chloropurine, Pd(PPh3 )4 .

A chiral auxiliary assisted asymmetric synthesis of carbovir and abacavir was accomplished by the same group (Scheme 1.36) [132]. Pentenoic pivalic mixed anhydride was coupled with 279 to obtain 280, which was subjected to the syn aldol condensation to provide the diene 281. A metathesis/reduction/esterification sequence afforded 284, which underwent Pd(0) catalyzed a coupling reaction and a base derivation to smoothly generate abacavir or carbovir. More recently, Crimmins and coworkers optimized the selectivity of the coupling reaction by a solid phase synthesis [133]. Trost and coworkers reported improvements in the asymmetric desymmertrization reaction in 1996 (Scheme 1.37) [134]. A unique ligand 288, a tertiary amine base (pempidine), and a modified guanine equivalent 289, were employed in the coupling step. By this method, both enantioselectivity and regioselectivity were significantly improved.

Scheme 1.37 Improved asymmetric synthesis of carbovir [134]. Reagents and conditions: (a) (C3 H5 PdCl)2 , pempidine, 288, 289, DMSO/THF; (b) [Pd2 (dba)3 ]-CHCl3 , Ph3 P; (c) (i) tetramethylgunidine; (ii) tetrabutylammonium oxone, Na2 CO3 ; (iii) Ca(BH4 )2 ; (iv) NH4 OH.


41

Riera and coworkers developed a new enantioselective approach to (-)-carbovir and (-)-abacavir from the Pauson–Khand (PK) adduct 292 (Scheme 1.37) [135]. The chiral cyclopentenone 292 is readily accessible in enantiomerically pure form via PK reaction of trimethylsilylacetylene 291 and norbornadiene, using N benzyl-N -diphenylphosphinotert-butyl-sulfinamide as a chiral P,S ligand, showing its usefulness as a cyclopentenone synthon [136]. Starting from PK adduct 292, the hydroxymethyl group was efficiently added via photochemical conjugate addition. Protection as a triisopropylsilyl ether followed by the retro-Diels-Alder reaction afforded the protected hydroxymethyl cylopentenone 295. Diastereoselective reduction to the corresponding allyl alcohol and palladium catalyzed substitution afforded an advanced intermediate 297, which was converted into enantiomerically pure (-)-carbovir and (-)-abacavir as shown in Scheme 1.38 [135]. Chu and coworkers developed another sequence to prepare l-carbovir analogs as shown in Scheme 1.39 [137]. Starting from l-enone 16, which could be readily

Scheme 1.38 Synthesis of (-)-carbovir and (-)-abacavir from enantiomerically pure Pauson-Khand (PK) adduct 292 [135]. Reagent and conditions: (a) (i) KCN, NH4 Cl, DMF, H2 O, rt, 3h; (ii) 2,2-Dimethyl-1,3-propanediol, p-TsOH cat., toluene, reflux, 12h; (b) (i) DIBAL-H, THF, 0◦ C, 1h; (ii) NaBH4 , MeOH, 0◦ C, 2h; (iii) HCl (1M), acetone, rt, 3h; (iv) TIPS-Cl, imidazole, DMF, rt, 3h; (c) AlMeCl2 , CH2 Cl2 , 55◦ C, 2h; (d) (i) DIBAL-H, THF,−78◦ C, 1h; (ii) ethyl chloroformate, pyridine, CH2 Cl2 , rt, 40 min; (e) 6chloro-9H-purin-2-amine, NaH, Pd(PPh3 )4 , DMF, 45◦ C, 3h; (f) cyclopropyl amine, EtOH, reflux, 4h; (g) TBAF, THF, rt, 1h; (h) NaOH, H2 O, 100◦ C, 5h.

42


Scheme 1.39 Synthesis of l-carbovir analogs [137]. Reagents and conditions: (a) (i) tert-butyl methyl ether, sec-BuLi, t BuOK, CuBr · Me2 S; (ii) NaBH4 , CeCl3 · 7H2 O; (iii) BzCl; (iv) HCl; (b) (i) CH(OMe)3 /Py/p-TSA; (ii) Ac2 O, 120◦ C–130◦ C; (iii) NaOH; (c) Mitsunobu couplings and base derivation.

synthesized from d-ribose in several steps (vida supra, Scheme 1.2), the diol 300 was obtained via a four-step sequence in high yield. Treating the diol 300 under pyrolytic elimination condition successfully provided the allylic alcohol 301. Condensation of the allylic alcohol 301 with proper base moieties, such as 6-chloropurine, under the Mitsunobu conditions provided desired nucleosides, which was subjected to base derivation and deprotection steps to furnish the target l-carvovir analogs (302). This scheme is very straightforward and suitable for scale up. 1.2.4.2. Carbovir and cbacavir analogs In light of the fact that noraristeromycin and norneplanocin increased selectivity, Huang and coworkers [138] prepared racemic 5 -norcarbovir 303 and 5 -norabacavir 304 with the anticipation that these two desmethylene derivatives might have anti-HIV activity similar to carbovir and abacavir. However, it was found that only norabacavir 304 showed moderate anti-HIV-1 activity with an EC50 5.0 μg/mL, but it was toxic to host cells. The synthesis of both compounds started from an epoxide 337, which underwent the Trost type coupling reaction to give nucleoside 338. Further base derivation afforded desired 303 and 304 (Scheme 1.40). Katagiri and Kaneko reported (-)-BCA 305, an unnatural l-carbocylic nucleoside as a potent anti-HIV-1 agent with an EC50 of 0.71 μM in MT-4 cells [139–141]. However, no updated information of this compound is available (Figure 1.13). The enzymatic resolution by Rhizopus delemar lipase (RDL) was applied in the synthesis of optically pure intermediate 339, which was then oxidized and underwent Curtius rearrangement to give carbamate 341. A deprotection step, followed by a well-known base construction procedure, afforded the target

Scheme 1.40 Synthesis of norcarbovir and norabacavir [138]. Reagents and conditions: (a) Pd(OAc)2 , TPP, base; (b) NaOH; (c) EtOH, cyclopropylamine.


43

Figure 1.13 Carbovir and abacavir analogs.

nucleoside 305 (Scheme 1.41). Nucleosides having a 4 -carbon-substituent have attracted much attention due to the reported potent anti-HIV activity of 4 -cyanothymidine and 4 -ethynyl-2 -deoxycytidine. Also, the 4 -ethynyl analogue (4 -Ed4T) of anti-HIV agent stavudine (d4T) was found to show a higher activity against HIV than the parent compound, stavudine. Based on

Scheme 1.41 Synthesis of (-)-BCA [141]. Reagents and conditions: (a) Rhizopus delemar lipase; (b) (i) MOMCl; (ii) K2 CO3 /MeOH; (iii) PCC and then NaClO2 ; (iv) DPPA; (c) (i) KOH; (ii) Base construction; (iii) NH3 .

44


these facts, Kumamoto and his coworkers synthesized 4 -branched (±)-BCA derivatives (4 -hydroxymethyl 306, 4 -vinyl 307, and 4 -ethynyl 308) [142] that can be regarded as a hybrid of 305 and 4 -carbon-substituted nucleosides (4 -cyanothymidine, 4 -ethynyl-2 -deoxycytidine, and 4 -ethynyl analogue (4 Ed4T)). Compounds 306–308 were tested for their potential to inhibit replication of HIV-1 and HCV in cell culture, but no significant inhibition was observed. Toyota and coworkers reported the synthesis of 3 -fluorine substituted carbovir analog 309 in 1998; however, no biological data were provided [143]. Since then, Chu and coworkers have accomplished the asymmetric synthesis of both 2 - and 3 -fluorine substituted analogs including d- and l-nucleosides, and antiviral data were reported (Table 1.15) [144,145]. Among 2 -F substituted nucleosides, ladenine derivative 310 was the most potent anti-HIV agent with an EC50 of 0.77 μM without toxicity at a concentration up to 100 μM. In the other series, D-3 -F guanosine analog 311 exhibited marked anti-HIV activity (EC50 0.41 μM) with marginal toxicity. Interestingly, this compound (311) showed significant crossresistance to the HIV M184V mutant (FI 38.8; Table 1.15), which was believed to be the result of template/primer realignment as indicated in the molecular modeling studies [145]. To synthesize 2 -F compounds, 2 -F-allylic alcohol 347 was prepared from a common enone intermediate 14 in 13 steps (Scheme 1.42). Compound 14 was then condensed with bases under Mitsunobu conditions and subjected to base derivations to afford target nucleosides. However, the 3 -F analog of 347 was difficult to prepare due to its instability. Therefore, in this series the double bond was generated in the final stage of the whole sequence by microwave-assisted reactions. The l-series compounds were prepared in the same manner. Modifications of 4 - and 6 -position of carbocyclic ring have also generated compounds 314 [146,147] and312–313 [148] (Scheme 1.43). However, neither of them showed significant biological activity. Two-directional ring-closing metathesis (RCM) was applied successfully for the synthesis of 4 -vinylated carbocyclic nucleoside analogues from the trivinyl intermediate 362, which was readily made using a sequential Claisen rearrangement and RCM starting from Weinreb amide 360, as depicted in Scheme 1.44 TABLE 1.15 Antiviral activity of compounds 310 and 311 against HIV-wild type (xxBRU) and M184V mutant [144,145] Compds

xxBRU

M184V

EC50 (μM) EC90 (μM)

EC50 (μM) EC90 (μM)

FIa

310 3TCb

0.77 0.027

8.34 0.25

75.3 >100

>100 >100

98 >100

311 Carbovirc

0.098 0.087

0.58 0.27

3.8 0.20

14.9 1.1

38.8 2.3

a

FI is the fold increase (EC50 HIV-1M184V /EC50 HIV-1xxBRU. ) Positive control for compound 310. c Positive control for compound 311. b


45

Scheme 1.42 Synthesis of 2 -F and 3 -F d4 carbocyclic nucleosides [144,145]. Reagents and conditions: (a) (i) tert-butyl methyl ether, sec-BuLi, t BuOK, CuBr · Me2 S; (b) (i) NaBH4 , CeCl3 · 7H2 O; (ii) BnBr; (iii) HCl; (c) AIBBr, K2 CO3 ; (d) LAH; (e) (i) TrCl, Py; (ii) PDC; (iii) DAST; (f) (i) t BuOK, THF; (ii) HCl; (iii) TBSCl, Im; (iv) Na/NH3 (liq.); (g) Mitsunobu conditions and base derivations; (h) TrCl, DMAP; (i) Super-hydride; (j) PDC; (k) neat DAST; (l) (i) TMSI; (ii) TBDPSCl, Im; (m) (i) Mitsunobu conditions and base derivations; (ii) deprotection; (n) microwave assisted elimination, t BuOK, DMF.

Scheme 1.43 General schemes for the synthesis of compounds 312–314 [146,148].

46


Scheme 1.44 An efficient synthesis of 4 -vinylated carbocyclic nucleosides [149]. Reagents: (a) Grubbs’ catalyst (II), CH2 Cl2 α-isomer:X=H, Y=OH, β-isomer: X=OH, Y=H; (b) ClCO2 Et, pyridine, DMAP; (c) nucleosidic bases, Pd2 (dba)3 · CHCl3 , P(O-i-Pr)3 , NaH, THF/DMSO; (d) (i) TBAF, THF; (ii) 2-mercaptoethanol, NaOMe, MeOH; (iii) CH3 COOH; (ii) and (iii) applicable only for compound 317.

[149]. An antiviral evaluation of the compounds 315–317 against various viruses such as HIV-1 (MT-4 cells), HSV-1 and HSV-2 (CCL 18 cells), and HCMV (AD-169) revealed that the guanine analogue 317 exhibited moderate anti-HIV activity in the MT-4 cell line (EC50 = 10.2 μM; Table 1.16) [149]. It is believed that the arrangement in the carbocyclic guanine nucleoside analogue 317 may be conformationally similar to that in natural nucleosides containing ribose. Hence, this arrangement will enhance the level of phosphorylation by kinase to produce the active monophosphate form. Based on these findings of branched nucleosides, a class of nucleosides comprising 4 α-quaternary carbocyclic nucleosides with an additional methyl group at the 6 -position was synthesized (Scheme 1.45) [150]. The quaternary carbon at the 4 -position of carbocyclic nucleoside was installed successfully via a Claisen rearrangement. The stereocontrolled construction of a methyl group in the 6 α-position was directed through the Felkin-Anh rule. A Bis-vinyl compound 368 was cyclized successfully using Grubbs’ catalyst II to provide a carbocycle

Scheme 1.45 Stereocontroled synthesis of quaternary carbon containing novel abacavir analogue 318 [150]. Reagents and conditions: (a) second-generation Grubbs’ catalyst, CH2 Cl2 , reflux, overnight, major 369; (b) (i) ClCO2 Et, DMAP, pyridine, rt, overnight; (ii) 2-amino-6-chloropurine, Pd2 (dba)3 · CHCl3 , P(O-i-Pr)3 , NaH, THF/DMSO, reflux, overnight; (iii) TBAF, THF, rt; (c) cyclopropylamine, EtOH, reflux.


47

TABLE 1.16 Antiviral activity of compounds 315–318, 321–324, and 326–329 [149,150,152,154] Compounds 315 316 317 318 321 322 323 324 326 327 328 329 AZT GCV ACV

HIV-1 EC50 (μM)

HSV-1 EC50 (μM)

HSV-2 EC50 (μM)

HCMV EC50 (μM)

Cytotoxicity CC50 (μM)

95 77.9 10.2 10.67 90 45.7 99 11.91 13.1 72.1 66.2 34.8 0.01 ND ND

>100 >100 65.8 >100 >100 98 >100 88 67.3 >100 99 >100 ND 2.2 0.2

>100 90.5 >100 >100 >100 >100 >100 >100 >100 >100 99 >100 ND 2.2 ND

>100 23.7 41.5 >100 >100 19.3 99 36.4 ND ND ND ND ND 0.8 ND

95 >100 99 >100 90 98 >100 99 >100 >100 99 >100 4.50 >10 >100

Note: AZT, azidothymidine; GCV, ganciclovir; ACV, acyclovir. ND, not determined. EC50 (μM). concentration required to inhibit 50% of the virus induced cytopathicity. CC50 (μM), concentration required to reduce the cell viability by 50%.

nucleus for the target compound. The antiviral evaluations against various viruses such as HIV-1, HSV-1, HSV-2, and HCMV were performed. The synthesized compound 318 showed moderate anti-HIV activity (EC50 10.67 μM, MT-4 cell lines) without any cytotoxicity up to 100 μM (Table 1.16) [150]. Chu and coworkers reported the hitherto unknown synthesis of 3-deazacarbovir 319 [151] and its adenosine analogue 320 as shown in Schemes 1.46 and 1.47 [151]. The major highlight in the synthesis of adenosine analogs was to use 6-N,N -diboc protected 3-deazapurines for regioselective Mitsunobu coupling as well as unexplored palladium catalyzed coupling with these substrates. Synthesis

371

372

373

319

Scheme 1.46 Synthesis of 3-deazacarbovir 319 [151]. Reagents and conditions: (a) (i) Diphenyl carbamoyl chloride, diisopropyl amine, pyridine, 1 h, 85%; (ii) K2 CO3 , MeOH, 45 min; (b) 374, Pd(PPh3 )4 , THF/DMSO (1:1), rt, 45 min–1 h; (c) NH3 , MeOH, 80◦ C, 16 h.

48

374


375

320

Scheme 1.47 Synthesis of 3-deazaadenosine analogue 320 [151]. Reagents and conditions: (a) N,N -di-Boc-3-deazaadenine, Pd(PPh3 )4 , THF/DMSO (1:1), rt, 1 h; (b) (i) DCM, TFA, rt, 5–7 h; (ii) K2 CO3 /MeOH, 1 h, rt.

of 3-deazacarbovir 319 has been accomplished by the regioselective palladium catalyzed coupling of 6-N,N -diphenylcarbamoyl protected 3-deazaguanine base 372 with dicarbonate 374. All the target nucleosides were screened for anti-HIV1 activity, and none of them have significant activity as well as toxicity up to 100 μM. Hong and coworkers developed a synthetic route for carbocyclic versions of stavudine analogues (321–324; Scheme 1.48) [152]. The construction of an ethynylated quaternary carbon at the 4 -position of carbocyclic nucleosides was accomplished using Claisen rearrangement and ring-closing metathesis (RCM) of dienyne 377 as key transformations. The synthesized compounds 321–324 were evaluated against HIV-1 (MT-4 cells), HSV-1 (CCL-81 cells), HSV-2 (CCL81 cells), and HCMV (AD-169, Davis cells). Among them, only the guanine analogue 324 is moderately active against HIV-1 in the MT-4 cell line (EC50 11.91 μM), and the thymine analogue 322 showed weak antiviral activity against HCMV (Table 1.16).

Scheme 1.48 Synthetic route of carbocyclic versions of stavudine analogues (321–324) [152]. Reagents: (a) Grubbs’ catalyst (II), CH2 Cl2 ; (b) ClCO2 Et, pyridine, DMAP; (c) (i) pyrimidine nucleosidic bases, Pd2 (dba)3 · CHCl3 , P(O-i-Pr)3 , NaH, THF/DMSO; (ii) TBAF, THF; (d) 2-amino-6-chloropurine, Pd2 (dba)3 · CHCl3 , P(O-i-Pr)3 , NaH, THF/DMSO; (e) (i) TBAF, THF; (ii) 2-mecaptoethanol, NaOMe, MeOH; (iii) CH3 COOH.


49

Synthesis of (±)-4 -ethynyl-5 ,5 -difluoro-2 ,3 -dehydro-3 -deoxy-carbocyclicthymidine (325) [153] was carried out by Kumamoto and coworkers, as shown in Scheme 1.49. The difluoromethylylidene group of 325 was constructed by the electrophilic fluorination to the cyclopentenone 382 by using Selectfluor to the enolate derived from 381. The resulting difluoroenone 383 was stereoselectively reduced to the allyl alcohol followed by manipulation of the methoxycarbonyl group of 383 allowed to prepare 384. Introduction of thymine base was investigated based on the Mitsunobu reaction by employing cyclopentenyl allyl alcohols variously substituted at the 4-position. It was found the 4-methoxycarbonyl derivative 383 gave the highest selectivity in terms of both regio- and stereochemistry. A brief conformational analysis of 325 was also carried out based on its X-ray crystallographic data. Hong and coworkers [154] synthesized 4 -modified cyclopentenyl pyrimidine C-nucleosides via C–C bond formation using SN2 alkylation via the key intermediate mesylates 388 and 389 (Scheme 1.50), which were prepared from acyclic ketone derivatives. When antiviral evaluation of synthesized compound was performed against various viruses such as HIV-1, HSV-1 and HSV-2, isocytidine analogue 326 found moderate active against HIV-1 in CEM cell line with an EC50 13.1 μM [154] (Table 1.16). Qing and coworkers stereoselectively synthesized 3 ,3 -Difluoro-2 hydroxymethyl-4 ,5 -unsaturated carbocyclic nucleosides 330–332 [156] from ester 400, which can be conveniently prepared from 2,3-isopropylidened-glyceraldehyde 398 (Scheme 1.51). The whole synthesis highlighted the stereoselective Reformatskii-Claisen rearrangement, ring-closing metathesis (RCM), and palladium-catalyzed allylic alkylation, in which the regioselectivity was reversed from that of nonfluorinated substrates.

Scheme 1.49 Synthesis of (±)-4 -ethynyl-5 ,5 -difluoro-2 ,3 -dehydro-3 -deoxy-carbocyclic-thymidine (325) [153]. Reagents and conditions: (a) PDC, CH2 Cl2 (78%); (b) (i) TMSCl, Li/HMDS, THF,−78◦ C; (ii) Selectfluor, MeCN; (c) (i) NaBH4 , CeCl3 .7H2 O, MeOH, THF,−78◦ C; (ii) DIBAL-H, CH2 Cl2 ,−78◦ C; (iii) P(O)(OMe)2 C(N2 )COMe, K2 CO3 , MeOH; (d) (i) N3 -benzoylthymine, DIAD, Ph3 P, THF; (ii) NH3 /MeOH.

50


Scheme 1.50 Synthesis of 4 -modified cyclopentenyl pyrimidine C-nucleosides via C–C bond formation using SN2 alkylation via the key intermediate mesylates 388 and 389 [154,155]. Reagents and conditions: (a) MsCl, TEA, CH2 Cl2 ; (b) (i) NaH, CH2 (CO2 Et)2 , THF; (ii) LiCl, DMSO; (c) (i) LDA/THF, HCOEt; (ii) H2 NC(=NH)NH2 .carbonate, NaOEt/EtOH, for comp 394 and 396; (d) (i) LDA, HCOEt; (ii) MeI, DMF; (e) NH2 CONH2 , t-BuOK, THF, for comp 397 and 395; (f) TBAF, THF.

Boojamara and his coworkers have discovered a diphosphate of a novel cyclopentyl based nucleoside phosphonate with potent inhibition of HIV reverse transcriptase (RT) (336, IC50 = 0.13 μM) [157]. In cell culture the parent phosphonate diacid 335 demonstrated antiviral activity EC50 = 16 μM, a prodrug of which is currently under clinical investigation and within fivefold of tenofovir (PMPA). A favorable resistance profile toward K65R was achieved but was offset by a decreased susceptibility by the prevalent M184V mutant. Further development of 335 toward optimal phosphonamidate prodrugs will likely allow efficient delivery the phosphonate to the lymphatic system and provide a novel nucleotide RT inhibitor for the treatment of HIV. 1.2.5. Entecarvir and Analogs 1.2.5.1. Entecavir Because of the slow kinetics of viral clearance and the spontaneous genetic variability of hepatitis B virus (HBV), antiviral therapy of chronic hepatitis B remains a clinical challenge. Despite the recent development of lamivudine, adefovir dipivoxil, and pegylated interferon alpha for the treatment of chronic HBV infection, there are still critical need for new antiviral compounds. Entecavir (416) (2 -deoxy carbocyclic guanosine analog with an exocyclic double bond on the 6 -position; Figure 1.14) has been approved in United States for the therapy of chronic hepatitis B [158]. Extensive studies have been performed to characterize its antiviral activity in enzymatic and tissue culture models, as well as in animal models of HBV infection. In clinical trails, entecavir administration was associated with a significantly more potent viral


51

Scheme 1.51 Synthesis of 3 ,3 -Difluoro-2 -hydroxymethylunsaturated carbocyclic nucleosides (330–332; R = Me) [156]. Reagents and conditions: (a) (i) (EtO)2 POCH2 CO2 Et, NaH, THF; (ii) AcOH/H2 O; (iii) Ag2 O, BnBr, CH2 Cl2 ; (b) (i) DIBAL-H, CH2 Cl2 ,−78◦ C; (ii) ClF2 COOH, CH3 Cl, 90◦ C; (c) (i) Zn, TMSCl, MeCN, 105◦ C; (ii) EtOH, H2 SO4 , 50◦ C; (d) HN(Me)OMe.HCl, n-BuLi, THF,−78◦ C; (e) (i) allylmagnesium chloride, THF,−78◦ C; (ii) Et3 N, THF; (iii) NaBH4 , CeCl3 .7H2 O, MeOH, 0◦ C; (f) Grubbs Catalyst II, toluene, 100◦ C; (g) (i) MeOCOCl, pyridine, CH2 Cl2 , 0◦ C; (ii) 3-benzoyaluracil or 3benzoyalthymine, Pd(PPh3 )4 , PPh3 , THF, 60◦ C; (h) (i) NH3 , MeOH; (ii) BCl3 , CH2 Cl2 , −40◦ C; (i) (i) NaIO4 , MeOH, H2 O; (ii) NaBH4 , 0◦ C. O N HO

5′

N

6′

NH N

NH2

1′

4′ 3′

2′

OH 416 Entecavir

Figure 1.14 Structure of entecavir (416).

52


suppression compared to lamivudine and a significant advantage in terms of biochemical and histological improvement in comparison to lamivudine. Entecavir was tolerated as well as lamivudine in the phase III trials. No case of resistance was detected after two years of therapy in nucleoside na¨ıve patients. Treatment of patients with lamivudine failure requires a higher dosage of entecavir (1.0 mg vs. 0.5 mg for drug na¨ıve patients) and induces a significant decline in viral loads. The availability of entecavir as a new treatment option is providing clincians more choice to keep both viral replication and liver disease under control. This provides new hope for improved treatment concepts for chronic HBV infection. Entecavir undergoes rapid intracellular phosphorylation to the active triphosphate form [159], which inhibits HBV replication by acting as a nonobligate chain terminator in priming, at the RNA-dependent DNA synthesis as well as DNA-dependent DNA synthesis stages [160]. In vitro studies demonstrated that entecavir was the most potent inhibitor of HBV replication in comparison to other anti-HBV agents (EC50 3.75 nM in HepG 2.2.15 cell assay) [161–164]. Resistance studies indicated that 3TC/FTC-double mutant (rtM204V/I and rtL180M) reduced the viral susceptibility to entecavir by 20 to 30-fold (Table 1.17), whereas adefovir-resistant mutant (rtN236T) retained full susceptibility to this compound [165,166]. In the clinical trial, a small proportion (6%) of patients developed entecavirassociated mutation after a long-term administration. However, most of them did not experience confirmed virological rebounds [167,168]. The main resistance mutations of entecavir are rtT184G, rtS202I, and rtM250V on a background of lamivudine-resistant mutations [169,170]. Phase II trials showed that entecavir administration at dose of 0.5 or 1.0 mg/day for 4 weeks produced significant reduction of serum HBV DNA in both nucleoside-na¨ıve and lamivudine-experienced patients, and the viral load rebound was slower than lamivudine treatment after cessation of the therapy [171–174]. In three randomized, multicenter phase III trials, nucleoside-na¨ıve or -experienced patients (HBeAg positive or negative) were included. After 2 years of administration, 81% of entecavir recipients (0.5 mg/day) had a viral load below 300 copies/mL versus only 39% in the lamivudine recipients

TABLE 1.17 Antiviral activity of entecavir (416) and 3TC against HBV (wild type and mutations) [165] EC50 (μM) HBV Wild type Mutants

rtL180M rtM204I rtM204V rtL180M/rtM204I rtL180M/rtM204V

Entecavir (416)

3TC

0.0004 0.0005 0.06 0.003 0.25 0.28

0.56 >10 >80 33 >10 >80


53

(100 mg/day), whereas ALT normalization ratio and clearance of HBsAg ratio were 79% versus 68% and 5% versus 3%, respectively. In addition, entecavir administration at 1.0 mg/day produced significant viral load reduction in lamivudine refractory patients in comparison to the control group [172]. Based on these impressive results from the clinical trials, the U.S. FDA has approved 0.5 and 1.0 mg doses of entecavir as an oral, once-daily drug (Baraclude®) for the treatment of chronic hepatitis B infection. Recently Huang and his coworkers studied genotypic evoluation of HBV quasi species in nucleoside-/nucleotide-na¨ıve patient who developed resistance to entecavir [175]. The lamivudine resistant quasi species (rtM204V ± rtL180M), absent at baseline, were emerged as early as 48 weeks after entecavir administration. Entecavir-resistant quasi species (rtM204V ± rtL180M plus S202G) were found after week 112 and gradually became the predominant mutations afterward. The lamivudine- and entecavir-resistant mutations emerged closely in combination with the rtV207L, rtA222T, rtP237T, or rtI163V substitutions. Their results indicated that the lamivudine-resistant mutations were developed first and may serve as a prequisite for subsequent entecavir-resistant mutations in this nucleoside/nucleotide-na¨ıve patient. The first synthesis of entecavir was accomplished by Bisacchi et al . [161], in which chiral epoxide 418 was prepared from sodium cyclopentadienide 417 by an asymmetric hydroboration/epoxidation/protection sequence (Scheme 1.52). Treatment of epoxide 418 with a purine salt provided nucleoside 419 in 60% yield with desired regioselectivity. After the protection of the amino group of 419, Dess-Martin oxidation and Nysted methylenation afforded the exocyclic double-bond compound 422, which was converted to entecavir (416) after base derivation and deblocking steps. Ziegler and Sarpong studied the radical cyclization of the important intermediate 428 toward the synthesis of entecavir (Scheme 1.53) [176]. Compound

Scheme 1.52 Synthesis of entecavir (416) [161]. Reagents and conditions: (a) (i) BnOCH2 Cl; (ii) diisopinylcampheylborane; (iii) NaOH, H2 O2 ; (iv) VO(acac)2 , t-BuOOH; (v) BnBr, NaH; (b) NaH, Base; (c) MMTrCl; (d) Dess-Martin reagent; (e) Nysted reagent, TiCl4 ; (f) base derivation and deprotection.

54


Scheme 1.53 Synthesis of carbocyclic core 428 as an intermediate for preparing entecavir [176]. Reagents and conditions: (a) (MeO)2 POCN2 COMe, K2 CO3 ; (b) (i) TBSOTf, 2,6lutidine; (ii) mCPBA; (c) Cp2 TiCl; (d) PivCl, DMAP; (e) HOAc.

423 was prepared from d-diacetone by a known procedure [177]. Ohira’s protocol [178] was applied to convert unsaturated 423 into terminal acetylene 424, which was protected with a TBS group and treated with mCPBA to provide epoxide 425. Compound 425 underwent intramolecular radical cyclization in the presence of Cp2 TiCl. Desired carbocyclic intermediate 428 was obtained after standard protection group manipulations. An alternative route to prepare entecavir was developed by Chu and coworkers starting from enone 14, which was transformed to 342 using 1,4-addition method (Scheme 1.54) [50]. The exocyclic double bond was constructed under standard Mannich reaction/Hoffman elimination protocol, which led to α, βunsaturated ketone 429. Compound 429 was subjected to reduction, protection, and deprotection steps afforded triol 430. The protection of the 3 - and 5 -hydroxyl

Scheme 1.54 New schemes toward the synthesis of entecavir by Chu and coworkers. Reagents and conditions: (a) tert-butyl methyl ether, sec-BuLi, t BuOK, CuBr · Me2 S; (b) (i) LDA, Eschenmoser’s salt; (ii) MeI, NaHCO3 ; (c) (i) NaBH4 , CeCl3 · 7H2 O; (ii) BnBr, NaH; (iii) HCl; (d) TIPDSCl2 /Imidazole; (e) (i). NaH, CS2 , MeI; (ii) Bu3 SnH, AIBN; (iii) Na/Liq NH3 ; (f) DIAD, TPP, base; (g) base derivation and deprotection.


55

groups was followed by Barton-McCombie deoxygenation and Birch reduction to yield key intermediate 432. Standard Mitsunobu coupling, base derivation, and protecting-group manipulations furnished entecavir (416). Despite the significant successes in the area of anti-HBV agents, resistance and cross-resistance against available therapeutics are the major hurdles in drug discovery. Chu and his group studied the molecular basis of drug resistance conferred by the B and C domain mutations of HBV-polymerase on the binding affinity of entecavir [179]. In this regard, homology modeled structure of HBV-polymerase was used for minimization, conformational search and induced fit docking followed by binding energy calculation on wild type, as well as on mutant, HBV-polymerases (L180M, M204V, M204I, L180M+M204V, L180MM204I) shown in Table 1.18. Their studies suggest a significant correlation between the fold resistances and the binding affinity of entecavir [179]. The binding mode studies reveal that the domain C residue M204 is closely associated with sugar/pseudosugar ring positioning in the active site, and further mutation of M204 to V204 or I204 reduces the final binding affinity, which leads to the drug resistance. The domain B residue L180 is not directly close ˚ to the entecavir, but indirectly associated with other active-site hydropho(∼6A) bic residues such as A87, F88, P177, and M204. These five hydrophobic residues can directly affect the incoming nucleoside analogs in terms of its association and interaction that can alter the final binding affinity. There was no carbocyclic ring shifting observed in the case of entecavir. The exocyclic double bond of entecavir occupied in the backside hydrophobic pocket (made by residues A87, F88, P177, L180, and M204), which enhances the overall binding affinity, as shown in Table 1.18. Further molecular modeling studies have been carried out with 3TC-resistant HBV to investigate the active site interactions to understand the resistant profile of ETV. The 3TC-associated L180 mutant disfavors the shifting of oxathiolane ring, whereas there is no significant movement of the carbocyclic ring of ETV in comparison to dGTP. Thus, it is obvious that the L180M mutation will not affect its binding to the active site. In addition, the mutant M204V does not have much of an effect on the binding mode of ETV as well. The mutation of M204 to V204 can result in only partial filling of the small hydrophobic pocket, which may be the reason for the reduction of potency of ETV in M204V HBV RT. There is a possible partial steric clash between the exocyclic alkene and the I204 residue, which supports the reduction of potency of ETV in comparison to M204V HBV. Despite of the partial steric clash, there is no forward movement observed for the exocyclic sugar ring of ETV as it was clearly observed in the case of 3TC. 1.2.5.2. Entecavir analogs During the course of the development of entecaivr, its regio-isomers, 434 and 435, were synthesized and screened against HBV. Unfortunately, both compounds proved to be inactive against HBV [180] (Figure 1.15). The antiviral activity of (±)-436, a carbocyclic adenine analog with exocyclic double bond, was first described in 1988 [82]. It was found that this compound

56


TABLE 1.18 Multiligand bimolecular association with energetic (eMBrAcE) calculation of entecavir triphosphate (ETV-TP) in comparison to dGTP after induced fit docking and minimization in HBV-polymerase [179] Energy Difference results (E: kJ/mol) HBV-polymerase

Electrostatic

Vdwa

Total

E b

Wild type (WT) dGTP ETV-TP

–5161.7 –5465.8

139.5 196.6

–366.8 –404.7

–37.9

L180M dGTP ETV-TP

–5010.2 –5009.4

173.7 163.1

–422.7 –420.1

2.6

M204V dGTP ETV-TP

–4977.5 –4972.7

179.7 163.3

–417.4 –427.7

–10.3

M204I dGTP ETV-TP

–5233.9 –5277.4

169.0 180.3

–429.9 –438.5

–8.6

L180M-M204V dGTP ETV-TP

–5293.3 –5321.3

179.5 170.7

–449.1 –452.1

–3.0

L180M-M204I dGTP ETV-TP

–5267.1 –5297.6

172.2 185.0

–438.5 –437.5

1.0

a b

van der Waals interaction. E = energy difference (-ive: favorable) in comparison to the dGTP.

Figure 1.15 Entecavir analogs.

was active against vaccinia virus. Recently, Chu and coworkers have accomplished the asymmetrical synthesis of the whole series of d-form compounds for a complete SAR study. However, no interesting biological activity of target nucleosides were observed except marked cytotoxicity effect of (-)-436. The synthesis of 436 in Chu’s protocol utilized the allylic alcohol 438, which was obtained by reducing α, β-unsaturated ketone 429 depicted in Scheme 1.55. The Mitsunobu coupling of alcohol 438 with proper base equivalents, followed by base derivations and deprotection steps, yielded the desired nucleosides.


57

Scheme 1.55 Synthesis of (-) 436. Reagents and conditions: (a) NaBH4 , CeCl3 · 7H2 O; (b) DIAD, Ph3 P, Base; (c) base derivation and deprotection.

During the course of our drug discovery programs, introduction of fluorine atom onto the sugar moiety generated a number of novel nucleosides with interesting biological interesting nucleosides. Therefore, it is of great interest to explore the substitution of fluorine atom on the carbocyclic nucleosides with a 6 -exocyclic alkene (6 -methylene) (437) by Chu and coworkers, as shown in Scheme 1.56 [181]. Fluorinated nucleoside 437 was synthesized according to the newly developed procedure. Interestingly, adenine derivative 437 was not only active against HBV-WT but also retained full potency against lamivudine- and adefovir-resistant mutants as shown in Table 1.19. Such potent anti-HBV activity, as well as excellent resistance profile, makes this type of nucleosides attractive candidates as potential anti-HBV agents. Further structure-activity studies of this class of 2 -F carbocyclic nucleosides are in progress. 1.2.6. Carbocyclic Arabino- and Xylo-Nucleosides

Cyclaradine 444, a carbocyclic analog of ara-A, was discovered by Vince and coworkers [182]. It was resistant against adenosine deaminase and exhibited antiviral activity against HSV-1 (EC50 2.8–9.0 μM) and vaccinia virus (EC50

Scheme 1.56 Synthesis of 2 -F-6 -methylene carbocyclic nucleoside (437) [181]. Reagents and conditions: (a) DAST, CH2 Cl2 , rt; (b) (i) TBAF/HOAc, THF, rt; (ii) Bz Cl, Py, rt; (c) BCl3 , CH2 Cl2 ,−78◦ C; (d) DIAD, Ph3 P, 6-chloropurine, THF, rt.

58


TABLE 1.19 In vitro anti-HBV activity of 437 against lamivudine and adefovir drug-resistant mutants on the intracellular HBV DNA replication assay [181] Anti-HBV activity 437 (μM) Strain WT rtM204V rtM204I rtL180M rtLM/rtMVa rtN236T a

Lamivudine (μM)

Adefovir (μM)

EC50

EC90

EC50

EC90

EC50

EC90

1.5 1.8 1.0 2.1 2.2 1.7

4.5 4.7 5.0 5.1 5.5 4.6

0.2 >100 >100 1.5 >100 0.2

0.6 >100 >100 22 >100 0.9

1.3 1.6 1.9 5.5 2.1 7.8

7.1 7.0 8.0 7.7 8.5 36

rtLM/rtMV = rtL180M/rtM204V double mutant.

9.0 μM) [182]. Carbocyclic xylo-nucleosides 445 was reported to exhibit potent antitumor activity with EC50 0.38 μM. Its guanine analog 446 was active against HSV-1 (EC50 1.8–3.0 μM) (Figure 1.16). The synthesis of both classes of compounds started from the same epoxide 447, which was hydrolyzed to yield two products: arabino-type 448 and xylo-type 450 (Scheme 1.57). Two intermediates were deprotected and further converted to the desired arabino- and xylo-nucleosides by known chemistry.

Figure 1.16 Biological active carbocyclic arabino- and xylo-nucleosides.

Scheme 1.57 Synthesis of carbocyclic arabino- and xylo-nucleosides [182]. Reagents and conditions: (a) (i) H2 SO4 , (ii) Ac2 O; (b) (i) HCl, (ii) OH− ; (c) bases construction.


59

1.2.6.1. Carbocyclic 2 -Deoxy-Nucleosides and Related Nucleosides (E)-5-(2-Bromovinyl)-2 1.2.6.1.1. Carbocyclic 2 -deoxy nucleosides deoxyuridine (452, BVDU) is highly active against anti–herpes zoster [183]. However, the fast degradation to (E)-5-(2-Bromovinyl)-2 -deoxyuracil (BVU) catalyzed by pyrimidine nucleoside phosphorylases limits the therapeutic usage of BVDU [184]. The carbocyclic counterpart, C -BVDU (453), however, is no longer a substrate for phosphorylases while it maintains the antiviral potency. Its analog, (E)-5-(2-iodovinyl)-2 -deoxyuridine (454, C -IVDU), exhibits similar selectivity as well as antiviral activity [185] (Figure 1.17). The synthesis of C -BVDU and C -IVDU started from aminotriol 5, which was converted to anhydrouridine 475 in three steps (Scheme 1.58). Treatment of 475 with acetyl bromide followed by dehalogenation, and deprotection provided carbocyclic 2 -deoxyuridine 476. Introduction of 5-vinylbromide was accomplished via an iodination/coupling reaction/hydrolyzation/bromination sequence. C -IVDU was prepared in a similar manner [185].

Figure 1.17 Biologically active carbocyclic 2 -deoxy nucleosides.

60


Scheme 1.58 Synthesis of C -BVDU and C -IVDU [185]. Reagents and conditions: (a) (i) silver cyanate, β-methoxyacryloyl chloride; (ii) NH4 OH; (iii) diphenyl carbonate; (b) (i) acetyl bromide; (ii) Bu3 SnH, AIBN; (c) (i) I2 , nitric acid; (ii) methyl acrylae, Pd(OAc)2 , Ph3 P, Et3 N; (iii) KOH; (iv) NBS for 453 or I2 , iodic acid, K2 CO3 for 454.

Another approach to the synthesis of C -BVDU was described by Wyatt et al . as outlined in Scheme 1.59, which is comparably concise with the original one [186]. Another interesting compound in this series is carbocyclic 2 -deoxyguanosine (455, C -dG), which demonstrated potent antiviral activity against herpes simplex virus (HSV-1 and 2) [187], human cytomegalovirus (HCMV), and HBV [188]. C dG apparently is activated by virus-encoded kinase to exhibit anti-herpes activity, although it is a poor substrate for cellular phosphorylating enzymes [189]. Racemic C -dG was synthesized by a linear method (Scheme 1.60) [187]. It is noteworthy that the enantiomeric synthesis of C -dG was accomplished by Liang and Moser via an enzymatic approach [190]. Protection and hydroformylation of a racemic vinyl diol 482 led to an aldehyde 483, which was reduced to an alcohol 484. A standard protecting-group manipulation provided compound 485. Enzymatic resolution of 485 with Pseudomonas fluorescens lipase (PFL) in the presence of vinyl acetate followed by the deprotection of the acetate group furnished optically pure compound 487. Cyclic sulfate 488 was then prepared and condensed with base moieties to provide the target carbocyclic nucleosides. Borthwick et al . described another enzymatic resolution method soon after Liang and Moser’s report [191]. Chu and coworkers developed a solid phase synthesis of L-C -dG as well as its analogs (Scheme 1.61) [192]. Standard protectinggroup manipulation and radical deoxygenation led to compound 494, which was

Scheme 1.59 Alternative route of the synthesis of C -BVDU [186]. Reagents and conditions: (a) (i) Et3 N, (ii) HCl; (b) Ac2 O, DMAP; (c) NBS or Br2 ; (d) NaOH.


61

Scheme 1.60 Synthesis of carbocyclic 2 -deoxynucleoside [187]. Reagents and conditions: (a) (i) (t-Bu)2 SiOTf2 , 2,3-lutidine; (ii) [RhCl(PPh3 )3 ], H2 /CO, 80 bar; (b) NaBH4 ; (c) (i) TrCl, DMAP, Et3 N; (ii) TBAF; (d) PFL, vinyl acetate; (e) ethylenediamine; (f) (i) SOCl2 , Et3 N; (ii) RuCl3 , NaIO4 ; (g) (i) NaH, base; (ii) deprotection.

Scheme 1.61 Solid phase synthesis of l-carbocyclic 2 -deoxynucleosides [192]. Reagents and conditions: (a) TIPDSCl2 , Py; (b) (i) NaH, CS2 , MeI; (ii) Bu3 SnH, AIBN; (c) (i) Pd(OH)2 , H2 ; (ii) DHP, PPTS; (d) (i) TBAF; (ii) TBDMSCl, Im; (e) (i) BzCl, Py; (ii) TBAF; (f) 498, DMAP, DIPEA; (g) PPTS, 1-butanol/1,2-dichloroethane; (h) DIAD, Ph3 P, Bases; (i) K2 CO3 .

coupled with p-nitrophenyl carbonate resin 498 to yield compound 495. Fully protected 495 was subjected to the acidic hydrolysis to remove the THP group to provide alcohol 496 ready for Mitsunobu coupling reaction. In the coupling reaction, it was found that both regioselectivity and yield were generally improved under the solid phase condition in comparison with solution phase synthesis [192].

62


The most interesting analogs of carbocyclic 2 -deoxyribonucleosides are a series of 2 -fluoro substituted compounds. Among these analogs, the adenine derivative 461 was approximately 10-fold more active than cyclaridine (444) against herpes viruses (HSV-1 and 2) [193]. Guanine derivative 460 initially displayed significant antiviral activity against HSV-1 and 2 with EC50 s of 0.006 and 0.05 μg/mL, respectively, and did not show any toxicity up to 300 μg/mL [194]. Unfortunately, compound 460 was found to be toxic in later studies. Soon after this discovery, the same group in Glaxo described the synthesis and antiviral activity of fluorinated pyrimidine analogs [195]. However, only C -FMAU (457) and C -FIAU (458) exhibited moderate anti-HSV-1 activities, which were both significantly lower than that of the parent compound FMAU (456). Also, 6 -Fluoro substituted guanosine analogs were prepared. The β-F analog 463 (EC50 0.16 and 0.77 μg/mL for HSV-1 and 2, respectively) was approximately 50- to 100-fold more potent than α-F isomer 462. The synthesis of these fluorinated carbocyclic nucleosides all followed a linear methodology via 499, 500, or 501 as key intermediates that can be prepared from compound 5 by standard methods (Figure 1.18) [196]. The replacement of the 2 -hydrogen of the natural ribonucleosides with a methyl group yielded compounds with excellent chain-terminating properties. Among them, 2 -C -methyladenosine and 2 -C -methylcytidine demonstrated potent anti-HCV agents in clinical trials [79,80]. More recently, 2 -C -fluoro-2 C -methylcytidine was discovered as a hepatitis C virus RNA-dependant RNA polymerase (HCV RdRp) inhibitor and showed better inhibitory activity in the HCV replicon assay than 2 -C -methylcytidine, with low cellular toxicity. On the basis of potent anti-HCV activity of 2 -modified nucleosides, Hong and coworkers designed and synthesized 2 (α)-C-fluoro-2 (β)-C -methyl carbodine derivatives from 2-methyl cyclopentenone 502 [197]. Geminal substitution at the 2 -position might impose favorable steric as well as electronic effect on the interaction with HCV polymerase. The key fluorinated intermediate 504 was prepared from the epoxide intermediate 503 via selective ring-opening fluorination of epoxide using hydrofluoric acid. Coupling of 504 with nucleosidic bases under the Mitsunobu reactions followed by deprotection afforded the target carbocyclic nucleoside analogues 464 and 465 (Scheme 1.62). These compounds were evaluated as inhibitors of the hepatitis C virus (HCV) in Huh-7 cell line in vitro. The cytosine analogue 464 weakly inhibited the replication of the replicon, NK-R2AN, in huh-7 cells by 50% at 18.2 μM [197].

499

500

501

Figure 1.18 Key intermediates for the preparation of fluorinated nucleosides.


63

Scheme 1.62 Preparation of 2 (α)-C -fluoro-2 (β)-C -methyl carbodine derivatives (464–465) [197]. Reagents: (a) (i) BnBr, NaH, DMF; (ii) 47% HF, (NH4 )2 SiF6 , CsF; (b) N6 -bis-Boc-adenine, PPh3 , DIAD, 0◦ C; (c) (i) TFA, DCE/MeOH, rt; (ii) Pd(OH)2 , cyclohexene, MeOH, reflux; (d) N4 -Bz-cytosine, PPh3 , DIAD; (e) (i) NaOMe/MeOH; (ii) Pd(OH)2 , cyclohexene, MeOH, reflux.

Hughes and coworkers [198] analyzed novel nucleoside reverse transcriptase (RT) inhibitors (NRTIs) for their ability to inhibit DNA synthesis of excisionproficient HIV-1 RT mutants. A major pathway for HIV-1 resistance to NRTIs involves reverse transcriptase mutations that enhance ATP-dependent pyrophosphorolysis, which excises NRTIs from the end of viral DNA. d-Carba-thymidine (466) [198] is a carbocyclic nucleoside that has a 3 -hydroxyl on the carbocyclic ring. The 3 -hydroxyl group allows RT to incorporate additional dNTPs, which should protect d-carba TMP from excision. d-Carba thymidine (466) can be converted to the triphosphate form by host cell kinases with moderate efficiency. d-Carba thymidine-TP is efficiently incorporated by HIV-1 RT; however, the next dNTP is added slowly to a d-carba TMP at the primer terminus. d-carba thymidine (466) effectively inhibits viral vectors that replicate using NRTI-resistant HIV-1 RTs, and there is no obvious toxicity in cultured cells as shown in Table 1.20. Meier and his coworkers synthesized several carbocyclic l-nucleosides (e.g., l-carbadU 467, l-carba-dT 468, l-carba-FdU 469, l-carba-BVDU 470, and

TABLE 1.20 Antiviral efficacy of AZT and d-carba T vs wild-type and select NRTI resistant HIV-1 viral vectors [198] d-carba T, 466 (μM) HOS Wild type M184V AZT-R SSGR + T215Y

3.2 1.8 1.3 4.6

± ± ± ±

1.7 1.0 0.4 1.2

AZT (nM)

HOS 313

HOS

± ± ± ±

1.4 ± 0.1 3.6 ± 0.3 20.1 ± 1.8 39.6 ± 7.7

0.83 0.19 0.87 1.0

0.21 0.03 0.13 0.1

64


l-carba-dC 471) [199] by coupling a cyclopentane-system with heterocycles according to a modified Mitsunobu-protocol (Scheme 1.63). This reaction gave two regioisomers, the N1 -alkylated product and an unwanted O2 product. A simple SN 2-reaction has been investigated as an alternative for such couplings. The key step is the stereoselective synthesis of the cyclopentane system 509. Starting from cyclopentadiene an alkylation with BOMCl followed by a stereoselective hydroboration introduces the 3 -hydroxy group of the needed nucleoside sugar moiety [199]. This step allows the subsequent synthesis of lor d-nucleosides using (+)-diisopinocampheylborane or (-)-(ipc)2 -borane. Carbocyclic nucleoside analogues are catabolically stable because these are resistant to the phosphorolytic cleavage by pyrimidine nucleoside phosphorylase. The carbocyclic analogue (C-BCNA, 473) [200] of a highly potent and selective anti-VZV bicyclic nucleoside analogue (BCNA, 472) 6-pentylphenylfuro[2,3d]pyrimidine-2 -deoxyribose was synthesized using carbocyclic 2 -deoxyuridine as starting material by McGuigan and his coworkers [200]. C-BCNA (473) was found to be chemically more stable than the furano lead (472); however, it was shown to be significantly less antivirally active than its parent nucleoside analogue, as shown in Table 1.21. It was noted to have a 10-fold lower inhibitory activity against the VZV-encoded thymidine kinase. This reduction of activity may be attributed to the different conformation of the sugar and base, as predicted by computational studies and supported by NMR studies. However, other factors besides affinity for VZV-TK may account for the greatly reduced antiviral potency. Imidazolone (dIz) nucleoside is an abundant, highly mutagenic, and rather unstable DNA lesion that can cause dG →dC transversion mutations. dIz is generated in DNA by a variety of oxidative processes such as type I photooxidation. Carell and his coworkers synthesized the carbocyclic imidazolone nucleoside (cdIz, 474) [201], which is a stabilized version of the oxidatively generated

Scheme 1.63 Synthesis of 2 -deoxy-l-carbonucleosides (453–457) [199]. Reagents and conditions: (a) (1) (+)- (ipc)2 BH or (2) (-)- (ipc)2 BH, −60◦ C to rt, 15 h 45%; (b) (i) NaH, BnBr, TBAI, THF, rt, 12 h, 91%; (ii) 9-BBN, THF, rt, 12 h, 3N NaOH, 30% H2 O2 , 0◦ C, 79%; (iii) PPh3 , DIAD, benzoic acid 0◦ C to rt, 96%; (c) (i) PPh3 , DIAD, N3 benzoylphyrimidines, CH3 CN, −40◦ C to rt, 15 h, 1% NaOH in CH3 OH, rt, 4 h; (ii) Pd/C, H2 , EtOH.


65

TABLE 1.21 Antiviral and cytostatic activity of 452, 472–473 compounds [200] EC50 a (μM) VZV MCC50 b (μM) CC50 c (μM) IC50 d (μM) TK+ OKA TK+ YS TK− 07 TK− YS HEL HEL VZV-TK 472 473 452

0.0003 0.49 0.005

0.0001 0.28 0.005

>5 >50 >200

>5 — >200

>20 >50 >200

>50 >50 >200

4.9 44 1.3

a The 50% effective concentration, or compound concentration, required to inhibit VZV-induced cytopathicity by 50%. b Minimal inhibitory concentration, or compound concentration, required to cause a microscopically visible alteration of cell morphology. c The 50% cytostatic concentration, or compound concentration, required to inhibit HEL cell proliferation by 50%. d The 50% inhibitory concentration, or compound concentration, required to inhibit VZV-TKcatalyzed dThd (1 μM) phosphorylation by 50%.

DNA lesion imidazolone (dIz). The carbocyclic modification protects this lesion analogue from anomerization. Replacement of the 2 -deoxyribose moiety by the cyclopentane unit stabilizes this lesion analogue against cleavage by base excision repair (BER) enzymes because of the lack of a glycosidic bond, but this modification does not prevent degradation of the heterocycle, for example, to give the oxazolone (dZ) hydrolysis product. 1.2.7. Conformationally locked carbocyclic nucleosides

Neplanocin C (511), a naturally occurring carbocyclic nucleoside isolated from Ampullariela regularis in the early 1980s, provided a prototype of conformationally locked nucleoside with a cyclopropane ring fused on the 4 , 5 position of a cyclopentane ring [202] (Figure 1.19). The [3,1,0]-bicyclic system adopted a predominant Northern conformation as indicated in the X-ray analysis [203]. The synthesis of the neplanocin C started from a known cyclopentenol 24 (Scheme 1.64) [117]: The cyclopentenol 24 was condensed with 6-chloropurine and deblocked the acetonide group to provide nucleoside 542, which was further subjected to the epoxidation, amination followed by debenzylation to furnish the target neplanocin C. Inspired by the novel structure of neplanocin C, a number of carbocyclic analogs have been prepared. Among these compounds, the most systematically and extensively studied are those 1 ,5 -methano (522–526) as well as the 4 ,5 methano carbocyclic nucleosides (517–521) as shown in Figure 1.19. d-form adenine derivative 512 was found to adopt a typical Northern conformation and exhibited moderate anti-HIV activity while its enantiomer was devoid of the antiviral activity. The synthesis of the target nucleosides was accomplished via a cyclopropane fused cyclopentanal 545, which was prepared by treating compound 544 with chloroiodomethane in the presence of samarium (+2) at −78◦ C (Scheme 1.65). Condensation of various base moieties with 545 followed by base derivation provided the desired nucleosides 512–516 [204,205].

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Figure 1.19 Conformationally locked carbocyclic nucleosides.

Scheme 1.64 Synthesis of neplanocin C [117]. Reagents and conditions: (a) (i) DIAD, Ph3 P, 6-chloropurine; (ii) AcOH; (b) m-CPBA; (c) (i) NH3 /MeOH; (ii) H2 , Pd/C.

Soon after the report of conformationally locked carbocyclic dideoxynucleosides as described earlier, Altmann et al . also accomplished the synthesis of both 4 ,5 - and 1 ,5 -methano-2 -deoxy carbocyclic thymidine (519 and 524, Scheme 1.66a; and 1.66b) [206]. The synthesis of 519 was accomplished starting from allylic alcohol 24, which was subjected to the Simmons-Smith cyclopropanation to give ring fused compound 547 with the desired stereochemistry due to the


67

Scheme 1.65 Synthesis of compounds 512–516 [204,205]. Reagents and conditions: (a) CH2 ICl, Sm, HgCl2 ; (b) DIAD, Ph3 P, Bases; (c) Base derivation and deprotection.

Scheme 1.66 Synthesis of compounds 519 and 524 [206]. Reagents and conditions: (a) Zn/Cu, CH2 I2 ; (b) (i) TsCl, Et3 N, DMAP; (ii) NaN3 ; (iii) H2 , Lindar’s catalyst; (c) base construction; (d) (i) HCl; (ii) H2 , Pd/C; (e) (i) TIPDSCl2 , Im; (ii) BOM-Cl, DBU; (f) (i) CH3 C6 H4 OC(S)Cl, DMAP, Et3 N; (ii) Bu3 SnH, AIBN; (g) (i) TBAF; (ii) H2 , Pd/C; (iii) NaOMe; (h) (i) TMSBr, ZnBr2 ; (ii) N -methyl acetamide; (i) t BuOK, t-BuOH; (j) (i) KOH; (ii) DPPA, Et3 N; (iii) H2 , Pd/C; (k) base construction and deprotection.

directing effect of the allylic hydroxyl group [207]. After the alcohol 547 was converted into the amine 548, the heterocyclic moiety was constructed under standard conditions to give nucleoside 550, which was protected and subjected to the Barton–McCombie deoxygenation to afford target compound 519 (Scheme 1.66a). On the other hand, the synthesis of 1 ,4 -methano-2 -deoxy carbocyclic thymidine utilized the bicyclic lactone 553 as a key intermediate, which was treated with TMSBr followed by N -TBDMS to provide compound 554 (Scheme 1.66b). Formation of the three-membered ring went smoothly under basic condition. Subsequent deprotection, Curtius rearrangement, deprotection and base

68


construction furnished target nucleoside 524. The X-ray analysis indicated that 4 ,5 -methano-2 -deoxy carbocyclic thymidine 519 preferred boatlike Northern conformation, whereas the isomer 524 existed predominately as boatlike Southern conformation. In the series of Northern 2 -deoxy nucleosides, the adenine analog 516 was prepared by Siddiqui and Marquez et al . (Scheme 1.65) and showed good antiviral activity against HCMV and EBV [208]. To explore the full potential of this class of molecules, a series of nucleoside bases (adenine, uracil, cytosine, and guanine) built on the Northern bicycle[3,1,0]hexane pseudo-sugar ring were synthesized using a convergent approach [209]. Nucleoside 519 displayed excellent antiherpes activity with EC50 of 0.03 and 0.09 μg/mL against HSV-1 and HSV2, respectively. It was nontoxic to host cells at concentration up to 100 μg/mL. Interestingly, the isomer of compound 524, Southern bicycle[3,1,0]hexane thymidine, was devoid of antiviral activity. Conformational analysis revealed that not only was the ring pucker of these two conformationally rigid nucleosides quite different (519: Northern, 524: Southern), but also the base rotation angle (χ) of compound 524 was more stiff in comparison to 519. All these disparities together might explain, to some extent, the difference in the antiviral activity of these two compounds. Based on these findings, Marquez and coworkers performed a systematic SAR study of a number of conformationally locked carbocyclic nucleosides (527–529) [210–214]. Their data showed that herpes thymidine kinase had a strong preference to the Southern conformation and antibase disposition in the monophosphorylation step but insensitive to the presence or absence of 3 OH. However, in the diphosphorylation step, the 3 -OH was extremely important to this enzyme, and Southern conformation was still preferred. Cellular DNA polymerase and HIV reverse transcriptase favored exclusively the triphosphate of the Northern conformers [210,212,214]. Marquez and his coworkers found that chemically synthesized 5 -triphosphates of bicyclo-[3.1.0] hexane models of 2 -deoxynucleosides that were restricted in the North conformation (517–520) [209] were readily incorporated by HIV-1 RT and other polymerases, none of the nucleosides were effective anti-HIV agents due to inefficient cellular phosphorylation. On the other hand, the conformational constraint in South-bicyclo [3.1.0] hexane nucleosides, which influences the nucleobase to adopt the biologically unsuitable syn conformation that is favored by intramolecular hydrogen bonding in a hydrophobic environment, such as the enzyme’s binding site, resulted in compounds 522–525 that were not phosphorylated by cellular kinases, despite their excellent substrate recognition by the more tolerant, viral HSV-1 kinase. The incorporation of the main structural feature of D4T, which is its planarity, into the structure of the very potent anti-HSV compound, Northmethanocarbathymidine (519) resulted in the very effective anti-HIV thymidine analogue (532) with a novel bicycle[3.1.0]hexene pseudosugar that included the critical double bond. The 4- to 10-fold decrease in potency of 532 relative to D4T was tentatively correlated with the nearly 10-fold reduction in the level of planarity (νmax = 6.818) of the embedded five-membered ring segment of the


69

bicyclo[3.1.0]hexene template relative to the nearly flat pseudosugar ring of D4T (νmax = 0.618) [215]. As each of the cellular kinases that perform the critical first phosphorylation step have different preferences for the various nucleobases, we decided to complete our study by incorporating the rest of the nucleobases (A, C, and G) to the same bicyclo[3.1.0]hexene template (530–533) (Scheme 1.67) derived from the conformationally locked North-bicyclo [3.1.0]hexane nucleosides 517–520 [215]. It is important to remember that in terms of sugar conformation, once the amplitude of the puckering measured by νmax reaches a small value, such as ∼6◦ in 530–533, the designation of North and South loses its significance. Based on that argument, they decided to investigate the synthesis and anti-HIV activities of the complementary set of bicyclo[3.1.0]hexane nucleosides (534–537) (Scheme 1.68) derived from the antipodal, conformationally locked, South-bicyclo[3.1.0]hexane nucleosides 522–525 bearing the four natural nucleobases. The transformation of a bicyclo[3.1.0]hexane nucleoside into a bicyclo [3.1.0]hexene nucleoside flattens the five-membered ring of the bicyclic system and rescues anti-HIV activity for North-D4T (532), North-D4A (530), and South-D4C (537). The relationship between planarity and the anti/syn disposition of the nucleobase that is favored by a particular pseudosugar platform are proposed as key parameters in controlling biological activity. 2 -C -methyladenosine and 2 -C -methylguanosine showed potent anti-HCV activity in a cell-based HCV replicon assay, in which 2 -methyl group prevents the incorporation of incoming nucleosides triphosphates [80]. These nucleosides were reported to adopt a Northern C3 -endo conformation (pseudorotation angle,

Scheme 1.67 Synthesis of north-bicyclo-[3.1.0]hexene nucleoside 530–533 [215]. Reagents and conditions: (a) DIAD, PPh3 , base THF, 0◦ C → rt; (b) Et3 N · 3HF, MeCN, .

Scheme 1.68 Synthesis of south-bicyclo-[3.1.0]hexene nucleoside 534–537 [215]. Reagents and conditions: (a) TBDPSCl, imidazole, DMF or TBDPSCl, DMAP, pyridine; (b) MsCl, pyridine; (c) (i) 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU), PhMe or DMF, Base; (ii) TBAF, THF.

70


P = 15.6◦ ). Carbocyclic nucleosides in which the cyclopropane ring is fused between C4 and C6 also fix the conformation of the carbasugars to a Northern C3 -endo conformation (P = 0±18◦ ). Thus, this conformational information prompted Moon and his coworkers to design and synthesize stereoselectively cyclopropyl-fused-carbanucleosides 538–541 (Scheme 1.69) [216]. The cyclopentenone 567 was stereoselectively reduced to α-allylic alcohol followed by modified Simmons–Smith cyclopropanation gave bicyclo[3.1.0]hexane derivative 568 as a single stereoisomer. Selective protection of the least hindered alcohol in diol with a TBDPS group followed by Swern oxidation of the remaining alcohol afforded the ketone 569. The cyclic sulfate was obtained by stereoselective Grignard reaction, removal of both silyl protecting groups followed by SOCl2 treatment in presence of triethyl amine, and oxidation of the resulting cyclic sulfite with sodium periodate in presence of RuCl3 -3H2 O. Compounds 538–541 were synthesized by utilizing regioselective cleavage of the isopropylidene group and cyclic sulfate (571) chemistry as key steps [216]. Antiviral assay of 538–541 against HCV was performed, but these compounds did not show any significant anti-HCV activity in a cell-based HCV replicon assay.

Scheme 1.69 Synthesis of 2-C -methylcarbanucleoside 538–541 [216]. Reagents and conditions: (a) (i) NaBH4 , CeCl3 .7H2 O, MeOH, 0◦ C, 30 min.; (ii) Et2 Zn, CH2 l2 , CH2 Cl2 , rt, 5 h; (b) (i) Me3 AI, CH2 Cl2 , rt, 4 d; (ii) TBDPSCl, imidazole, CH2 Cl2 , rt, 20 min.; (iii) (COCl)2 , DMSO, TEA, CH2 Cl2 , rt, 1 d; (c) (i) MeMgl, Et2 O, rt, 2.5 h; (ii) TBAF, THF, rt, 1 d; (iii) TBDMSCI, imidazole, CH2 Cl2 , 0◦ C, 2 h; (d) (i) SOCl2 , TEA, CH2 Cl2 , 0◦ C, 10 min.; (ii) RuCl3 -3H2 O, NalO4 , CCl4 : CH3 CN:H2 O = 1 : 1 : 1.5, rt, 10 min.; (e) (i) adenine or 2-amino-6-chloropurine, NaH, DMF; (ii) 20% aq H2 SO4 ; (f) 70% CF3 COOH, rt, 50 min.; (g) NaNO2 , AcOH, rt, 3 h; (h) 3N aq HCl, rt, 2 d; (i) cyclopropylamine, ethanol, rt, 1 d.


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1.2.7.1. Conformationally locked norborane and Isoxazoline-based carbocyclic nucleoside A modification, which increases resistance against enzymatic degradation, is substitution of the furanose ring of the sugar moiety by a carbocyclic ring. Many of such modified analogues carbocyclic nucleosides exhibit interesting antiviral activity. Recently, a series of carbocyclic analogues containing bicycloalkanes, bicycloheteroalkanes or tricycloheteroalkanes with activity against Coxsackie viruses was prepared by Sala and coworkers as shown in Figure 1.20 [217,218]. They were reported synthesis of novel racemic conformationally locked nucleosides with bicyclo[2.2.1]heptene (norbornene) or heptane (norbornane) ring system substituted with nucleobase at position 7 with syn-configuration as well as with anticonfiguration. Two methodologies were employed for introduction of the chloropurine moiety to the scaffold: (i) the Mitsunobu reaction of the chloropurine nucleobase with appropriate alcohols and (ii) a built-up strategy from appropriate amines. These compounds could be considered as the 6-chloropurines substituted at position 9 with different substituted bicyclic scaffolds (bicyclo[2.2.1]heptane/ene-norbornane or norbornene). These bicyclo systems (norbornene or norbornane), like the oxabicyclo[2.2.1]heptane, represent conformationally locked carbapentofuranose ring systems. All compounds were evaluated for antiviral activity against Coxsackie virus B3, as shown in Table 1.22. Most analogues showed activity in the low micromolar range with minimal cytotoxicity to the cell line used in this study. Studies on the mode of action of the most promising compounds are in progress.

Figure 1.20 Conformationally locked norborane and Isoxazoline-based carbocyclic nucleoside.

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TABLE 1.22 Antiviral evaluation against CVB3 of the 9-substitutedpurines in Vero cells, all data are mean values ± standard deviation for at least three independent experiments [217,218] Compounds

IC50 (μM)

EC90 (μM)

CC50 (μM)

574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591

1.13 ± 0.33 1.86 ± 1.13 1.00 ± 0.04 0.88 ± 0.11 1.00 ± 0.01 0.98 ± 0.17 0.91 ± 0.07 1.10 ± 0.03 0.81 ± 0.20 1.05 ± 0.01 0.95 ± 0.02 1.00 ± 0.02 1.16 ± 0.24 0.83 ± 0.05 46.9 0.90 ± 0.04 1.03 ± 0.20 0.66 ± 0.35

NDa

>50 >50 >50 >50 >50 >50 >50 47.85 ± 3.05 >50 >50 >50 >50 >50 >50 >326 >50 >50 >50

a

ND ND ND ND ND ND ND 1.82 ± 0.91 ND 1.96 ± 0.98 ND 34.40 ± 27.97 1.70 ± 0.85 ND 1.78 ± 0.89 3.46 ± 2.91 >50

ND: not determined.

Quadrelli and coworkers synthesized the isoxazoline-based carbocyclic nucleosides 592 and 593 by the linear construction of the desired purine and pyrimidine bases on the regioisomeric aminols 600 (Scheme 1.70) [219] obtained through elaboration of the hetero-Diels–Alder (HDA) cycloadducts 598 of cyclopentadiene with nitrosocarbonyl intermediates (RCONO). The complex course of 1,3-dipolar cycloaddition between benzonitrile oxide and the cyclohexadiene adduct 598 of (nitrosocarbonyl) benzene afford regioisomeric mixtures of exo and endo cycloadducts. The exo cycloadducts of 599 are suitable starting materials for isoxazoline-cyclohexane nucleoside synthesis. Detachment of the benzoyl group and reductive cleavage of the N–O bond provided the stereodefined aminols 586, which were used for the linear construction of purine nucleosides by well-established synthetic protocols. Substitution with 5-amino-4,6-dichloropyrimidine and subsequent condensation with orthoformates afford the chloropurines 592 and 593 [219]. These were further derivatized by replacement of the chlorine with amines and alkoxides to give suitable samples for antiviral tests in very good yields. The same group was synthesized isoxazoline-carbocyclic nucleosides having a hydroxymethylene side chain, and a variety of analogues was attained starting from the stereodefined heterocyclic aminol 605, which are readily available through exo selective 1,3-dipolar cycloadditions of benzonitrile oxide to 2-azanorborn-5-enes 601 and elaboration of the cycloadducts 602 (Scheme 1.70).

THREE-MEMBERED CARBOCYCLIC NUCLEOSIDES

73

Scheme 1.70 Preparation of aminols for the synthesis of isoxazoline-carbocyclic nucleosides (592–596) [219,220].

The stereodefined heterocyclic aminols 605 afford the carbocyclic skeleton for the linear construction of the purine rings. Functionalization of the chloropurines 594–596 [220] with a variety of amines extended the synthetic potential of this strategy, allowing for a fine-tuning of their biological and antiviral activity as well as comparison with the corresponding nor-nucleosides. Biological evaluation of the newly obtained compounds 594–596 is in progress. 1.3. THREE-MEMBERED CARBOCYCLIC NUCLEOSIDES

In general, three-membered carbocyclic nucleosides can be divided in two classes. The first ones have the base moiety directly attached to the ring, whereas the other ones have a spacer between the base and the ring (Figure 1.21). Chu and coworkers accomplished the first asymmetric synthesis of d- and l-cyclopropyl nucleosides, which belong to the first category (Scheme 1.71) [221]. Protected d-mannitol was converted to the vinyl alcohol 618 by standard oxidation/wittig reaction/reduction sequence. The requisite cyclopropyl ring was installed by Simmons-Smith cyclopropanation following oxidation, Curtius rearrangement and deprotection protocol to yield cyclopropyl amine 620. The target d-nucleosides 606 were obtained by a linear methodology. l-Cylcopropyl nucleosides 607 were synthesized in similar fashion using l-gulonic γ-lactone as chiral starting material. Unfortunately, no significant biological activity was exhibited by synthesized nucleosides. On the other hand, in the second category, several biologically interesting nucleosides were discovered. Ashton et al . reported that the conformationally constrained acyclovir analog 608 showed similar anti-HSV-1 and 2 activities to the parent nucleoside [222,223]. Tsuji and coworkers explored extensive SAR of carbocyclic nucleosides bearing a methylene spacer between the base and carbocyclic ring [223,224]. The guanine derivative 609 was active against HSV-1

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Figure 1.21 General structures of three-membered ring carbocyclic nucleosides and some representative molecules.

Scheme 1.71 Synthesis of cyclopropyl nucleosides 606 and 607 [221]. Reagents and conditions: (a) (i) Pb(OAc)4 ; (ii) Ph3 P = CHCOOMe; (iii) DIBAL-H; (b) ZnEt2 , CH2 I2 ; (c) (i) RuO2 /NaIO4 ; (ii) ClCO2 Et, Et3 N; (iii) NaN3 ; (iv) BnOH, heat; (v) H2 , Pd/C; (d) base construction, deprotection.

THREE-MEMBERED CARBOCYCLIC NUCLEOSIDES

75

and HSV-2 with EC50 s of 0.0093–0.035 and 0.12–0.24 μg/mL, respectively, in comparison to 0.27–1.0 and 0.25–1.3 μg/mL for acyclovir and 0.54–2.0 and 1.2–2.7 μg/mL for penciclovir. Furthermore, this nucleoside was 8- to 20-fold more potent than acyclovir and penciclovir against VZV, and the selectivity index of nucleoside 609 was also high. Studies demonstrated that 609 can be phosphorylated by HSV-1 thymidine kinase (TK) very efficiently. As an extension of the research, a series of 5-substituted uracil derivatives were prepared, and some of the target nucleosides exhibited potent anti-VZV activity (Scheme 1.72) [225]. Particularly, 5-bromovinyl nucleoside 610 was about 40-fold more potent than acyclovir and had good oral bioavailability in rats (68.5%). The enantiomeric syntheses of compounds 609 and 610 were accomplished using chiral cyclopropane lactone 621 as starting material. The key intermediate was condensed with base moiety via classic SN2 reaction followed by deprotection/derivation to afford target nucleosides [225]. Zemlicka and coworkers described another type of interesting carbocyclic nucleosides, in which the spacer between the base and the ring is an unsaturated double bond [226,227]. Compounds, such as 611 and 612 Figure 1.21), displayed broad-spectrum antiviral activity. The pair of enantiomers (611 and 612) was synthesized through enzymatic as well as chemical resolutions (Schemes 1.73a and 1.73b). It was interesting to find that nucleosides 611 and 612 exhibited equipotent anti-HCMV activity (EC50 2.9 and 2.4 μM, respectively). However, compound 611 was somehow more potent than 612 against HSV-1 and 2 with EC50 s of 8.8 vs. 38 μM and 35 vs.>50 μM, respectively. On the contrary, compound 611 was less effective (EBV) or devoid of activity (HIV-1) in comparison to 612. Further modifications of the spacer generated spiropentane nucleoside 613 (Scheme 1.73c). Although no antiviral activity was found at the nucleoside level, phosphoralaninate nucleotide of 613 showed significant antiviral activity against HCMV, HSV-1 and 2, VZV, and EBV HIV-1, as well as HBV, which indicated the inefficient phosphorylation of spiropentane of nucleosides in vitro [228]. The same group also synthesized chiral E and Z-stereoisomers of (1,2dihydroxyethyl) methylenecyclopropane analogues of 2 -deoxyadenosine and 2 -deoxyguanosine and evaluate for their antiviral activity (Scheme 1.74) [229]. (R)-Methylenecyclopropylcarbinol (636) was converted in few steps to reagents 642, which were used for alkylation-elimination of adenine and

Scheme 1.72 Synthesis of cyclopropyl nucleosides 609 and 610 [225]. Reagents and conditions: (a) (i) NaBH4 ; (ii) Ph2 CN2 , DDQ; (iii) LiBH4 ; (iv) CBr4 , Ph3 P, Et3 N; (b) BVU, K2 CO3 ; (c) HCl.

76


Scheme 1.73 Synthesis of cyclopropyl nucleosides 611–613 [226,227]. Reagents and conditions: (a) N2 CHCO2 Et, Rh(OAc)4 ; (b) K2 CO3 ; (c) K2 CO3 , heat; (d) adenosine deaminase, pH 7.5; (e) (i) Ac2 O, Py; (ii) [ Me2 N = CHCl]+ Cl− ; (f) NH3 /MeOH; (g) i -BuOCOCl, Et3 N; (ii) (R)-2-phenylglycinol, separation; (h) (i) H2 SO4 ; (ii) HCl, EtOH; (iii) Br2 ; (iv) DIBAL-H; (v) Ac2 O, Py; (i) (i) K2 CO3 , adenine; (ii) NH3 /MeOH; (j) (i) LAH; (ii) Ac2 O, Py; (k) N2 CHCO2 Et, Rh(OAc)4 ; (l) (i) NaOH, separation; (ii) Ac2 O, Py; (iii) (PhO)2 P(O)N3 , Et3 N, tBuOH; (iv) K2 CO3 , aq. MeOH; (v) separation; (vi) HCl, MeOH; (m) base construction and deprotection.

2-amino-6-chloropurine to get ultimately analogues from 614–617. The Z-isomer 615 was an inhibitor of plaque reduction assay against Towne and AD169 strain of human cytomegalovirus (HCMV) with EC50 of 6.8 and 7.5 μM [229]. It was also active in murine cytomegalovirus (MCMV) assay (EC50 of 11.5 μM). It was less active against HCMV with mutated gene UL97. It inhibited Epstein-Barr virus (EBV) with EC50 of 8 μM. It is not cytotoxic, and its efficacy is somewhat lower than that of ganciclovir. None of the E-isomers of the present analogues were effective against HCMV. 1.4. FOUR-MEMBERED CARBOCYCLIC NUCLEOSIDES

A natural nucleoside, oxetanocin A 644 (OXT-A, Figure 1.22) is a four-memberring nucleoside produced by Bacillus megaterium [230,231]. The broad-spectrum

FOUR-MEMBERED CARBOCYCLIC NUCLEOSIDES

77

Scheme 1.74 Synthesis of E and Z-(1,2-dihydroxyethyl)methylenecyclopropane Analogues of 2 -deoxyadenosine and 2 -deoxyguanosine by Zemlicka and coworkers [229]. Reagents and conditions: (a) (i) (COCl)2 , DMSO, CH2 Cl2 ,—60◦ C; (ii) NaCN, NH4 Cl, H2 O, CH2 Cl2 ; (b) Conc. HCl, CH2 Cl2 ; (c) BnBr, K2 CO3 , Bu4 NI, DMF; (d) DIBALH,THF, 0◦ C; (e) (i) Ac2 O, pyridine; (ii) pyridine.HBr3 , CH2 Cl2 ; (f) (i) adenine, K2 CO3 , DMF, 100–105◦ C; (ii) K2 CO3 , MeOH; (g) 2-amino-6-chloropurine, K2 CO3 , DMF, 100–105◦ C; (h) (i) NH3 , MeOH; (ii) 80% HCOOH, 80◦ C.

antiviral activity of the compound has prompted considerable attention to this class of nucleosides [232]. Preparation of carbocyclic analogs of the natural counterparts was first reported by Honjo [233]. A [2+2] formation provided cyclobutane intermediate 655, which underwent a series of manipulations to afford cyclobutylamines 656. The racemic C -OXT-A was constructed through a linear approach (Scheme 1.75a.). In the same year, the synthesis of optically pure C -OXT-G was accomplished by Narasaka and coworkers by using an asymmetric [2+2] addition as a key step (Scheme 1.75b) [234]. Among synthesized nucleosides, the guanine (646, C -OXT-G) and adenine (645, C -OXT-A) derivatives were active against HIV in ATH18 cells (EC 50 1–2 μM) [235]. In addition, the d-enantiomer of C -OXT-G (647, lobucavir, LBV) could be phosphorylated to its triphosphate by viral TK as well as protein kinase [236] and exhibited broad-spectrum antiviral activity against HBV and herpes viruses [237]. Lobucavir was advanced to clinical trials as an anti-HBV agent by the Bristol-Myers Squibb Phamaceuticals. However, the clinical studies were suspended due to oncogenicity in rodents. Further modifications based on the structure of C -OXTs generated series of interesting four-membered carbocyclic nucleosides. Monofluoro nucleoside (-)

78


Figure 1.22 Four-membered carbocyclic nucleosides.

648 showed significant antiviral activity against HSV-1 and 2 (EC50 0.7–1.8 μM), VZV (EC50 1.8–3.5 μM), and HCMV (EC50 3.5–35 μM); however, it was toxic to cells [238,239]. Removal of the 4 -methylene group of C -OXTs (649) resulted in a considerable decrease of anti-HSV and anti-VZV activity in comparison to the parent compounds [240]. Interestingly, the triphosphate of nucleoside 650, which did not have a 2 -hydroxylmethyl group, was reported to be active against wild-type HIV-RT as well as M184V mutant [241]. Novel spiro-carbocyclic nucleosides 651 and 652 have been prepared by Chu and coworkers via enzymatic resolution (Scheme 1.76). Both d- and l- nucleosides exhibited some anti-HIV activity with EC50 values of 22.4 and 48.6 μM, respectively, whereas l-enantiomer was less toxic than its d counterpart [242].

1.5. SIX-MEMBERED CARBOCYCLIC NUCLEOSIDES

Herdewijin and coworkers have prepared a number of cyclohexenyl and cyclohexanyl analogs, such as nucleosides 668–672 [243–246]. However, no biological

SIX-MEMBERED CARBOCYCLIC NUCLEOSIDES

79

(a)

(b)

Scheme 1.75 Synthesis of racemic and optically pure C -OXT-A and C -OXT-G [233,234]. Reagents and conditions: (a) CH3 CN, heat; (b) (i) LAH; (ii) BzCl, Py; (iii) p-TsOH, acetone; (iv) NH2 OH; (v) H2 , PtO2 ; (c) base construction and deprotection.

Scheme 1.76 Synthesis of optically pure spiro-carbocyclic nucleosides [242]. Reagents and conditions: (a) P. cepacia lipase, AcOCH=CH2 ; (b) (i) Amberlite IR-120; (ii) TrCl, Py; (iii) TBDPSCl, Im; (iv) BF3 • OEt2 ; (v) Me3 P(OPh3 )I; (vi) DBU; (vii) TBAF; (c) Et2 Zn, CH2 I2 ; (d) Mitsunobu coupling and base derivations and deptrotection; (e) Ac2 O, Py, then followed the procedure for compound 663.

activity was noticed with the exceptions of guanine derivatives of C3-hydroxyl cyclohexenyl 671 and672, which were shown to be potent and highly selective antiviral agents against herpes virus (HSV-1 and 2 and VZV) with EC50 vaules comparable to acyclovir and ganciclovir as shown in Table 1.23 [246]. The NMR conformational studies suggested that the nucleosides antiviral activity was correlated with their predominant conformation (Figure 1.23).

80


TABLE 1.23 Antiviral activity of d- and l-cyclohexenyl-G (671 and 672) in comparison with approved antiviral drugs [246] Virus HSV-1 (KOS)a HSV-1 (F)a HSV-1 (McIntyre)a HSV-1 (TK− KOS ACV)a HSV-1 (TK− /TK+ VMW1837)a HSV-2 (G)a HSV-2 (196)a HSV-2 (Lyons)a VZV (YS)c VZV (OKA)c VZV (TK− 07/1)c VZV (TK− YS/R)c CMV (AD 169)c CMV (Davis)c

671 (IC50 μg/mL)

672 (IC50 μg/mL)

Acyclovir (IC50 μg/mL)

Ganciclovir (IC50 μg/mL)

0.002b 0.002b 0.004b 0.38b

0.003b 0.003b 0.004b 1.28b

0.01b 0.003b 0.005b 9.6b

0.001b 0.001b 0.001b 0.48b

0.01b

0.01b

0.07b

0.01b

0.05b 0.07b 0.07b 0.49d 0.64d 2.1d 2.8d 0.6d 0.8d

0.07b 0.1b 0.07b 1.2d 1.9d 5.8d 6.8d 1.5d 1.7d

0.02b 0.02b 0.02b 1.1d 0.8d 13d 28d ND ND

0.002b 0.001b 0.001b NDe ND ND ND 0.6d 0.8d

a

Activity determined in E6SM cell cultures. Minimum inhibitory concentration (μg/mL) required to reduce virus-induced cytopathogenicity by 50%. Virus input was 100 50% cell culture infective doses (CCID50 ). c Activity determined in HEL cells. d Minimum inhibitory concentration (μg/mL) required to reduce virus plaque formation by 50%. Virus input was 20 plaque-forming units (PFU). e ND: not determined. The values are means of two independent determinations. b

Figure 1.23 Six-membered carbocyclic nucleosides.

SIX-MEMBERED CARBOCYCLIC NUCLEOSIDES

81

Recently, it has been described that both enantiomers of cyclohexenylguanine 671 and 672 display potent and selective anti–herpes virus activity (HSV-1, HSV2, VZV, CMV) [246]. It is well known that opposite enantiomers can display different pharmacological and toxicological properties. Hence, Alibs and coworkers report an enantiodivergent approach to d- and l-4-hydroxycyclohexenyl nucleosides 673–676 [247], starting from the common intermediate 679, which bears a hydrobenzoin moiety as the chiral auxiliary (Schemes 1.77 and 1.78). They

Scheme 1.77 Synthesis of Cyclohexenyl adenine nucleoside 673 and 674 [247]. Reagents and conditions: (a) (R,R)-hydrobenzoin, p-TsOH, benzene, reflux; (b) (i) Br2 , ether, from 10◦ C to 0◦ C; (ii) DBU, dioxane, 100◦ C; (iii) (S)-2-Me-CBSCB, CH2 Cl2 , −78◦ C to rt; (c) 6-chloropurine, DBAD, PPH3 , THF, −10◦ C to rt; (d) (i) TFA-H2 O (14:1), 0◦ C; (ii) (R)-2-Me-CBS CB, CH2 Cl2 , −78◦ C to rt; (e) (i) p-NO2 C6 H4 COOH, DBAD, PPH3 , THF, −78◦ C to rt; (ii) NH3 , MeOH, 90◦ C.

Scheme 1.78 Synthesis of Cyclohexenyl uracil nucleoside 675 and 676 [247]. Reagents and conditions: (a) N3-benzoyluracil, DBAD, PPh3 , THF,—10◦ C to rt; (b) (i) TFA-H2 O (14:1), 0◦ C; (ii) (S)-2-Me-CBS CB, CH2 Cl2 ,−78◦ C to rt; (c) (i) p-NO2 C6 H4 COOH, DBAD, PPh3 , THF,−78◦ C to rt; (ii) Me2 NH, EtOH; (d) (i) ClCO2 Et, Py, DMAP, CH2 Cl2 , rt; (ii) N3-benzoyluracil, (η3 − C3 H5 PdCl)2 , dppe, DMF, 80◦ C; (e) (i) TFA-H2 O (14:1), 0◦ C; (ii) (R)-2-Me-CBS CB, CH2 Cl2 ,−78◦ C to rt.

82


have designed a divergent synthesis that involves two main transformations: (1) introduction of the nucleobase with either inversion (Mitsunobu methodology) or retention (Pd-catalyzed coupling reaction) of configuration, followed by removal of the chiral auxiliary, and (2) stereoselective reduction of the carbonyl group to deliver the target cyclohexene nucleosides with the cis relative configuration. As a preliminary test, the new synthesized nucleoside analogues were evaluated on MT4 cells for anti-HIV-1 activity against wild-type NL4-3 strain as well as cytotoxicity using AZT and AMD3100 as the positive control. Unfortunately, none of the compounds was found anti-HIV active.

1.6. CONCLUSION

Carbocyclic nucleosides have been a subject of great interest in the synthetic medicinal chemistry for the past decades. Particularly, the discovery of abacavir and entecavir as clinically effective antiviral agents prompted the studies of various carbocyclic nucleosides. Although the synthesis of carbocyclic nucleosides has advanced dramatically, more efficient and practical methods are still in demand for the preparation of biologically active compounds as well as chiral key intermediates. In the future, novel biologically interesting carbocyclic nucleosides will likely be continuously discovered to improve the existing list of chemotherapeutic agents.

1.7. ACKNOWLEDGMENT

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CHAPTER 2

STRUCTURES AND FUNCTIONS OF NUCLEIC ACIDS MODIFIED WITH S, Se, AND Te AND COMPLEXED WITH SMALL MOLECULES WEN ZHANG, JIA SHENG, and ZHEN HUANG Department of Chemistry, Georgia State University, Atlanta, GA

2.1. INTRODUCTION

Nucleic acids are the storage of genetic information and directly participate in gene replication, transcription, and expression, and the control of nucleic acids can lead to modulation and regulation of genetic information flow and gene expression [1–3]. Moreover, many noncoding RNAs participate in signal transduction directly [4,5]. Therefore, nucleic acid-based therapeutic strategies, where the natural nucleic acids are recognized as drug targets and the modified oligonucleotides (DNAs and RNAs) are used as drug candidates, are effective tools in drug discovery and disease study at the molecular as well as the genetic level [6–8]. Because of the advantages of low toxicity, notable efficacy, and high specificity, numerous nucleic acid–based potential therapeutics, including antisense oligonucleotides, siRNAs, miRNAs, ribozymes and DNAzymes, and nucleic acid aptamers, are under development or in clinical trials [9–15]. Chemical modifications of natural nucleic acids have significantly reduced their limitations in stability and bioavailability, expanding their potentials in medical and biological applications. Because there are many oxygen atoms in nucleic acids, the atom-specific substitution of oxygen atoms with the other chalcogen atoms (Figure 2.1) in the same elemental family is an useful chemical strategy to allow tremendous improvements of nucleic acid properties (such as nuclease resistance and bioavailability) Medicinal Chemistry of Nucleic Acids, First Edition. Edited by L.H. Zhang, Z. Xi and J. Chattopadhyaya. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

101

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STRUCTURES AND FUNCTIONS OF NUCLEIC ACIDS MODIFIED 5′

X N N

NH N

5′ O

O O P O

NH2

ribose or deoxyribose

O

–

X O

X H (or CH3)

O O P O O

B sugar modification

O

O

O

O O P O –

5′

–

O

R′

5′

R′

X 3′

B

X O

O

O

NH


B

R

R

X ′3

5′

O O P O

B O

O

terminal modification

N

N

R (H or OH)

3′

NH2

O

X–R

O ′3 5′

X N ribose or deoxyribose

X N ribose or deoxyribose

B O

–

O P O –

R

O

5′

phosphate backbone modification

NH

R X

X

'3

O nucleobase modification

O

H

R

5′

O

B

O

O

NH


(or CH3)

P

–

3′

N

H

O

B

R

O – O P O O

O O P X –

B O

O O

3′

R

′3

Figure 2.1 DNAs and RNAs modified with sulfur or selenium by atom-specific replacement. X represents S or Se.

without causing significant alterations in native structures, functions, and protein interaction (such as interaction with RNase H) [6–18]. Sulfur (S), selenium (Se), and tellurium (Te) are the elements following oxygen in the VIA group of the periodic table. As an element discovered in ancient times, sulfur is currently applied in many fields. Selenium and tellurium were discovered in 19th century, and little systematic research was conducted at that time due to their toxicity. With modern chemical and biological technologies, the existence of sulfur, selenium, and tellurium in natural organisms was demonstrated. Moreover, their functions in biomolecules have attracted the increasing attention of scientific communities. For example, selenium was found to exist in natural tRNA [19] in the form of 2selenouridine and 5-[(methylamino)methyl]-2-selenouridine with the corresponding natural 2-thiouridine as a precursor. Interestingly, this 2-Se-derivatization may provide the translation process with higher accuracy and efficiency [20]. As they are in the same elemental group, oxygen, sulfur, selenium, and tellurium have closely related chemical and electronic properties. This allows almost the complete retention of the native structures, functions, and protein recognitions after the nucleic acid modification by replacing oxygen atoms with the other atoms. This atom-specific replacement allows many modification choices in introducing the chalcogen elements because of the multiple oxygen

SULFUR-DERIVATIZED NUCLEIC ACIDS

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atoms in nucleic acids. By screening for the ideal substitution sites, the multiple choices allow minimization of the potential perturbations. Furthermore, because ˚ Se: 1.16 A; ˚ Te: 1.40 A), ˚ of their larger atomic sizes (S atomic radius: 1.02 A; ˚ and their higher electron comparing with oxygen (atomic radius 0.73 A), densities, S-, Se-, and Te-derivatized nucleic acids may possess many unique properties. In this review, the syntheses, structure and function studies, and therapeutic potentials of the S-, Se-, and Te-modified nucleic acids are discussed.

2.2. SULFUR-DERIVATIZED NUCLEIC ACIDS 2.2.1. Modifications

The replacement of oxygen with sulfur 2.2.1.1. Sugar Modification in nucleic acids likely provides more structural and functional information, including duplex recognition and base-pairing stability and fidelity [21,22]. Furthermore, because of its larger atomic size than oxygen, sulfur derivatization in nucleic acids can potentially afford unique properties, such as nuclease resistance [23], which is extremely useful in therapeutic design [24]. In addition, the replacement of oxygen with sulfur is an advantageous strategy for mechanistic and structural investigation to better understand nucleic acids [25–27]. Several review papers have extensively discussed the subject of the sulfur-modified nucleic acids [28,29] and their applications [30–32]. Currently, the thio-modification of nucleic acid sugars focuses on the 2 , 3 , 4 or 5 positions. In the past few decades, researchers have successfully synthesized numerous sugar modified nucleoside analogues and nucleic acids, most of which have unique biological functions [33]. The syntheses of 2 -thio-containing nucleosides started in the 1970s [34–36], and in the following decades more and more syntheses of 2 -deoxy-2 -thio-pyrimidine and -purine nucleoside analogs were reported by different groups [37–39]. The convenient approaches included the thiophosphorylation of nucleoside analogues by P2 S5 to generate predominant 3 -O-phosphorothioate product, followed by an intramolecular replacement, leading to the 2 -thio modified product [36,37], or involving the nucleophilic displacement of leaving groups at 2 position by a soft nucleophile, such as methanethiol [40]. Compared with the other positions, the syntheses of the 3 -thio-containing nucleosides and nucleic acids attracted little attention until late 1980s [41]. Because of the interest in the achirality of this phosphorothioate modification, the synthesis strategies of the 3 -thio-modification, also known as the bridging phosphorothioate modification, were developed for functional studies [42,43]. After the accomplishment of the 3 -thio-modification-containing dithymidine phosphate analogues [44,45] and automated solid-phase synthesized oligonucleotides [42,44,46,47] assisted by Michaelis-Arbusov reaction [48,49], it is easy to obtain the 3 -thio-containing nucleic acids as potential therapeutics [50] and mechanism-studying probes [51,52]. The replacement of oxygen with sulfur at 4 -position was accomplished about four decades ago [53,54] with the initiation of synthesis of 4 -thio-ribofuranose [53,55], followed by its

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STRUCTURES AND FUNCTIONS OF NUCLEIC ACIDS MODIFIED

condensation reaction with nucleobases [54,56]. Currently, the 4 -thio-modified nucleic acids are usually applied for biochemical investigation, such as antiviral activity [56–58]. 2.2.1.2. Nucleobase Modification The sulfur modifications on the nucleobases have been studied as well in the past decades. In 1968, the 2-thiomodified nucleobase and nucleoside analogues, including 5-methylaminomethyl2-thiouracil [59], 2-thiocytosine [59], and 2-thio-uridine [60], were isolated from yeast and E. coli transfer RNAs and characterized. In addition, the chemical and enzymatic syntheses of 2-thio-containing nucleosides were developed, including 2-thiouridine [21], 2-thiocytidine [61,62], and 2-thiothymidine [63,64]. The strategy for solid-phase synthesis of 2-thio-containing oligonucleotides was demonstrated as well [65]. The 4-position of thymidine or uridine can also be replaced by a sulfur atom, either in nucleoside level [63,66] or by postsynthetic modification [67,68]. The successful protocol for the solid-phase synthesis and purification of 4-thio-modification containing nucleic acids has been published [69], and the sulfur atom was protected by cyanoethyl [69,70] or pivaloyloxymethyl [71] group. The 6-thio-guanine analogue was successfully synthesized by the activation of 6-position with pyridyl group [72]. The successful solid-phase synthesis of 6-S-G containing oligonucleotides was reported [73,74], and the postsynthetic modification to get the thio-derivatization is also available [75]. Besides the substitution of the exo-oxygen atoms, sulfur was successfully inserted into nucleobase as well. For example, Benner and coworkers incorporated the sulfur functionality into the 5-position of thymidine for some enzymatic investigation [76,77]. It is worthy to mention that nowadays the syntheses of the thio-nucleobase-modified nucleic acids has become relatively straightforward, and the S-nucleobase modifications are frequently used as tools for structural and functional studies [78,79] and drug discovery [80]. Fortunately, many S-nucleobase-modified phosphoramidites currently are also commercially available. 2.2.1.3. Phosphate Backbone Modification The thio-modification on the phosphate backbones of nucleic acids leads to the synthesis of phosphorothioate. This is probably the most frequently used S-modification, and it often results in some functional oligonucleotides for mechanism studies [81] and disease treatment [82]. Since the Eckstein group’s pioneering work on the synthesis of the nucleoside phosphorothioate in the 1960s [83], extensive studies of phosphorothioates in synthesis [84,85] and biological evaluation [86] have been performed. The phosphorothioate-containing oligonucleotides were usually prepared from the phosphate triester [81,87,88] or H-phosphonate [23,89], which could be oxidized by sulfur in pyridine [88,90]. Currently the solid-phase synthesis of this type of modified nucleic acids is availabe [91], greatly facilitating medicinal chemistry and drug design [92,93]. In addition to incorporation of the thio-functionality to the nonbridging positions, sulfur can also be used to replace the oxygen atoms on the bridging positions (i.e., the 3 - or 5 -oxygen). The successful incorporation of sulfur into


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the 5 -termini of oligonucleotides was accomplished 20 years ago [94], by reacting the 5 -amino-modified DNA with a thio-containing reagent and catalyst. The 5 -thio-functionality is currently applied to facilitate immobilization of oligonucleotides on gold [95] or glass [96] support, generating the biosensors for the DNA–ligand interactions [97,98]. Likewise, the 3 -thio-modified nucleic acids can be immobilized as well on gold-nanoparticle. Mirkin and coworkers demonstrated that the 3 -thio-functionality of a DNA can form a stable Au–S bond, thereby generating oligonucleotides containing gold assemblies and materials [99–102]. Considering the unique advantages of the constructed nanoparticles, such as relatively large surface-to-volume ratio, target binding properties, and structural robustness [103], these nano-architectures are extremely useful for gene expression regulation and biological substance detection [102]. 2.2.2. Potential usefulness

The researches on the thio-derivatized nucleic acids have attracted tremendous attentions, such as investigation of the insights into structures and catalytic functions of nucleic acids [104]. Experiments using molecular dynamics (MD) simulations demonstrated that the 2 -thiomethyl substitution in duplex favored C2 -endo puckering conformation, and potential structural properties of the modified duplex to activate RNase H recognition were predicted [105]. However, recently the crystal structures of 2 -SMe-containing duplexes from Egli’s group indicated the substitution adopted C3 -endo in both A- and B-form duplexes [106]. They revealed that even with the less electronegativity, 2 -SMeRNA, similar to 2 -OMe-RNA, would prefer C3 -endo with the 2 -substitution pointing to the minor groove [106]. Experiments also showed that the 2 -SMecontaining duplex could inhibit RNase H degradation [107]. These results were consistent with the thio-modification leading to the C3 -endo conformation. With the 2 -substitution pointing to the minor groove, which is critical to RNase H binding, the modified substrate was unable to interact with the enzyme [106]. The 4 -thio-derivatized nucleosides and nucleic acids are also useful and have been demonstrated to possess some interesting biological properties, including cytotoxicity [108,109], antiviral activity [109,110], resistance to methyltransferase [111], RNase H [112], and restrict endonuclease and exonuclease [113–115]. Therefore, this type of modification is a unique strategy to improve the stability of therapeutic oligonucleotides. The x-ray crystal structures of the 4 -thio-containing nucleic acids were also determined [116,117]. Clearly, there was a small, but detectable conformational difference on the backbone between the native and thio-modified duplexes [116]. It was most likely that this change caused the interference to the enzyme recognition. Moreover, the investigation on the 2-thio-modified nucleic acids, including melting studies [21,118], enzymatic studies [22], and x-ray crystallography [119], helped to better understand the base-pair recognition and wobble pairing. The experimental results suggested that the stabilities of 2-S-U:A or 2-S-T:A base pairs were slightly increased compared with the native ones [21,22,118], and the 2-S-T:G wobble pair was discriminated [22]. The

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experimental results from Benner’s group indicated that the replication fidelity of the novel six-letter genetic system (including A, T, C, G, isoG, isoC) was improved by applying the 2-thio-thymidine. Due to the steric and electronic effects of the sulfur atom, the mismatch between isoguanine and 2-thiothymine was discriminated, thereby enhancing the PCR amplification accuracy [120]. The most interesting property of the phosphorothioate-nucleic acids is their resistance to nucleases, including protein [121] and nucleic acid nucleases (e.g., hammerhead ribozyme [122,123]). Actually, phosphorothioate oligonucleotides were the first generation of antisense molecules that showed resistance to nuclease [124] and had been used as tools for studying biochemical processes of DNA and RNA cleavages [125,126]. Researchers from many laboratories had demonstrated that the sulfur-containing antisense compounds were agents to inhibit gene expression with longer lifetime [92,127,128]. Oligonucleotides with phosphorothioate modifications have high potentials to be applied in pharmaceutical design and drug discovery [24,129]. One indication is that the number of journal articles and patents related to “phosphorothioate antisense” has dramatically increased since the 1980s according to the recent statistical data from American Chemical Society [30]. The diastereomers generated by the chemical syntheses of the S-non-bridging phosphorothioates are also useful in catalytic mechanism studies, and the differences between these two phosphorothioate diastereomers are used to elucidate the substrate cleavage mechanisms [130]. For instance, phosphorothioate groups were incorporated into the cleavage sites of hammerhead ribozymes. Thus, the reactivity of the ribozyme can be tailored by changing with different cations, and one isomer may serve as a much better substrate than the other one [131,132]. These experimental results could provide insights into the ribozyme cleavage at the atomic level. Furthermore, another advantage of the phosphorothioate-containing therapeutic oligonucleotides is the enhanced bioavailability [133]. One example is Angiozyme, which is a chemically modified ribozyme targeting vascular endothelial growth factor receptor 1 (VEGF-1R) mRNA [134]. In phase I clinical trial, it was demonstrated that after intravenous infusion or subcutaneous bolus administration, this S-modified ribozyme generated good bioavailability, and the uptake was dramatically improved [135]. 2.2.3. Potential Therapeutic Applications

In the past three decades, the technology advancements on the S-modified oligonucleotides have provided excellent platforms for evaluating disease treatments and studying diseases, including cancer, cardiovascular, inflammation, and other diseases [129,136]. One successful example in therapeutic S-oligoncleotides is Vitravene, an antiviral drug discovered and developed by ISIS pharmaceuticals and approved by the Food and Drug Administration in 1998 [137]. As a 21mer DNA oligonucleotide modified with phosphorothioate groups for resistance to nuclease, Vitravene can bind to the coding region of the transcript of cytomegalovirus retinitis (CMV) gene and block its translation [10]. Vitravene is the first antisense drug that has met the FDA requirements for safety


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and efficacy. Furthermore, there are a number of antisense drug candidates, most of which are still in different clinical phases. Mipomersen, which is a recently developed cholesterol-reducing drug candidate [138], is currently in phase III clinical trial. The molecule has the phosphorothioate groups and the 2 -O-methoxyethyl-riboses at terminals to resist nuclease [139]. In 2009–2010, it already showed positive effects in the patient treatment with heterozygous familial hypercholesterolemia, a genetic disorder that causes exceptionally high levels of low-density lipoprotein cholesterol [140]. Other antisense drug candidates include OGX-011 from OncoGeneX [141,142], G3139 from Genta [143,144], and AEG35156 from Aegera [145]. More antisense drugs will most likely enter into the market in the near future. Since the siRNA strategy was discovered about 10 years ago [146], compared with antisense therapeutics, the S-containing siRNA therapeutics has not been utilized clinically yet, though several siRNA drug candidates are in clinical trials [147,148]. Many groups have investigated the applications of phosphorothioatemodified siRNAs and demonstrated their potential advantages [149]. In Corey’s lab, they demonstrated that the thio-containing siRNA duplexes could resist the degradation of serum up to 72 h [150]. Tuschl’s group found that the combination of the phosphorothioated backbone and the 2 -sugar modification largely increased the siRNA resistance to RNase without losing their gene silencing efficiency [151]. Moreover, several phosphorothioate-containing siRNAs are under development to treat cancers and age-related macular degeneration [152–154]. Recently, Alnylam Pharmaceuticals reported the application of a siRNA drug candidate containing phosphorothioate groups at the 3 end of duplexes, in addition to the 2 -O-methyl modification at some positions [155]. This drug candidate can mediate RNA interference and regulate lipoprotein receptor levels for treating hypercholesterolemia [156]. Due to the specific binding with the target RNA and the RNA cleavage function, ribozymes have been regarded as an efficient tool to inhibit the target gene expression [157]. Compared with antisense oligonucleotides, ribozymes have several unique advantages, such as cleavage of disease gene transcript and low concentration requirement due to the catalytic turnovers [158]. One successful example of the S-modified ribozyme therapy is Angiozyme mentioned earlier, which is a synthetic ribozyme containing four phosphorothioate groups [159]. As a drug candidate in phase I clinical trial, Angiozyme showed very high bioavailability and stability, and it represses the tumor growth by targeting mRNA of vascular endothelial growth factor receptor 1 (VEGF-1R) [134,160]. So far, most of the therapeutic ribozymes are in phase I or II clinical trials, and there are rooms to improve drug efficacy and reduce drug toxicity [158,161,162]. The ribozyme therapy has great potential to play a big role in human disease treatment in the future. Moreover, the S-modification methodology has been used to improve the stability of DNAzymes as potential therapeutics [163]. Researchers have shown that, by integrating the phosphorothioate linkages and some other modifications in binding arms or catalytic cores, the cleavage activity and nuclease resistance of DNAzymes were significantly improved [164]. Despite the

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advantages of DNAzymes in easy synthesis and lower cost, there are few clinical trials on DNAzymes as drug candidates [165,166]. Nevertheless, with careful design and modification, DNAzymes are potential therapeutics in the future. Another application of the S-derivatized nucleic acids is the development of nucleic acid aptamer-based drug candidates, such as the DNA aptamers to block tumor growth [167] and the RNA aptamers to treat inflammatory diseases [168]. To resist nucleases, chemical modifications are necessary, including the nucleobase and 2 sugar modifications, and the phosphorothioate modification is also an effective approach [169]. For example, in Gorenstein’s lab, a 22mer aptamer was synthesized by PCR using α-thio-dNTPs [170,171]. This phosphorothioate-containing aptamer can specifically bind to a leucine zipper transcription factor NF-IL6, resulting in blocking of inflammation [171]. Most nucleic acid thioaptamers were selected with the SELEX strategy [172]. E.g., Caughey and co-workers from NIH reported that the selected thio-containing aptamers could inhibit the conversion from protease-sensitive form of prion protein to the disease-specific protease-resistant form of prion protein [173]. These sulfur-containing aptamers are novel therapeutic compounds for treatment of transmissible spongiform encephalopathies (TSEs) [174].

2.3. SELENIUM-DERIVATIZED NUCLEIC ACIDS (SeNA) 2.3.1. Modifications 2.3.1.1. Sugar Modification Besides oxygen and sulfur, selenium is another chalcogen element from VIA group of the periodic table. Despite its discovery about 200 years ago, the scientific research on selenium in early stages was extremely limited because of its toxicity. With development of the technologies, the existence of selenium in biological molecules was demonstrated [175]. Nowadays selenium has been considered an essential element (i.e., the trace micronutrient) [176]. Two Se-containing special amino acids, selenomethionine and selenocysteine, have been found in nature [177,178]. Similar to sulfur, selenium is also discovered in natural tRNA anticoden in form of the 2-selenouridine and 5-[(methylami-no)methyl]-2-selenouridine (mnm5se2U) [20,175]. Although the detailed function of the seleno-derivatized tRNA still remains as a mystery, this 2-Se-functionality is most likely able to enhance the translation efficiency and accuracy [19]. In addition, because of its larger atomic size (atomic radius, ˚ S: 1.02 A, ˚ Se: 1.16 A) ˚ and its higher electron density, selenium O: 0.73 A, (Figure 2.2) can be used to derivatize nucleic acids, thereby generating potential nucleic acids with many unique and novel properties. Recently the synthesis, structure, and function studies of selenium-derivatized nucleic acids (SeNA) have been successfully pioneered by Huang and coworkers [179–186]. The Sefunctionalities at various positions, including the sugar 2 , 4 , and 5 positions, nucleobases, and phosphate groups, have been accomplished by Huang’s and

SELENIUM-DERIVATIZED NUCLEIC ACIDS (SeNA)

109

Se O (Or other nucleobases) N 5′

NH

O O

P O

N O

O– 3′

O

N

NH2

OH or (H)

Figure 2.2 Se-derivatized nucleic acids (SeNA).

other research groups [17,179,183,187,188]. SeNA provides extra research advantages in chemistry and biology areas, like chemical biology, molecular biology, and structural biology (i.e., x-ray crystallography) [182,186,189]. This atomic Se-substitution also provides the novel insights into structures and functions of nucleic acids, including base-pairing recognition, duplex stability and fidelity, catalysis, and noncoding RNA function [18,183,190]. In the sugar modification with selenium, the 5 -Me-Se-derivatized phosphoramidites, including A, C, G, T, and U, were first synthesized by Huang’s group [179]. This route was achieved by activation of the 5 -position by good leaving groups, followed by an SN2 substitution with the selenide reagents. Experiments showed that these 5 -Se-modified phosphoramidites are stable and can be incorporated into oligonucleotides with high yields. After the demonstration of the Se-functionality stability, a novel phosphoramidite compound (2 -α-methylseleno uridine phosphoramidite) was first synthesized by Huang’s laboratory [191], with the 3 ,5 -protected-2,2 -anhydrous uridine as the intermediate. It showed fine stability in solid-phase synthesis and offered a high coupling efficiency (99% yield) [191]. Later, all the other four nucleoside phosphoramidites (A, C, G, T) were synthesized [188,192]. The replacement of the sugar ring oxygen with the selenium generates the 4 -seleno nucleoside. The syntheses of the 4 -selenocytidine and 4 -selenouridine were first achieved by Matsuda’s group using stereoselective seleno-Pummerer reaction [187]. Later, a modified strategy was applied by Jeong’s group to achieve 4 -seleno nucleoside synthesis, and the crystal structure of the 4 -Se-nucleosides was also determined [193]. Later, Pinto’s laboratory applied the Pummerer rearrangement to synthesize both pyrimidine and purine nucleosdies containing the 4 -seleno functionality [194]. 2.3.1.2. Nucleobase Modification The Se-nucleobase modifications of nucleic acids were first accomplished by Huang’s laboratory recently [182,186]. The Se-functionality was introduced to the 4-position of thymidine (Scheme 2.1) [183] by selective activation of the 4-position with triazoyl group, followed by the introduction of 2-cyanoethyl selenide as the protected

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N N

N O

O

Se N

NH DMTr O

NC

N

O a b

N

DMTr O

N O

N

DMTr O

d

O

OH

c

O

O

OH

OH e NC

Se

Se N

NH

O N

5′-Oligonucleotide P O O–

O

N

DMTr O

O

O P

N P Oligonucleotide-3′

O

O

O

O –O

f

OCH2CH2CN

Scheme 2.1 Synthesis of the 4-Se-thymidine phosphoramidite and Se-DNAs. (a) TMS-Im and MeCN; (b) 1H-Triazole, POCl3 , Et3 N; (c) (NCCH2 CH2 Se)2 , NaBH4 , EtOH; (d) 10% Et3 N in MeOH; (e) 2-Cyanoethyl N,N-diisopropyl(chloro)phosphoramidite and EtN(i-Pr)2 in CH2 Cl2 ; (f) solid-phase synthesis.

selenium functionality, using the Huang’s reagent [(CNCH2 CH2 Se-)2 ] [183]. The corresponding 4-Se-T phosphoramidite was successfully synthesized and then incorporated into oligonucleotides in a high yield. Later, the 6-Sedeoxyguanosine phosphoramidite was also synthesized for the first time [184]. The 6-position of guanosine was activated by 2,4,6-triisopropylbenzene-1sulfonyl group, followed by the introduction of the Se-functionality. This novel 6-Se-G-phosphoramidite was successfully incorporated into oligonucleotides by solid-phase synthesis. Furthermore, selenium was inserted to the 5-position of thymidine [195]. This novel 5-Se-T-nucleoside was achieved with Mn(OAc)3 as a catalyst and CH3 SeSeCH3 as an electrophile. Then the first 5-Se-T-containing DNA was synthesized using the 5-Se-T-phosporamidite, and its structure was determined. Moreover, the 2-Se-T-phosphoramidite (Scheme 2.2) was also successfully synthesized for the first time [190]. After the methylation of 2-thiothymidine, the seleno-moiety was introduced by NaSeH, generating the 2-selenothymidine. The corresponding 2-Se-T-phosphoramidite was synthesized, followed by the synthesis of the 2-Se-T DNAs. The crystal structural and biophysical studies of the 2-Se-T-containing DNA demonstrated that the bulkiness and electronic effect of the selenium atom at the thymidine 2-exo position discriminate against the T:G wobble pair without significant impact on the T:A pair, thereby enhancing the high fidelity of A:T base pair with the Se-modification.

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SELENIUM-DERIVATIZED NUCLEIC ACIDS (SeNA) O

O

HO

S

N

O NH

NH DMTrO

a

O

N

N DMTrO

S

b

O

OH

OH c

O N N

CH3

O

OH

DMTrO

S

N

O

O

CN

Se

N

O

CN

e N

DMTrO

Se

NH

d DMTrO

N

Se

O

O

O N P O CH2CH2CN

OH

HO

f O O 5′-Oligonucleotide – P O O

NH N

Se

O

O O – P Oligonucleotide-3′ O

Scheme 2.2 Synthesis of the 2-Se-thymidine phosphoramidite and Se-DNAs. (a) DMTr-Cl, Pyridine, DMAP, rt; (b) DBU, DMF, CH3 I; (c) Se, NaBH4 , EtOH; (d) I-CH2 CH2 CN, i-Pr2 NEt, CH2 Cl2 ; (e) (i-Pr2 N)2 P(Cl)OCH2 CH2 CN, (i-Pr)2 NEt, CH2 Cl2 ; (f) solid-phase synthesis.

2.3.1.3. Phosphate Modification The phosphoroselenoates were synthesized via oxidative selenium incorporation [88], and the longer phosphoroselenoates were prepared for the antisense study [196]. Later, the phosphoroselenoates were used for the structural study by Egli’s group [189]. This Se-modification is achieved during the solid-phase synthesis by a selenizing reagent (such as potassium selenocyanate, KSeCN), replacing iodine. Subsequently, several other reagents were also developed to introduce the Se-functionality into phosphate groups. These reagents include benzothioselenol-3-one [197], organometallic reagent (iPrC5 H4 )2 TiSe5 [198], and selenium-cyanoethylphthalimide [199]. Though the chemical synthesis can successfully prepare phosphoroselenoates, the synthesized phosphoroselenoates are diastereomer mixtures. Separation of diastereomers is very challenging and impractical for oligonucleotides containing more than one phosphoroselenoate group. The enzymatic synthesis of phosphoroselenoates was first developed by Huang’s group, and the diastereomerically pure phosphoroselenoate oligonucleotides were synthesized enzymatically. Se-dNTP series was chemically synthesized, and the diastereomers were easily separated [180]. Interestingly, DNA polymerase can well recognize and incorporate both dNTP diastereomers, thereby synthesizing diastereomerically pure phosphoroselenoate DNAs.

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Later, phosphoroselenoate RNAs were also synthesized enzymatically with diastereomeric Se-NTPs [17,18], including α-Se-ATP, α-Se-CTP, α-Se-GTP, and α-Se-UTP. They were successfully incorporated into the hammerhead ribozyme to generate diastereomerically pure RNAs for the function study. Furthermore, the thymidine triphosphate containing the Se-moiety at the 4-position was synthesized as well, and it was recognized by the DNA polymerase as well as the native TTP [200]. 2.3.2. Potential Functions and Applications

Since the discovery of the naturally Se2.3.2.1. Crystallography Study containing tRNAs [175], the Se-modification has attracted scientists’ enthusiasm to study its unique properties in biochemical and biological systems. Due to ˚ and its signal for multiple wavelength anomalous disits K edge of 0.9795 A persion (MAD) and single wavelength anomalous dispersion (SAD) analysis, selenium is considered an ideal anomalous scattering center in crystal x-ray diffraction, which drastically facilitates crystallographic studies on biological macromolecules. Since the first successful structural determination of a protein with selenomethionine (replacing sulfur with selenium) and MAD technique developed by Hendrickson’s laboratory 20 years ago [201], more and more novel protein structures have been determined with the selenomethionine derivatization [202–204]. Fortunately, the sulfur substitution with selenium does not cause significant perturbations in protein structures and functions. Thus, it is not surprising that currently over two-thirds of the novel protein structures are determined by the selenomethionine strategy and MAD phasing (RCSB Protein Data Bank) [204]. Clearly, the selenomethionine and MAD strategies have revolutionized the protein x-ray crystallography. However, it was surprising that oxygen atoms in nucleic acids can also be stably replaced with selenium without causing significant perturbations in structures and functions [17,184]. This novel strategy with selenium-derivatized nucleic acids (SeNA) is changing the x-ray crystallography of nucleic acids and proteinnucleic acid complexes [186]. The classic strategy in nucleic acid crystallography relies on the halogen derivatization, such as Br or I derivatization [205,206]. Unfortunately, the I- or Br-derivatized nucleic acids are sensitive to lights, such as UV and x-ray lights. In addition, the modification sites for the halogen incorporations are very limited: primarily the 5-position of deoxyuridine, which may cause unavoidable perturbations in structures and functions [207]. In comparison, the Se-derivatization of nucleic acids offers better stability and much more diversities than the halogen-derivatization because in nucleic acids, there are many oxygen atoms in different chemical environments. Huang’s laboratory synthesized the first Se-containing nucleoside phosphoramidites and pioneered the chemical and enzymatic syntheses and the selenium-derivatization of nucleic acids for structure and function studies. Through the collaboration among Huang, Egli, and their coworkers, the first structure of the Se-derivatized nucleic acids was determined via the direct selenium derivatization and MAD phasing [208]. Over the


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past several years, many crystal structures of DNAs, RNAs, and protein–nucleic acid complexes have been successfully determined. This novel Se-derivatization strategy has received more and more attention. It will revolutionize x-ray crystallography of nucleic acids and protein–nucleic acid complexes, as well as small molecule ligand-nucleic acid complexes. The crystal structures of the selenium-modified nucleic acids provide lots of useful structural information on nucleic acids. The DNA crystal structures containing 4-Se-T [183] and 6-Se-G [184] indicated that the selenium modifications generated no obvious structural perturbation comparing with the native DNA structures (Figure 2.3), and that a novel hydrogen bond between the selenium atom and the amino group (Se . . . H–N) was formed. In addition, unique structural and functional properties of the selenium-modified nucleic acids were discovered.

(a)

(b)

3′

3′

Gse

A C

C T

A

Gse

G

T

C U

A 5′

5′

(c) 3.48

3.16 2.59

Figure 2.3 The superimposed global and local structures of 6-Se-G containing DNA/RNA duplexes of the nucleic acid–protein complex. (a) The structure of the Se-DNA sequence (2R7Y, in yellow) is superimposed over the corresponding native DNA (2G8U, in gray). (b) The structure of the RNA sequence (2R7Y, in green) is superimposed over the corresponding native (2G8U, in gray). (c) The Se-G3/C5 base pair (2R7Y) with the experimental electron density shows three H-bonds (exo-6-Se/exo-4-NH2 , 1-NH/N(3), and ˚ respectively. (Full-color exo-2-NH2 /exo-2-O) with bond lengths of 3.48, 3.16, and 2.59 A, version of the figure appears in the color plate section.)

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With an inserted atom at the 5-position of thymidine, it was unexpectedly found that the 5-methyl group is able to form a hydrogen bond with the 5 -phosphate. Another example is that after the replacement of oxygen with selenium at the 2-position of thymidine, the T:G wobble pair is largely discriminated against, whereas the T:A base pairing stability was not significantly affected. Probably because of the steric and electronic effects of the selenium atom, the fidelity of Se-T:A base pair is largely increased. Because high fidelity is essential for gene replication, transcription, and translation [190], this selenium modification provides a novel chemical approach to significantly increase the fidelity of the base pairing, which is critical in living systems. Unexpectedly, we found another useful advantage of the selenium modifications: the crystallization facilitation (Figure 2.4), especially by the 2 -Se derivatization. The exciting fact is that with the selenium functionality at the 2 -position of sugar, nucleic acids (especially DNAs) are able to crystallize much faster under broader buffer conditions and with higher diffraction qualities [207]. One example is the application of 2 -SeMe-dU. The DNA oligonucleotide (GdU2 -Se GTACAC)2 crystallized in a few days from 20 out of the 24 buffer conditions, while the corresponding native DNA (GTGTACAC)2 did not crystallize over 2 to 3 months [207,209]. The crystallization condition of the native DNA is quite narrow. Obviously, the selenium derivatization is advantageous. 2.3.2.2. Biochemical Applications Selenium modifications result in nucleic acids with unique properties that are particularly useful in biochemical and biological investigations. For instance, the nucleoside triphosphates derivatized with selenium at nonbridging α-position of phosphate were synthesized [18], and their two diastereomers were separated. It was demonstrated that only one of these two Se-modified triphosphate diastereomers is a good substrate for T7 RNA polymerase in vitro transcription, whereas the other diastereomer is not recognized by the enzyme: neither a substrate nor an inhibitor [17]. On the contrary, DNA polymerase recognizes both diastereomers [180]. The recognition differences of the Se-NTPs and Se-dNTPs can be used to study the catalysis and substrate interactions of DNA and RNA polymerases. Another discovery is that the Se-hammerhead ribozymes transcribed with the nucleoside 5 -(α-P-seleno)triphosphates could demonstrate a high catalytic activity, up to the native level [18], and could also display a low or no activity depending on

(a)

(b)

(c)

Figure 2.4 Photos of crystals of the native and Se-derivatized octamers. (a) Native octamer; (b) Se-octamer; (c) Se/Br-octamer sizes of the crystals are ranged from 0.1 × 0.1 to 0.4 × 0.4 mm.


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the Se-modified nucleotides, indicating their participation in the catalysis. These Se-NTPs offer a useful methodology for rapid screening of ribozymes and other noncoding RNAs. Furthermore, it is found that both phosphoroselenoate DNAs and RNAs resist nuclease digestion, which is especially useful in developing Se-oligonucleotide therapeutics. Moreover, the triphosphate with the Se-derivatization at the thymidine 4-postion was also synthesized, recognized by DNA polymerase, and incorporated into DNAs [200]. The enzymatic incorporation efficiency was as good as native TTP. Interestingly, this replacement of a single oxygen atom with selenium in the nucleobase generates yellow-colored DNA, which will be used as a unique probe for the enzymatic assay. 2.3.2.3. Therapeutic Development and Clinical Applications Due to the selenium toxicity and limitation of investigation strategies, in the past, the selenium modification was not significantly explored in drug discovery research, compared with the sulfur modification and gene therapy. Because recently selenium has been recognized as an essential element, drug discovery with the selenium modifications becomes an opportunity. There is no reason that all the approaches applied in the S-derivatized gene therapy cannot be applied in the Sederivatized gene therapy. As an element from the same elemental family as sulfur, selenium-functionalized nucleosides, nucleotides, and nucleic acids may possess ideal stabilities and chemical and biological functions. Furthermore, the special electronic and dimensional characteristics of selenium generate unique advantages, making SeNA an advanced approach for exploring disease mechanisms and treatments at the atomic level. As discussed previously, the Se-derivatization of nucleic acids is an advanced tool for structure determination. In Huang’s lab, a ternary complex with 6-Se-G containing DNA, RNA, and RNase H protein was crystallized, and their crystal structure was determined [184]. This is the first example to solve the protein structure with SeNA facilitation, and it opened a gate for the application of selenium-derivatized nucleic acids in drug design and discovery. Another advantage of SeNA is its resistance to nuclease in vivo or in vitro, which is the essential requirement for the oligonucleotide therapy. Similar to the thio-modified antisense oligonucleotides, we have demonstrated recently that the Se-modified nucleic acids, including the modifications on the backbone, sugar, and nucleobase, resist nuclease degradation. It is believed that this novel SeNA can also play important roles in drug discovery. Another important application of selenium in therapeutic development is the synthesis of nucleoside analogues as potential anticancer, antimicrobial, or antiviral drugs. Most of the therapeutic nucleosides contain chemical modifications disrupting nucleic acid synthesis or damage repair. Successful examples are the compounds used to block the human immunodeficiency virus replication to cure AIDS. The best-known approved drugs (Figure 2.5) include 3 -azido3 -deoxythymidine (AZT), 2 ,3 -dideoxycytidine (ddC), 2 ,3 -dideoxyadenosine (ddA), and 2 ,3 -didehydro-3 -deoxythymidine (d4T). Their efficient synthesis is critical in medicinal chemistry and drug manufactory. Researchers have developed some seleno-mediated synthesis routes to make reactions simple and efficient. Selenium functionalities were introduced to the nucleosides to generate

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NH2 N HO

N O

O

O

NH

NH O

N

HO

O

N

HO

O

O

O

N3 ddC

d4T

AZT

Figure 2.5 Structures of some FDA-approved anti-HIV drugs.

2 ,3 -unsaturated nucleosides. The phenylselenyl group was the most frequently used. Several laboratories [210–212] have achieved the syntheses of the 2 ,3 dideoxy- and 2 ,3 -didehydro-2 ,3 -dideoxynucleosides. After the synthesis of the 2 -phenylselenenyl nucleosides, 2 ,3 -unsaturated nucleosides were created by treatment with H2 O2 or n-Bu3 SnH. These methodologies offered the highly efficient synthesis. 2.4. TELLURIUM-DERIVATIZED NUCLEIC ACIDS (TeNA)

As an element from the oxygen family, tellurium hasn’t attracted much research attention, compared to sulfur and selenium. This is most likely due to the high reactivity and toxicity of the tellurium functionalities. As a nonmetal element but with strong metallic property, compared to selenium, tellurium is much more difficult to form a covalent bond with hydrogen or carbon, which leads to instability of the Te-modified organic compounds. Fortunately, the incorporation of tellurium into amino acids was attempted 20 years ago [213], and the telluromethionine was successfully introduced to a dihydrofolate reductase and other proteins [214]. The tellurium-derivatized proteins showed reasonable stability, and their crystal structures were determined on the basis of the Te-heavy atom derivatization. Thus, via the derivatization, tellurium is expected to also bring the native nucleic acids with useful and unique properties because of its high electron density and ˚ oxygen: 0.73 A; ˚ sulfur: 1.02 A; ˚ selelarge atomic size (Te atomic radius: 1.40 A; ˚ [215]. Fortunately, research on the Te-derivatized nucleic acids has nium: 1.16 A) begun recently. Huang and coworkers have pioneered this research area, and the Te-derivatized nucleic acids were first reported one year ago [216]. As expected, the Te-modified oligonucleotides have been found with many novel and unique properties [113,216]. Initially, tellurium element was applied in synthesis of the modified nucleosides with high efficiency and simple approach. As a strong nucleophile as well as an excellent leaving group, the tellurium functionality (such as the phenyl telluride) has been used to facilitate the elimination reaction, yielding the 2 ,3 -unsaturated nucleosides [217], which are the key intermediates of many nucleoside drugs (Figure 2.5). The pioneering research of introducing tellurium

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TELLURIUM-DERIVATIZED NUCLEIC ACIDS (TeNA)

functionality into nucleic acids was accomplished by Huang’s group. The nucleoside phosphoramidite and nucleic acids with Te-modification at the 2 -position were first synthesized [216]. With the protection by an alkyl or aryl group, the 2 -tellurium functionality is stable during the solid-phase synthesis, deprotection, and purification. Interestingly, the DNA damages (the nucleobase elimination or the DNA fragmentation) can be generated selectively by treating the 2 -Temodified DNAs under different conditions [216]. This 2 -Te-derivatization provides a potential strategy to investigate mechanisms of the nucleic acid damages. Furthermore, Huang’s laboratory has first synthesized the Te-nucleobase-modified nucleosides and nucleic acids [245]. The Te-functionality was successfully incorporated into the 5-position of deoxyuridine (Scheme 2.3). Interestingly, this Te-nucleobase derivatization is stable in the solid-phase synthesis, and the Te-DNAs showed the same thermostability as the corresponding native DNA. Moreover, the crystal structure of the Te-containing DNA was determined for the first time. The x-ray crystal structure indicated that the Te-modified DNA was virtually identical to the native DNA, and that the Te-modified thymidine could still pair with adenine by hydrogen bonds as well as the native pair, indicating that the Te-functionality caused no significant structural perturbation to both the DNA duplex and the T:A base pair (Figure 2.6). Furthermore, in the STM imaging, the Te-modified DNAs showed much stronger topographic and current peaks (Figure 2.7) than the corresponding native DNAs. This implies

O I N

DMTrO

I

NH O

a

DMTrO

O

O

O

Ph Te

NH N O

b

NH N

DMTrO

O-TBDMS

HO

O O

O O-TBDMS c

DNA

O P

–O

Ph Te

O

O

O

Ph Te

NH N

O

O

e

N

DMTrO

O

O

O

P DNA

P

O –O

N

Ph Te

NH O

d

O NH N

DMTrO

O

O HO

OCH2CH2CN

Scheme 2.3 Synthesis of the 5-Te-thymidine phosphoramidite and Te-DNAs. (a) TBDMSCl, Imidazole., DMF, rt; (b) (i) NaH, THF, 15 min, rt; (ii) n-BuLi, THF, 10 min., −78◦ C; (iii) Ph2 Te2 , 1 h, −78◦ C; (c) TBAF, THF, RT; (d) i -Pr2 NP(Cl)CH2 CH2 CN, DIEA, CH2 Cl2 , rt; (e) solid-phase synthesis.

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2.87 Å

2.77 Å (b) 2.37 (a)

2.10

(c)

Figure 2.6 The x-ray crystal structures of Te-DNA duplex [5 -G(2 -SeMedU)G(Te T)ACAC-3 ]2 . Red ball: Te, yellow ball: Se (a) superimposed structures of ˚ resolution; PDB ID: 3FA1) and native one (in Te-dsDNA structure (in cyan; 1.50 A magenta; PDB ID: 1DNS); (b) the local structure of the Te T/A base pair (in cyan) is superimposed over the native T/A base pair (in magenta); (c) the experimental electron density (2|Fo|—|Fc| map) of the Te T/A base pair (σ = 1.0). The green ball represents approximate 1/6 of phenyl group (1 carbon). (Full-color version of the figure appears in the color plate section.)

(a)

(b) nm

nm

20

35 nm

20

35 nm

Figure 2.7 The STM images of the Te-modified DNA duplex [5 - ATGG(TeT)-GCTC3 and 5 -(GAGCACCAT)6-3 ]. The arrows indicate the edges or current peaks of the measured molecules. (a) Topographic image of Te-duplex; (b) Current image of Te-duplex. The sample bias: 0.50 V, the current set point: 100 pA.

STRUCTURAL STUDIES OF NUCLEIC ACIDS–SMALL MOLECULE COMPLEXES

119

that electron-rich tellurium, with a high metallic character, is able to facilitate electron delocalization in the electron-deficient nucleobase, thereby improving the DNA conductivity. Obviously, the Te-modifications are extremely useful for the structural and functional studies, imaging and molecular wire exploration, and mechanistic investigation of DNAs as well as RNAs.

2.5. STRUCTURAL STUDIES OF NUCLEIC ACIDS–SMALL MOLECULE COMPLEXES

Structure-based drug design (SBDD) has been used in the pharmaceutical industry for over 25 years, which continues to play an important role in drug discovery, design, and optimization, especially with the development of sophisticated biophysical and computational methodologies. The biological macromolecules, such as proteins and nucleic acids, naturally become the ideal targets for identifying the “leading compounds” through structure studies. Comparing to proteins, nucleic acids have gained much less attention in this field, probably because one of the reasons is the difficulty to cocrystallize and solve the structure of the nucleic acid–drug (or small molecule) complexes. Considering the importance of nucleic acids in biological systems and their structure diversities (especially RNAs), there will be tremendous potential opportunities in this area. In addition, the discussed selenium and tellurium strategies are useful for crystallization and structure determination of nucleic acids. We are confident that this field will advance rapidly in the near future. Herein we briefly highlight the most recent progress in this area of structural studies of nucleic acid–small molecule complexes. This section offers general guidance for the readers who are interested in this field and provides a direction for the selenium- and tellurium-assisted structural determination of the complexes of small molecular ligands and nucleic acids. 2.5.1. Targeting DNA through the covalent interaction

Cisplatin [or Cis-diamminedichloroplatinum(II)] (Figure 2.8a) is one of the most extensively used antitumor drugs that has been confirmed to be effective in treating a variety of human malignancies, including testicular, ovarian, cervical, head and neck, esophageal, and nonsmall cell lung cancers, since its approval by the FDA in 1978 [218–220]. Tremendous following research has been carried out to investigate their mechanism of action, including the exploration of the biological targets. Although many other cellular components can react with cisplatin (e.g., phospholipids and phosphatidylserine) [221], it’s generally accepted that DNA is the primary target of cisplatin in cells. The research in this area has predominated [222]. To illustrate the detailed interaction mechanism and to reduce its high toxicity, a lot of research has been focused on the structure studies of cisplatin-DNA adducts by using NMR, molecular mechanics calculation, and more important, x-ray crystallography. The early crystallographic information of these adducts was mainly limited to the short single-stranded DNAs, and it

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had been concluded that the cisplatin reacted with DNA by covalently linking the N7 positions of the adjacent G or A. The following structure work of the cisplatin complexed with more complicated DNA and DNA-protein systems, accomplished by Lippard and coworkers, have significant contribution to this field [222–224]. Because their work has been extensively reviewed [225], we will just add two very recent examples, including the complexes of the DNA-cisplatin and protein-DNA-cisplatin. As shown in Figure 2.8b, Lippard’s ˚ to 1.77 A) ˚ of a cisplatingroup improved the structural resolution (from 2.6 A containing DNA duplex [d(CCTCTG*G*TCTCC).d(GGAGACCAGAGG)], where the G*G* means the intrastrand GG dimer cross-linked by cisplatin at N7 positions [226]. From this structure, it’s revealed that the major groove of this platinated DNA was compressed and the minor groove was widened, making the double-stranded DNA ready for the relevant protein recognition and binding in a structure-specific manner. In addition, the ammonia ligands and the cross-linked geometry with both platinum center and nucleobases are much more clearly visualized. Meanwhile, the deoxyribose sugar puckers are also better resolved, allowing reexamination of the global structure of the platinum-modified DNA. Furthermore, with this new model, it’s revealed that there are four octahedral [Mg(H2 O)6 ]2 + ions associated with bases in the DNA major groove and 124 highly ordered water molecules that participate in hydrogen-bonding interactions with either the nucleic acid or the diammineplatinum(II) moiety. It has also been known for a long time that some proteins can recognize the platinated DNA duplexes [227]. For instance, the high-mobility-group domain (HMG) binds to the platinated DNA duplexes. Its DNA complex structure was determined in 1999 by Lippard and coworkers [224]. Recently, it has been shown that two special polymerases (Pol η and Pol ζ) can also interact with

H3N

Cl Pt

H3N

Cl

Cisplatin (a)

(c)

(b)

Figure 2.8 (a) Chemical structure of cisplatin. (b) Complex structure image of cisplatin and DNA duplex d(CCTCTG*G*TCTCC).d(GGAGACCAGAGG), cisplatin ligand is showed in gray. (c) Image of G*G* platination sites. (PDB ID:3LPV).


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cisplatin during the cell replication process to bypass the platinum lesion, which reduces the therapeutic effect of cisplatin and contributes to tumor resistance. Very recently, Carell et al . [228] reported a novel complex crystallographic structure of yeast pol η, where the Pt–GTG lesion pairs with two nucleobases, providing the explanation of the partial bypass mechanism. As shown in Figure 2.9, the low-fidelity Pol η can place two nucleobases opposite the Pt–GTG lesion. The flipped-out central dT of the Pt–GTG cross-link cannot be positioned in the active site of the enzyme, thus blocking the enzyme movement along the duplex DNA. These structural examples discussed here provide an ideal model to design and modify the platinum- or other metal-containing compounds to develop better therapeutics. So far, over 3000 cisplatin analogues have been screened for the further clinical trials to overcome certain side effects, such as high toxicity and intrinsic or acquired resistance [229]. 2.5.2. Targeting DNA through the minor groove interaction

Netropsin, its close derivative distamycin, and Hoechst dye type molecules (Figure 2.10) are typical antiviral and antitumor antibiotics, which can interfere with both replication and transcription and have been studied extensively due to their specific binding with the minor groove of the B-form DNA duplex

Figure 2.9 The cartoon representation of overall crystal structure of a (DNA template)–primer duplex containing a cisplatin–(1,3-GTG) lesion in complex with Pol η. (PDB ID: 2WTF).

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NH2 +

(a)

N

N

H N

H

N

N

NH

NH

H N

N N H

N

CH2

C+

CH3

O

C

NH CH2

O

CH3

O

N H

N

N

O

CH3

O

N N H

C

H

(b)

CH3

O

N

C

H2N

(c)

H

H H

CH2

CH3

O

N

OH

CH2

C+ NH

N N

N

Figure 2.10 Chemical structures of Netropsin (a), Distamycin (b), and Hoechst 33258 (c).

[230,231]. Because they are too toxic for clinical use, the structure-based researchers will provide valuable information for their further optimization. Toward this goal, Dickerson, Sundaralingam, Dervan, Neidle, et al . and their coworkers have pioneered this research area by revealing the 3-D structures of DNA–Netropsin complexes, as well as the detailed molecular foundation of their interaction and nucleobase recognition [232–236]. The overall structures of some DNA-drug complexes are presented in Figure 2.11. Their detailed structure features as well as other DNA structures complexed with drugs, such as imidazole-pyrole-hydroxypyroole polyamide [237], benzimidazole [238,239], and tribenzimidazole [240], are not discussed here. In addition to the research on the drug–DNA complexes, the further mechanism studies based on the crystal structures of the drug–DNA–protein complexes have also been reported in literature [241]. In 2005, Georgiadis and coworkers cocrystallized the complex (Figure 2.12) containing dsDNA, Netropsin, and the N-terminal fragment of MMLV-RT (Moloney Nurine Leukemia Virus Reversetranscriptase) [242], which provided significant details on the drug–DNA–protein interactions. 2.5.3. Targeting DNA through the intercalation

In addition to the discussed covalent and noncovalent binding, the intercalation is another major noncovalent binding of small molecule drugs with nucleic acids, when they have proper size and chemical natures to fit into the stacked base pairs of DNAs or RNAs [242]. So far, a lot of DNA intercalators have been used as chemotherapeutics, such as Adriamycine, Daunomycin, Daunorubicin,


(a)

(b)

(c)

123

(d)

Figure 2.11 (a) Overall structure of the Netropsin-DNA complex (1:1 ratio), the DNA sequence is: 5 -d(CGCGAATT5-BrCGCG)2 -3 ; PDB ID: 6BNA. (b) Overall structure of Netropsin–DNA complex (2:1 ratio), the DNA sequence is: 5 -d(C5-BrCCCCIIIII)2 -3 ; PDB ID: 358D. The Netropsin molecules are showed in gray. (c) Overall structure of the Distamycin–DNA complex (2:1 ratio), the DNA sequence is: 5 -d(GTATATAC)2 -3 ; PDB ID: 378D. (d) Overall structure of Hoechst 33258-DNA complex (1:1 ratio), the DNA sequence is 5 -d(CGCGAATTCGCG)2 -3 ; PDB ID: 8BNA. The Distamycin and Hoechst molecules are shown in grey spheres.

Figure 2.12 The crystal structure of an RT fragment–DNA–Netropsin complex. The DNA sequence: 5 -d(CTTAATTCGAATTAAG)2 -3 ; PDB ID:1ZTT. The Netropsin molecule is showed in blue spheres. (Full-color version of the figure appears in the color plate section.)

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Dactinomycin, Echinomycin, and Ditercalinium, to inhibit the growth of cancer cells. Again, the structure-based researches of these DNA-drug complexes are invaluable for further design and optimization to achieve better specificity and low toxicity. The complex structures of Adriamycin, Daunomycin, Ditercalinium, and Echinomycin with DNAs are presented here as examples (Figures 2.13 and 2.14).

O

O

OH

OH

OH

OH OH O

O O

O

OH

O

O

(a)

O

CH3

O

O

OH

O

(b)

OH

OH NH2

NH2

N

N

N

N N

N

O

O

N

N N H O N

O O S S

O H N

O N O

O

O

N

N

O

O

O

N

O N

CH3

N N

(d)

(c)

Figure 2.13 Chemical structures of Adriamycin (a), Daunomycin (b), Ditercalinium (c), and Echinomycin (d).

(a)

(b)

(c)

(d)

Figure 2.14 (a) Overall structure of the Adrimycin-DNA complex (2:1 ratio), the DNA sequence is: 5 -d(CGATCG)2 -3 , (PDB ID: 1D10). (b) Overall structure of the Daunomycin-DNA complex (2:1 ratio), the duplex is a B-form DNA/RNA chimera (dC)(rG)d(ATCG) (PDB ID: 1JO2). (c) Overall structure of the Ditercalinium-DNA complex (1:1 ratio), the DNA sequence is: 5 -d(CGCG)2 -3 , (PDB ID: 1D32). (d) Overall structure of the Echinomycin -DNA complex (2:1 ratio), the DNA sequence is: 5 d(GCGTACGC)2 -3 , (PDB ID: 1PFE). All the drug molecules are shown in spheres.

PERSPECTIVE

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2.6. PERSPECTIVE

This strategy of the oxygen replacement with the other chalcogen elements (S, Se, and Te) opens novel research avenues of nucleic acids, including therapeutic discoveries, structure and function studies, mechanism and catalysis investigations, and material and nanoscience research. Currently the thio-modification is the most promising strategy for designing and developing the nucleic acid therapeutics due to the stability and bioavailability of the S-modified oligonucleotides in vivo. It is expected that with the high specificity, low toxicity, and efficient delivery, more S-modified therapeutic oligonucleotides will enter into clinical trials and eventually drug market in the future. Because selenium has the unique application in x-ray crystallography, the Se-nucleic acids have great potentials in facilitating phasing and crystallization [183,185,186,190,192,207,244,245] for 3-D structure determination of nucleic acids, protein–nucleic acid complexes, as well as small molecule–nucleic acid complexes. In addition to the regular duplexes of DNAs and RNAs, nucleic acids can have highly diversified secondary and tertiary structures, such as bulges, junctions, i-motifs, triplexes, and quadruplexes. Thus, small molecules can interact with nucleic acids in many different ways (i.e., much more than the three classical binding modes discussed in this chapter). The further advances in this field, including rationally designed interactions (e.g., major groove recognitions by small molecular ligands), rely on the new structure determination. As discussed in this chapter, we are confident that our novel Se-derivatization strategy will significantly facilitate the process of structure determination and revolutionize the structure-based drug design and discovery to conveniently target nucleic acids and protein-nucleic acid complexes with diversified interaction modes and ligand structures. The results of the structural studies will provide direct guidance and useful platforms for further optimization of existing drugs and the discovery of novel therapeutics with high specificity, potency, and low toxicity. Furthermore, selenium is an essential element to human health, and the Semodified DNAs and RNAs show strong resistance to nucleases and cause no significant structural perturbations. Thus, the Se-nucleic acids may have tremendous potentials in therapeutic development and drug discovery as well. In addition, because selenium can form strong Au–Se bond, selenium may be useful in gold nanoparticle derivatization and property tailoring. Regarding electron-rich tellurium, the electron-deficient nucleobases can stabilize the Te-functionality, and in return metallic tellurium largely increases the electron delocalization of the nucleobases, thereby significantly improving the nucleic acid conductivity [245]. This unique property via the Te-modification is particularly important and useful for imaging and nanostructure construction with nucleic acids. This atomspecific modification of nucleic acids with the chalcogen elements opens many novel doors for fundamental studies of structures, functions, and applications of nucleic acids. We are confident that this novel strategy also has great potential in investigating structures, functions, and drug discovery of small molecular ligands complexed with nucleic acids.

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ACKNOWLEDGMENT

This work was financially supported by the Georgia Cancer Coalition (GCC) Distinguished Cancer Clinicians and Scientists and by the U.S. National Science Foundation (NSF MCB-0824837).

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233. Schultz, P. G., Dervan, P. B., Distamycin and penta-N-methylpyrrolecarboxamide binding sites on native DNA. A comparison of methidiumpropyl-EDTA-Fe(II) footprinting and DNA affinity cleaving. Journal of Biomolecular Structural Dynamics 1984, 1 (5), 1133–1147. 234. Nunn, C. M., Garman, E., Neidle, S., Crystal structure of the DNA decamer d(CGCAATTGCG) complexed with the minor groove binding drug netropsin. Biochemistry 1997, 36 (16), 4792–4799. 235. Chen, X., Mitra, S. N., Rao, S. T., Sekar, K., Sundaralingam, M., A novel end-to-end binding of two netropsins to the DNA decamers d(CCCCCIIIII)2 , d(CCCBr5CCIIIII)2 and d(CBr5CCCCIIIII)2 . Nucleic Acids Research 1998, 26 (23), 5464–5471. 236. Tevis, D. S., Kumar, A., Stephens, C. E., Boykin, D. W., Wilson, W. D., Large, sequence-dependent effects on DNA conformation by minor groove binding compounds. Nucleic Acids Research 2009, 37 (16), 5550–5558. 237. Kielkopf, C. L., White, S., Szewczyk, J. W., Turner, J. M., Baird, E. E., Dervan, P. B., Rees, D. C., A structural basis for recognition of A.T and T.A base pairs in the minor groove of B-DNA. Science 1998, 282 (5386), 111–115. 238. Squire, C. J., Baker, L. J., Clark, G. R., Martin, R. F., White, J., Structures of m-iodo Hoechst-DNA complexes in crystals with reduced solvent content: implications for minor groove binder drug design. Nucleic Acids Res 2000, 28 (5), 1252–1258. 239. Neidle, S., Mann, J., Rayner, E., Baron, A., Boahen, Y., Simpson, I. J., Smith, N. J., Fox, K. R., Hartley, J. A., Kelland, L. R., Symmetric bis-benzimidazoles: new sequence-selective DNA-binding molecules. Chemical Communications. 1999, 929–930. 240. Aymami, J., Nunn, C. M., Neidle, S., DNA minor groove recognition of a nonself-complementary AT-rich sequence by a tris-benzimidazole ligand. Nucleic Acids Research 1999, 27 (13), 2691–2698. 241. Neidle, S., Crystallographic insights into DNA minor groove recognition by drugs. Biopolymers 1997, 44 (1), 105–121. 242. Goodwin, K. D., Long, E. C., Georgiadis, M. M., A host-guest approach for determining drug-DNA interactions: an example using netropsin. Nucleic Acids Research 2005, 33 (13), 4106–4116. 243. Richards, A., Rodger, A., Synthetic metallomolecules as agents for the control of DNA structure. Chemical Society Reviews 2007, 36 (3), 471–483. 244. Salon, J., Sheng, J., Gan, J.-H., Huang, Z., Synthesis and crystal structure of 2 -Semodified guanosine containing DNA. Journal of Organic Chemistry 2010, 75 (3), 637–641. 245. Sheng, J., Hassan, A. E. A., Zhang, W., Zhou, J., Xu, B., Soares, A. S., Huang, Z., Synthesis, structure and imaging of oligodeoxyribonucleotides with telluriumnucleobase derivatization. Nucleic Acids Research 2011, 39 (9), 3962–3971.

CHAPTER 3

UNRAVELING THE NAD CYCLIZING AND CALCIUM SIGNALING FUNCTIONS OF HUMAN CD38 HON CHEUNG LEE Department of Physiology, University of Hong Kong, Hong Kong

3.1. INTRODUCTION

It is now well established that CD38 is a signaling enzyme that is important in regulating a wide range of physiological functions. The advance in elucidating the functions of CD38 is especially remarkable when one considers that CD38 was originally identified, not purposely, but as the result of random typing using monoclonal antibodies to catalog the hundreds of molecules expressed on the surface of lymphocytes [1]. CD38 appeared interesting because its expression on lymphocytes showed variations depending on the developmental and functional stages of the cells and had thus been called a lymphocyte differentiation antigen [1,2]. Therefore, it was highly unexpected when it was later found to be a signaling enzyme responsible for synthesizing two novel Ca2+ messenger molecules, cyclic ADP-ribose (cADPR) from NAD and nicotinic acid adenine dinucleotide phosphate (NAADP) from NADP and nicotinic acid [3–5]. These two novel molecules have been well established to serve as Ca2+ messengers in a variety of cellular functions, including fertilization [6–10], receptor activation in lymphocytes [11,12], insulin secretion in pancreatic islets [13–15], hormonal signaling in pancreatic acinar cells [16,17], chemotaxis in neutrophils [18], and abscisic acid signaling in plants [19] and sponges [20] (reviewed in 21–24). Most recently, the cADPR-pathway is shown to be involved in regulating neuronal oxytocin secretion and social behavior of mice [25]. Medicinal Chemistry of Nucleic Acids, First Edition. Edited by L.H. Zhang, Z. Xi and J. Chattopadhyaya. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

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This chapter is focused on this latter aspect of CD38, the unraveling of the NAD cyclizing and Ca2+ signaling functions of the molecule. The wide range of functions regulated by CD38 documents its central role in physiology. In fact, CD38 is so critical for humans that individuals lacking CD38 have not been found despite extensive screening of more than 5000 blood samples from newborns, indicating its absence in humans may well be lethal [26].

3.2. ANTIGENIC FUNCTIONS OF CD38

CD38 is not simply a marker passively expressed at various stages of lymphocyte differentiation, but is also actively regulating cell functions. This was first indicated by the observation that ligation of CD38 using specific monoclonal antibodies can lead to activation and proliferation of lymphocytes [27]. A natural ligand of CD38 was later identified as CD31 expressed preferentially on vascular endothelial cells, which upon binding to CD38 can effect many of the cellular functions seen with agonistic antibody-ligation [28]. The antigenic function of CD38, however, requires the incorporation of other surface antigens. For example, in natural killer cells, antibody ligation of CD38 activates their lytic function, if and only if the CD16-signaling pathway is also present and operational in the cells [29]. This suggests that CD38 may not have its own antigenic signaling function, but instead may transmit the ligation effect indirectly to other signaling pathways via interaction with their surface antigens. In addition to CD16, CD38 has been found to interact with other antigens, including CD4, a receptor for the human immunodeficient virus (HIV). In the early stage of infection, the majority of the T-cells infected are not expressing CD38, suggesting that CD38-expression can exert a protective effect [30]. Transfection of CD38 in cells likewise protects them against de novo infection with HIV-1IIIB [31]. It has been proposed that CD38 is endogenously associated with CD4 and, in doing so, inhibits the binding of gp120 of the HIV to CD4 and thus protects the cells from HIV infection [32]. This inhibitory effect on HIV infection is, perhaps, the clinically most relevant aspect of the antigenic functions of CD38. Brain tissues from HIV-positive patients show that the CD38 gene is activated and the protein is highly expressed [33]. Increased CD38 expression on CD8-positive T-lymphocytes is also linked to immune system activation, progression of HIV infection, and increased death rate in adult patients [32]. This and many other facets of the antigenic function of CD38 has recently been reviewed [26]. The rest of this chapter will focus, instead, on the Ca2+ signaling functions of CD38.

3.3. ENZYMATIC FUNCTIONS OF CD38

The first indication that CD38 is, in fact, an enzyme came from sequence comparison [34] with the ADP-ribosyl cyclase from Aplysia [35,36], a sea snail. The Aplysia cyclase, the first cADPR synthesizing enzyme ever identified, cyclizes

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Figure 3.1 Structures of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). The cyclization site in cADPR is between the anomeric carbon (C1 ) of the terminal ribose and the N1 of the adenine ring.

NAD, a linear molecule, in a head-to-tail fashion, to a cyclic molecule and releases the nicotinamide group of NAD in the process [35]. As shown in the top panel of Figure 3.1, the cyclization site, as determined by x-ray crystallography, is at the N1-atom of the adenine ring of NAD, which is linked back to the C1-atom of the terminal ribose group, forming a complete circle [4,5]. The sequence of human CD38 is about 29% identical to the cyclase [34]. Moreover, there are 10 cysteine residues in the cyclase, and all can be aligned with those in CD38. The Aplysia cyclase, however, is a soluble protein, whereas the human CD38 has a single trans-membrane segment starting at residue 22 from the N-terminus [2]. CD38 also has four glycosylation sites distributed in the C-terminal domain.

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That CD38 is indeed capable of producing cADPR from NAD was shown using recombinant CD38 from mice [37] and humans [38]. In the case of murine CD38, the purified C-terminal domain of the protein was shown to have all the enzymatic activity [37], which was later found to be also the case for human CD38 [39]. Unlike the cyclase, CD38 catalyzes the formation of only small amounts of cADPR from NAD. The great majority of the enzymatic product is ADP-ribose (ADPR) instead [37,38,40]. More surprising, CD38 was found to use cADPR as substrate and hydrolyzes it to ADPR [37,38,41,42]. CD38 is thus a highly unusual enzyme that can take two completely different molecules as substrate, a linear NAD and a cyclic cADPR, and produce two equally distinct products, a cyclic cADPR and a linear ADP-ribose, respectively. Its catalytic novelty goes much further. In fact, CD38 can also use NADP as substrate and, at acidic pH and in the presence of nicotinic acid, catalyze a base exchange reaction, exchanging the nicotinamide group of NADP with nicotinic acid [43]. The product is nicotinic acid adenine dinucleotide phosphate (NAADP; bottom panel in Figure 3.1) [3,43]. Moreover, CD38 can take NAADP as substrate, again at acidic pH, and hydrolyze it to ADPR-2 -phosphate (ADPRP) [44]. Figure 3.2 illustrates the enzymatic reactions catalyzed by CD38. At neutral or alkaline pH, CD38 mainly catalyzes the synthesis and hydrolysis of cADPR, while at acidic pH, it catalyzes the synthesis and hydrolysis of NAADP. The symmetry of its catalysis is remarkable and suggests that CD38 may be specially designed to metabolize cADPR and NAADP. CD38 could be passed off as a freak of nature, a promiscuous enzyme without specificity, if it were not because of the extensive and important signaling functions regulated by its catalytic products. As will be described in more detail later, both cADPR and NAADP are messenger molecules mediating mobilization of

Figure 3.2 The multiple reactions catalyzed by CD38. At neutral or alkaline pH, CD38 cyclizes NAD to small amounts of cADPR, whereas the majority of the substrate is hydrolyzed to ADP-ribose (ADPR). CD38 is also the only known enzyme that can hydrolyze cADPR to ADPR. At acidic pH, CD38 catalyzes a base-exchange reaction, exchanging nicotinic acid with the nicotinamde group of NADP to produce NAADP. CD38 can also hydrolyze NAADP to ADP-ribose phosphate (ADPRP).

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intracellular Ca2+ stores; cADPR targets the endoplasmic stores while NAADP targets the lysosomal stores (reviewed in 22,24,45). ADPR, on the other hand, targets a Ca2+ influx channel, the TRPM2 (reviewed in 46). Ca2+ signaling is crucial for essentially all aspects of life, from fertilization, the beginning of life, to apoptosis, programmed cell death, and CD38 is central to Ca2+ signaling. This is well established by studies ablating the CD38 gene in mice. Multiple defects are documented, which include those in insulin secretion [47], hormonal signaling in pancreatic acinar cells [48], migration of dendritic cell precursors [49], bone resorption [50], airway responsiveness [51], α-adrenoceptor signaling in the aorta [52], cardiac hypertrophy [53], susceptibility to bacterial infection [54], as well as social behavior in mice through modulation of oxytocin secretion [25]. In humans, the deletion of the CD38 gene may well be lethal because no CD38-negative individual has yet been identified [26].

3.4. Ca2+ MOBILIZATING FUNCTIONS OF THE CATALYTIC PRODUCTS OF CD38

The Ca2+ signaling activities of cADPR and NAADP were first discovered in sea urchin eggs [3,5,55]. Both have since been shown to be active in a wide range of biological systems from plants [19] and sponges [56] to a variety of mammals, including human systems (reviewed in 21–24,45). The pharmacological profiles of cADPR and NAADP, analyzed extensively in sea urchin eggs, are distinct, indicating separate receptors are involved [55,57,58]. The profiles are likewise distinct from that of inositol trisphosphate (IP3 ), the first Ca2+ messenger ever identified [59]. That cADPR targets not the IP3 -receptor but the ryanodine receptor (RyR) was first implicated by pharmacological approaches [58,60,61] and was subsequently confirmed by single channel recordings using RyRs reconstituted into lipid bilayer [62–64]. Like the IP3 -receptor, the RyRs are also located in the endoplasmic reticulum (ER). Thus, cADPR is a Ca2+ messenger, together with IP3 , responsible for mobilizing the major intracellular Ca2+ stores in the ER. NAADP not only targets a separate receptor but the responsive Ca2+ stores are also different from the ER. This was first shown by separating and stratifying cellular organelles inside live sea urchin eggs [65] and by organelle fractionation [66]. The organelles responsive to NAADP were physically separable from the ER and identified as the reserve granules, lysosome-related organelles in sea urchin eggs [9]. That NAADP mobilizes Ca2+ from the lysosomal stores has also been shown in a variety of mammalian cells (reviewed in 67). Most directly, the NAADP-activated Ca2+ currents have now been recorded from individual and isolated lysosomes using a glass chip-based method [68]. The identity of the Ca2+ channel specifically activated by NAADP has been controversial. Both TRP-ML1 [69], a lysosomal protein, and skeletal RyR [70] have been proposed as targets of NAADP. Subsequently, a series of studies from several laboratories has identified the lysosomal two-pore channels (TPCs) as the target (71–73 and reviewed in 74). Direct measurements on individual

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lysosomes isolated from cells overexpressing TPCs indeed exhibit the presence of the NAADP-dependent Ca2+ currents [68]. It has been suggested that RyR could be interacting with TPCs through conformational coupling, analogous to the coupling between RyR and the dihydropyridine-receptor in muscles, or through Ca2+ -induced Ca2+ mechanism [75], thus accounting for the apparent involvement of RyR in the NAADP-dependent Ca2+ signaling. The Ca2+ signaling through mobilization of specific internal Ca2+ stores by both cADPR and NAADP is stimulus dependent. A wide range of stimuli and agonists can elevate the endogenous concentrations of cADPR and NAADP and activate specific cellular functions. These include the plant hormone, abscisic acid [19,76–79], fertilization [6,10,80], acetylcholine [81], cholecystokinin [17,82], insulin [83], Glucagon-like peptide-1 [84], retinoic acid [85], interleukin 8 [86], and angiotensin II [87], just to list a few. Both cADPR and NAADP thus satisfy all criteria of being universal messengers [88,89].

3.5. Ca2+ INFLUX ACTIVATING FUNCTIONS OF THE CATALYTIC PRODUCTS OF CD38

There are two sources of Ca2+ for signaling in cells. In addition to mobilizing internal stores, stimuli and agonists can also stimulate influx through activation of Ca2+ channels in the plasma membrane. One of the influx channels of particular relevance is the TRPM2 channel. It is a member of the M-family of transient receptor potential (TRP) channels named after the first identified member of the family, the tumor suppressor melastatin, now TRPM1 (reviewed in 46). It is widely expressed in many tissues, including the brain, bone marrow, spleen and pancreas. They are best recognized for their contributions to sensory transduction, including response to temperature, touch, light, and other stimuli. The 3-D reconstruction of the TRPM2 channel using transmission EM reveals a bell-shaped structure with two large cytoplasmic domains [90]. The last segment of 267 residues at the C-terminal cytoplasmic tail is homologous to human nucleoside diphosphate-linked moiety X-type motif 9 (NUDT9), a mitochondrial ADP-ribose hydrolase that degrades ADPR to AMP and ribose 5-phosphate (reviewed in [46]). This segment is termed the NUDT9-H domain. Evidence indicates that ADPR binds to this domain and activates the opening of the TRPM2 channel [91,92]. ADPR is the hydrolysis product of cADPR and NAD, both substrates for CD38. Indeed, cADPR also can activate the TRPM2 channel in a synergistic manner such that, in the presence of subthreshold concentrations of ADPR, the EC50 value of cADPR for activating the channel is reduced more than 10-fold [93]. The cADPR-dependent activation of the TRPM2 channels is found to be remarkably sensitive to temperature, being essentially quiescent at room temperature, but greatly enhanced at body temperature or higher, suggesting that it can be involved in heat sensing [94]. This is consistent with the known function of TRPM2 in sensory transduction [46].

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In addition to functioning as a Ca2+ influx channel at the plasma memebrane, TRPM2 has also been detected in the lysosomes of the pancreatic β cells [95]. Indeed, the channel is found to be sensitive to NAADP as well, although not as responsive as to ADPR [96,97]. TRP-ML1, another lysosomal channel mentioned earlier, is also sensitive to NAADP [69], suggesting that NAADP is capable of activating multiple lysosomal channels to varying degree, consistent with it functioning as a mobilizing messenger of lysosomes. The cytoplasmic domain of the TRPM2 channel is critical to gating because various deletions or mutations in it have been shown to render the TRPM2 channel incapable of being regulated by these ligands [94,98]. 3.6. STRUCTURE AND CATALYTIC MECHANISM OF CD38

Understanding how CD38 can use multiple substrates and produces three different Ca2+ messengers is of importance not only because of the obvious physiological relevance but also because it is catalytically intriguing. A unified scheme has been proposed to account for the novel multifunctionality of CD38 as an enzyme [99]. For CD38 to produce cADPR, its active site must be able to bind and fold the linear NAD into a cyclic conformation so that the two ends can be linked. The next step is likely the cleavage of the glycosidic bond between the nicotinamide ring of NAD and the anomeric carbon (C1 ) of the terminal ribose, releasing the nicotinamide and forming an intermediate with the catalytic residue in the active site. This would then allow the N1 of the adenine ring to link with the C1 of the ribose, producing cADPR as depicted in Figure 3.3. Successful competition by water molecules with the intramolecular adenine ring for reaction with the C1 of the intermediate would result in hydrolysis, producing ADPR instead. Like water, nicotinic acid too can react with the intermediate, resulting in base exchange and the production of NAADP. This catalytic scheme has now been verified by a series of studies using x-ray crystallography. The Aplysia cyclase is the first cADPR-synthesizing enzyme crystallized and its structure solved [100], which serves as a model for CD38. Cocrystallization of the cyclase with nicotinamide, a substrate for the base-exchange reaction, shows that the active site of the enzyme is in a pocket near the middle of the protein [101]. Systematic studies using site-directed mutagensis of residues in the pocket identify the catalytic residue of the cyclase [101]. Mutagenizing the corresponding residue in CD38, Glu226, likewise inactivates all enzymatic activities of CD38, indicating it is the catalytic residue [102]. For crystallization, recombinant CD38 is produced in yeast that has the tranmembrane segment and the N-terminal tail deleted [39]. CD38 is a bean-shaped molecule with a central cleft separating the C- and N-terminal domains, near which the active site pocket is located [103; Figure 3.4). The N-terminal domain is composed mostly of αhelixes, whereas the β-structures are mainly in the C-terminal domain. All 12 cysteines of CD38 are paired up as disulfide linkages. Changing the catalytic residue, Glu226, to glutamine inactivates all the enzymatic activities of CD38 and allows the structure of CD38 complexing with NAD,

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Figure 3.3 A unified catalytic scheme for CD38. The substrate NAD (R = OH) or NADP (R = Pi) binds to the active site in a folded conformation and interacts with the catalytic residue Glu226, resulting in the cleavage of the glycosidic bond and the release of the nicotinamdie group. An intermediate is formed with the anomeric carbon (C1 ) of the terminal ribose in an activated state. Both covalent and noncovalent intermediates have been observed. The former shows a covalent bond between C1 and the carboxylate group of Glu226. Linkage between C1 with the N1 of the adenine ring results in cyclization and produces cADPR. Alternatively, reaction of C1 with water results in hydrolysis and produces ADP-ribose (ADPR), whereas reaction with nicotinic acid (NA) results in base exchange and produces NAADP. (Full-color version of the figure appears in the color plate section.)

its main subtrate, to be determined [104]. NAD enters the active site pocket with the nicotinamide end first, which interacts with the site through both hydrophobic stacking with Trp189 and by hydrogen bonding with Glu146 and Asp155. The catalytic residue, Glu226, forms two hydrogen bonds with the hydroxyl groups of the nicotinamide ribose [104]. This interaction appears to strain the ribose ring. Likewise, the strong interactions between the nicotinamide and the active site leads to a rotation of the plane of the carboxamide group by 22◦ from the plane of the pyridine ring, straining the nicotinamide moiety as well. These distortions are the main driving force for the cleavage of the glycosidic bond between the nicotinamide and the ribose, instead of direct attack on the anomeric C1 of the nicotinamide ribose [104]. After the release of the nicotinamide group, an intermediate is formed, which is stabilized by the hydrogen bonding between Glu226 and the ribosyl OH-groups.

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Figure 3.4 The structure of CD38. The secondary structure shows the concentration of β-sheets (yellow) in the carboxyl domain and α-helixes (red) in the amino (N) domain. The six disulfides are colored cyan. The bound substrate NAD is shown in the stickrepresentation: Carbon in green, nitrogen in blue, oxygen in red, and phosphorus in orange. The surface view of CD38 is shown on the right, with NAD bound to the active site pocket at the middle of the molecule. The single transmembrane segment is represented by a golden helix, whereas the N-terminal tail is modeled as a random coil (gray). The lipid bilayer is shown in green. (Full-color version of the figure appears in the color plate section.)

Both noncovalent and covalent intermediates have been seen by crystallography, depending on the substrate used. Using NAD and nicotinamide mononucleotide as substrates, noncovalent intermediates can be trapped by a short co-incubation of CD38 crystals with the substrates and high concentrations of guanine containing compounds [105,106]. On the other hand, a covalent intermediate is observed when CD38 is cocrystallized with an inhibitory analog of NAD, ara-2 F-NAD. The C1 of the ribose of the substrate forms a covalent bond with the carboxylate group of Glu226. The linkage anchors one end of the substrate and allows the observation of the folding back of the other end of the substrate, the adenine ring, toward the C1 [105,106], which would lead to cyclization and the formation of cADPR. Water molecules are seen bound at the active site close to the carboxylate group of Glu226 [104,107], which should be able to compete readily with the adenine ring for interacting with the intermediate, leading to hydrolysis and the production of ADPR.

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For CD38 to hydrolyze cADPR, the cyclic molecule must bind to the active site. Crystallography shows that the same active site pocket of CD38 that binds NAD can readily bind cADPR as well [108]. Like NAD, the bound cADPR also forms two hydrogen bonds between the two hydroxyl groups of one of its ribose and Glu226 [108]. It is thus expected that the same intermediate as that formed by NAD would also be formed with cADPR, accounting for the cADPR-hydrolyzing activity. Alternatively, if the intermediate is attacked by nicotinic acid, NAADP will be formed. One remarkable feature is that it occurs mainly in acidic conditions [43]. This may be relevant because NAADP targets lysosomes, an acidic organelle. The acidic dependence is shown to be due to the electric charge repulsion between the substrate, nicotinic acid, and acidic residues, Asp155 and Glu146, at the active site, prohibiting the entry of the substrate into the site [44]. Mutating the residues to neutral amino acids, such as alanine or glutamine, indeed eliminates the acidic dependence [44]. These results provide structural evidence at atomic resolution supporting the catalytic scheme shown in Figure 3.3, which accounts for how a single active site of CD38 can catalyze three different reactions; cyclization, hydrolysis or base exchange. Finally, structural evidence also pinpoints the residues responsible for the catalytic differences between CD38 and the Aplysia cyclase. When using NAD as substrate, the cyclase produces only cADPR, whereas CD38 forms mainly ADPR [35,37]. Crystallography shows that during cyclization, hydrophobic stacking between the adenine ring and Phe174 at the active site of the cyclase facilitates the folding back of ring [109]. The corresponding residue in CD38 is Thr221. Mutating Phe174 in the cyclase to threonine indeed turns it into a CD38-like enzyme, producing large amounts of ADPR from NAD. Conversely, mutating Thr221 in CD38 to phenylalanine turns it into a cyclase like enzyme. Another residue in CD38 important in controlling the hydrolysis reaction is Glu146. A double mutation, changing Thr221 to phenylalanine and Glu146 to alanine, turns CD38 into a complete cyclase, producing over 94% of its catalytic product from NAD as cADPR [109]. The results demonstrate that catalysis by CD38 or the cyclase is controlled by one or two critical residues, and mutating them can interconvert the reactivities of the two enzymes. The stage is set for engineering the enzymes with specific activity toward cADPR for expression in cells, which should be valuable tools for manipulating the function and metabolism of this novel Ca2+ messenger.

3.7. CELLULAR LOCATION AND MEMBRANE TOPOLOGY OF CD38

The cellular location of CD38 is controversial. The confusion is mainly derived from the fact that CD38 was historically identified as a lymphocyte antigen of unknown function [1]. It has now been established that it is neither uniquely found in lymphocytes, nor exclusively expressed on cell surfaces (reviewed in [110,111]). Virtually all mammalian tissues ever examined have been

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shown to express CD38. Immunoelectronmicroscopic, immunofluorescence, and fractionation studies indicate it is expressed intracellularly in various organelles [33], including the ER [112] and the nucleus [113–115] as well. Most important, induced expression of CD38 increases intracellular cADPR levels [85,116]. Conversely, knocking out CD38 in mice leads to a dramatic decrease in the endogenous cADPR in many tissues [54,117]. It is thus clear that the site of catalytic synthesis of cADPR by CD38 in mammalian cells is intracellular. It is also true that in some cell types, particularly blood cells, CD38 is expressed on cell surfaces as a type II membrane protein with its catalytic domain located outside the cell. How the cytoplasmic NAD gets out of the cells and the product cADPR back inside to mobilize intracellular Ca2+ stores is a topological paradox [118]. Elaborate mechanisms have been described as shown in Figure 3.5. NAD is released from cells via the connexin hemichannels, and the product, cADPR, is transported across the cytoplasmic membrane by nucleoside transporters [119–121]. Likewise, a type II membrane protein expressed in organelles would have its catalytic domain facing the lumen of the organelles and not the cytoplasm, where the substrate NAD is. Similar mechanisms would have to operate for intracellular CD38 to effect Ca2+ signaling as well.

Figure 3.5 Two alternate signaling schemes for CD38. On the left, CD38 is depicted in type-II orientation only. The catalytic domain (light blue) faces outside or the organellar lumen. The connexin channels mediate the movement of NAD, whereas cADPR is transported by the nucleoside transporter (grey spheres). The type-II CD38 on the cell surface interacts with agonistic antibody and serves antigenic function. On the right, CD38 is depicted in both type-II and type-III orientations. The type-III CD38 serves the Ca2+ signaling function, converting cytosolic NAD to cADPR, whereas the type-II CD38 on the surface serves the antigenic functions. The type-III CD38, with its catalytic domain inside the cytosol, is amenable to a wide range of regulation mechanisms, such as phosphorylation or interaction with various protein factors.

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Perhaps the most perplexing aspect of this scheme concerns how the activity of CD38 can be regulated. The cellular regulation mechanisms would have to target the short N-terminal tail of CD38 because it is the only portion inside the cells, making it unclear how the regulatory signal can be transmitted from the tail to the catalytic domain outside the cells. The fact is that the membrane topology of CD38 has never been seriously investigated, especially for intracellular CD38. It is an assumption that all CD38 molecules should have type II membrane orientation. The possibility that two forms of CD38 with opposite membrane polarity may actually exist in cells deserves careful consideration. This possibility is novel and not only may offer a much more straightforward resolution to the topological paradox, but also provides more understandable ways for regulating the activity of CD38. With the catalytic domain being in the cytoplasm, it would be amenable to a wide range of regulation mechanisms, such as phosphorylation or interaction with various protein factors. That a membrane protein can adopt two opposite orientations has precedence. The bacterial multidrug transporter EmrR is a protein with four transmembrane helixes. As a functional transporter, it is a homo-dimer. The two identical monomers, however, are in opposite orientation, with the N-terminus of one mono-dimer facing the cytoplasm, whereas the other faces outside. A singleresidue mutation can change the membrane orientation of the monomer [122]. It is proposed that the mutation can actually allow the protein to flip within the membrane [122,123]. Another example is the prion protein, a type II protein similar to CD38, which can be synthesized not only in two transmembrane orientations, but also in a glycosyl-phosphatidylinositol-linked form. Transacting protein factors have been described that can direct prion proteins toward different topologic fates [124–127]. It is generally believed that the polarity of membrane proteins is determined prominently by charged residues flanking the hydrophobic core of the transmembrane segment (reviewed in [128]). The most positive end is generally cytoplasmic (“positive inside rule”). For CD38, both sides have four positive charged residues. The N-terminal side has: Lys13, Arg17, Arg20, and Arg21, whereas the C-terminal side also has four Arg45, Arg47, Lys57, and Arg58. The charge distribution being equal on both sides of the transmembrane segment suggests that CD38 may well express both opposite polarities in the membrane. Indeed, the type III orientation, with the catalytic domain facing the cytoplasm, where the substrates and the targets of the products are, may well be the relevant form of CD38 as a signaling enzyme. The type-II orientation would, on the other hand, subserve the antigenic functions of CD38. One final hurdle for the type III CD38 to function as a signaling enzyme is related to the disulfide linkages of the molecule. It is generally believed that disulfide formation occurs only inside the ER and that cytoplasmic proteins, or cytoplasm-facing proteins, do not have disulfide linkages. Systematic studies show that it is not the case. A wide range of cytosolic proteins, including those involved in chaperone function, signal transduction, and cell growth, have

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been found to contain inter- as well as intramolecular disulfide bonds in both mammalian cells and bacteria [129–131]. More intriguing, perhaps, is the recently hypothesized involvement of the formation of lipid droplets to facilitate the escape of polypeptides from the endoplasmic reticulum, the site of the synthesis of membrane proteins [132]. This mechanism has been invoked to account for the presence of a fully glycosylated transmembrane protein, Class I Major Histocompatibility Complex, in the cytoplasm [133]. Thus, CD38 could be fully formed in the ER, with disulfide linkages and glycosylation, and transported out of the ER with this mechanism. Fusion of the lipid droplets carrying the extracted CD38 with the inner surface of the plasma membrane can potentially allow its insertion in an alternate orientation. The discovery of the Ca2+ signaling function of CD38 was totally unanticipated and truly a remarkable feat. It began with the even more remarkable discovery of the Ca2+ signaling functions of its catalytic products, cADPR and NAADP, in 1989 [4,5,55]. Two decades have passed, and much is now well established for CD38, including physiological functions, structure, and catalytic mechanism. The next major step is the resolution of its membrane topology, which will provide important clues on how the enzymatic functions of CD38 are regulated in vivo, a largely uncharted territory.

ACKNOWLEDGMENTS

My current research is supported by grants from the National Science Foundation of China and Research Grants Council of Hong Kong.

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CHAPTER 4

DNA AND RNA BINDING SMALL MOLECULES SHIBO LI and ZHEN XI Department of Chemical Biology and State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, China

DNA is the major target of a lot of antitumor drugs, as well as many antiviral and antibacterial agents, whereas ribosomal RNA is one main target of clinically used antibiotics. DNA replication and transcription and RNA translation are crucial to cell survival and proliferation. The tertiary structures of DNA or RNA are recognized by small molecules through all kinds of interactions that can be found in other protein–protein, DNA (or RNA)–protein, drug–protein interactions, including hydrogen binding, van der Waals interaction, metal ion coordination, and electrostatic interaction, in different combinations. To design new small molecules with higher binding affinity and specificity to a determined DNA or RNA, we must discuss the structures of drug–DNA (or RNA) complexes that have been determined by a variety of physicochemical and biochemical techniques.

4.1. DNA-BINDING DRUGS

DNA in the double-helix form is the main target of DNA binders. Here the DNA binders are classified by the mode of interactions by which small molecules may bind to DNA. All these forms of interaction are also employed by RNA binders and can also be utilized in designing new nucleic acid–binding molecules for research or therapy applications. Some publications have reviewed DNA-binding drugs and methods to study the drugs and their targets [1–3].

Medicinal Chemistry of Nucleic Acids, First Edition. Edited by L.H. Zhang, Z. Xi and J. Chattopadhyaya. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

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4.1.1. Intercalation

Intercalation is one of the most common modes of interaction for DNA-binding drugs. In the 1960s, Lerman and Pritchard [4,5] introduced the concept of intercalation. Flat polycyclic aromatic ring molecules can be inserted between two consecutive base pairs in the DNA double helix, such as acridine dyes and ethdium bromide (Figure 4.1). Proflavine have also been known to bind RNA since the late 1960s [6,7]. The main binding force is van der Waals interaction between the π-electron systems of the dye molecule and the heterocyclic rings of the base pairs. In 1975, Seemen [8] reported the first detailed structure of an intercalation complex of 9-aminoacridine with r(ApU). The base pairs between adenine and uracil residues were stacked parallel to each other, with 9-aminoacridine sandwiched directly between them. When a drug molecule is intercalated between two base pairs, the DNA base step at the binding site is stretched and causes the increase of the overall length of the double helix. The intercalation of drug molecules is limited by the neighbor exclusion rule, which states that drug molecules cannot occupy two successive base steps. Ethidium bromide can inhibit DNA-dependent nucleic acid synthesis by efficiently intercalating into the DNA [9,10]. X-ray analyses of ethidium bromide complexed with dinucleoside monophosphate revealed that the detailed conformations of the sugar–phosphate backbone and the ethidium intercalative geometry were almost identical in complexes both containing iodine and containing no iodine [11–13]. Aclacinmycin A (Figure 4.2), first found in 1975 [14], has been used worldwide as an antitumor drug with low cardiac toxicity. NMR solution structures revealed the intercalation geometry of aclacinomycin in the CpG dinucleotide.

Figure 4.1 Structures of acridine dyes and ethidium bromide.

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Figure 4.2 Structures of aclacinmycin A.

The D ring of alkavinone moved toward the minor groove direction by about 1.2A and results in crowding between the drug sugar (deoxyfucose ring) and the adenine deoxyribose, the reason for the DNA kinking at the TpA step. Distrisarubicin (Figure 4.3), a member of anthracyclines [15,16], preferentially binds the dinucleotide step GpT located in regions of alternating purines and pyrimidines. The primary reason for this sequence specificity of drug–DNA interaction is the sequence-dependent conformation of the DNA double helix. Calladine [17] and Dickerson [18] proposed a series of generalized rules to describe the standard structure of duplex DNA. The elements of a drug intercalation site are not only the two successive base pairs into which the intercalator is inserted, but also the third base pair located next to the two base pairs. The third base pair seems to interact with the other portion of the drug molecule better than the chromophore of the drug. Camptothecin, indolocarbazoles, and indenoisoquinolines (Figure 4.4) bind to the topoisomerase I and DNA complex (the covalent complex), resulting in a ternary complex, and thereby stabilizing it. This prevents DNA religation and therefore causes DNA damage, which results in apoptosis. Staker [19] determined the ternary complex structure of Camptothecin and representative members of the indenoisoquinoline and indolocarbazole with human topoisomerase I and singlestrand cleaved DNA duplex. The planar nature of all three structurally diverse classes allows them to intercalate between DNA base pairs at the site of singlestrand cleavage and stabilize topoisomerase I-DNA covalent complexes. Drugs containing two chromophores joined by a molecular linker were synthesized [20–26], because the antitumor efficiency for several families of intercalating drugs is correlated with their DNA binding affinity [27–29]. The most widely studied class of potential bis-intercalating agents was bis(acridines) (Figures 4.5a

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Figure 4.3 Structures of anthracycline antibiotics.

Figure 4.4 Structures of camptothecin, indolocarbazoles, and indenoisoquinolines.

and 4.5b) as antitumor agents [30–32] and as probes of bifunctional ligand–DNA interaction [33,34]. Both the length and the nature (flexibility, polarity, hydrogen bond ability, etc.) of the linker are important for binding mode and biological activities of the bisintercalator. Hansen [35] compared the DNA binding of the three acridine derivatives in Figure 4.3b. The results suggest tris-intercalation of the trimer. However, biological activities of the three compounds did not differ obviously from each other. Ditercalinium in Figure 4.5c is a DNA bis-intercalator with high DNA affinity and strong antitumor activities. The structures of the complexes formed by a

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(a) (b)

(c)

Figure 4.5 Structures of bis-intercalators and tris-intercalators.

self-complementary tetranucleotide with ditercalinium and with a related monomer were investigated by NMR [36,37]. The monomer appears to monointercalate and the dimer to bis-intercalate into the teranucleotide duplex. 4.1.2. Groove-Binding

The DNA double helix has two grooves. The major and minor grooves have different width and depth, and one face of the base pair is exposed to the major groove, wherease the other face is exposed to the minor groove. As shown in Figure 4.6, natural antibiotics such as distamycin and netropsin are long, flexible, and crescent-shaped molecules. They can fit tightly into the minor groove in the AT-rich region of B-DNA, stabilized by hydrogen bonding, electrostatic, and van der Waals interactions. Dickerson [38] determined the crystal structure of the 1:1 complex of netropsin complexed to a dodecamer duplex. The drug molecule binds to the AATT region in the minor groove. As seen in Figure 4.7, Netropsin amide NH form hydrogen bonds with DNA adenine N3 and thymine O2 atoms occurring on adjacent base pairs and opposite helix strands. The narrowness of the groove forces the molecule to sit symmetrically in the center, with its two pyrrole rings slightly noncoplanar so that each ring is parallel to the walls of its respective region of the groove. Drug binding neither unwinds nor elongates the double helix, but it does force open the minor groove by 0.5–2.0 A. The sequence specificity is provided by close van der Waals contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the drug molecule. Substitution of one or more pyrroles by imidazole could permit recognition of G:C base pairs as well, and it could lead to a class of synthetic “lexitropsins,” capable of reading any desired short sequence of DNA base pairs. This structure was confirmed by molecular dynamics simulation and NMR of netropsin complexed to the dodecamer duplexes also

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Figure 4.6 Structures of distamycin and netropsin.

Figure 4.7 Schematic drawing of netropsin in 1:1 complex.

[39,40]. Similar “bifurcated” hydrogen bonds were found by x-ray analysis of distamycin bound to DNA duplex [41]. Distamycin can also form a 2:1 complex with a 11-mer duplex [42]. As shown in Figure 4.8, two distamycin molecules bind simultaneously in the minor groove of the central 5 -AAATT- 3 segment and are in close contact with both the DNA and one another. With the side-by-side antiparallel binding orientation, the positively charged propylamidinium groups are directed toward opposite ends of the helix. Energy calculations suggest that electrostatic interactions, hydrogen bonds, and van der Waals forces contribute to the stability of the complex. Molecular ˚ relative to the 1:1 modeling shows that the minor groove must expand (3.4 A) complex to accommodate both drugs [43].

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Figure 4.8 Complex of distamycin with duplex in 2:1.

Many works were reported on the sequence specificity and design of new molecules that exhibit preferences for other and longer sequences. A number of derivatives of netropsin and distamycin were synthesized. Dervan [44,45] carried out extensive investigations of oligopeptides containing both pyrrole and imidazole rings (Figure 4.9). They synthesized 2-ImN and 2-ImD and determined the solution structure of the 1:1:1 adduct (2-ImN/distamycin/DNA) [45] and the 1:1:1 adduct (2-ImD/distamycin/DNA) with 11-mer duplexes (Figure 4.9) [46]. Geierstanger [47] summarized the preferential interaction of pyrrole and imidazole with A/T and G bases, respectively, and synthesized the oligopeptide consisting of two pyrrole and two imidazole rings (Figure 4.10d; ImPImP). As expected, it forms the 2:1 adduct with each ligand bonded to the 5-(A,T)GCGC(A,T)-3 sequence as shown in Figure 4.10. To design sequence-specific drugs, White [48] connected two peptide chains at the ends via a linker to form a hairpin containing pyrrole (Py), 3-hydroxypyrrole (Hp), and imidazole (Im). Hydroxypyrrole–imidazole–pyrrole polyamides form four ring-pairings (Im/Py, Py/Im, Hp/Py, and Py/Hp), which distinguish all four Watson–Crick base pairs in the minor groove of DNA. Many other synthetic drugs replacing N -methylpyrrole rings of distamycin and netropsin with a variety of aromatic or heterocyclic rings, such as Hoechst 33258 (Figure 4.11) [49], can also bind to AT-rich region in the minor groove of DNA.

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Figure 4.9 Structures of distamycin, 2-ImN, 2-ImD, and ImPImP.

Figure 4.10 2:1 complexes of 2-ImN, 2-ImD, Diatamycin and ImPImP with mixed A, T and G, C sequences.

Antitumor antibiotics Chromomycin A3 and mithramycin (Figure 4.12) bind to the GC-rich region of an oligonucleotide in the minor groove with a stoichiometry of 2:1 in the presence of a divalent metal ion such as Mg2+ . The GC specificity is suggested to be associated with hydrogen bonding between the C8 OH group of the aglycon ring and the N3 atom (as acceptor) or NH2 group (as donor) of guanosine. Quinolones and fluoroquinolones are chemotherapeutic bactericidal drugs, eradicating bacteria by interfering with DNA replication. Sandstrom [50] studied

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Figure 4.11 Structure of Hoechst 33258.

Figure 4.12 Structures of Chromomycin A3 and mithramycin.

Figure 4.13 Structure of norfloxacin.

the interactions of norfloxacin (Figure 4.13) with different decamers and found that the planar two-ring system of norfloxacin is partially intercalated at the central 5 -CpG-3 steps in the minor groove. Combination of different interaction modes into one molecule is a simple and helpful way to design new DNA binder. Bailly synthesized a variety of hybrid drugs in which groove binders are connected covalently to intercalators via linkers combining two intercalations and groove-binding in one molecule (Figure 4.14). [51–53]. 4.1.3. Covalent Bonding

A number of antitumor drugs can covalently bond to DNA bases to inhibit DNA replication and transcription. These drugs are also called alkylating agents

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Figure 4.14 Structure of thianet-GA.

Figure 4.15 Structure of anthramycin.

because nucleotides are alkylated by the drug molecules on adduct formation. Here, we list some alkylation mechanisms including imine intermediate, cyclopropane opening, and reductive alkylating. Covalent bonding drugs containing metals such as cisplatin and its derivatives are reviewed by others [54–58] and not included here. Anthramycin (Figure 4.15) inhibits the DNA-dependent RNA and DNA polymerase reactions by binding to the DNA template. One drug molecule bond within the minor groove at each end of the DNA helix is covalently bound through its C11 position to the N2 amine of the guanine of the chain. The reaction is slow, and the suggested mechanism [59,60] involves the imine intermediate. The origin of anthramycin specificity for three successive purines arises not from specific hydrogen bonds but from the low twist angles adopted by purine–purine steps in a B-form DNA helix. The origin of anthramycin’s preference for adenines flanking the alkylated guanine arises from a netropsin-like fitting of the acrylamide tail into the minor groove. Kamal [61] synthesized a series of unsymmetrical molecules exhibiting significant DNA minor groove binding affinity (Figure 4.16). This study reveals the significance of noncovalent interactions in combination with covalent bonding aspects when two moieties of structural similarities are joined together. The

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Figure 4.16 Structures of pyrrolobenzodiazepine dimmers.

Figure 4.17 Structures of CC1065 and its adenine adduct.

largest increase in melting point suggests that n = 5 stabilizes the DNA adduct the most. Molecular modeling studies on the adducts of these drugs with the 15-mer DNA containing a central AGA as the preferred binding site indicate that further increase in the linker length decreases the stability. (+)CC-1065 (Figure 4.17) [62], a highly potent antitumor drug, displayed a remarkable curvature with hydrogen-bond donors and acceptors on the outer periphery of the curve and with a lipophilic surface on the inner periphery. The shape and charge density is peculiarly efficient for binding in the grooves of the DNA double helix. When reacted with DNA, the C4 atom forms a covalent bond (C–CH2 –N) with the N3 atom of adenine or guanine by opening the cyclopropane ring. Alkylation of guanine is much slower than that of adenine. Reynolds [63] had shown that the thermally induced DNA strand breakage by CC-1065 occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3 side. Park [64,65] proposed two possible mechanisms involving either the tautomerization of the G-C base pair to give the enol-G-imino-C tautomer (Figure 4.18, pathway a) or a concerted mechanism (Figure 4.18, pathway b). Because protonated cytosines are deaminated to uracils with a much higher rate than the unprotonated cytosines, a (+)-CC-1065-N3-guanine DNA adduct that results in

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Figure 4.18 Proposed mechanisms for the alkylation of N3 of guanine.

Figure 4.19 Structures of (+)-duocarmycin A (DA) and (+)-duocarmycin SA.

a cross-strand protonated cytosine may, with increased frequency, produce a C → U transition mutation on the opposite strand. This may contribute to the mutagenic and carcinogenic potential of drugs that alkylate N3 of guanine in DNA. Indeed, in a mutagenicity study with (+)-CC-1065, G:C to A:T transitions were observed as minor mutagenic events [66]. Similar to (+)-CC1065, (+)-duocarmycin A (DA) and (+)-duocarmycin SA(DSA) (Figure 4.19), contain a cyclopropane ring that alkylates the N3 atom of adenine or guanine in minor groove. Becausee duocarmycin A and its synthetic analogs produce higher levels of N3 guanine alkylation [67], these compounds may represent a higher mutagenic risk than (+)-CC-1065. Bizelesin [68], a bifunctional DNA cross-linking antineoplastic agent, consists of two open-ring homologs of the (+)-CC-1065 subunit connected by a

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Figure 4.20 Structure of bizelesin bonded to adenines.

Figure 4.21 Probable mechanism of alkylation to form mono- and bifunctional adducts by reduced mitomycin C.

rigid linker moiety (Figure 4.20). It binds to two adenine bases on opposite strands. Bizelesin’s size, rigidity, and cross-linking properties restrict the DNA adduct’s range of motion and freeze out DNA conformations adopted during the cross-linking process. Because of its capacity to induce stable Hoogsteen base pairing with only minimal distortion of base–base stacking, bizelesin affords an opportunity to explore this unusual DNA conformation. Mitomycin C (Figure 4.21) contains an aziridine ring, an indoloquinone ring, and a carbamate group. Unlike alkylating drugs discussed earlier, mitomycins are reductive alkylating agents. The reduction is carried out chemically or enzymatically. The ratio of mono- and bifunctional adducts depends on the DNA base sequence. It is known that the first alkylation step prefers guanine at d(CpG) sequence over other G-containing sequences. Tomasz proposed a probable mechanism of alkylation by Mitomycin C that is triggered by the reduction of the quinone ring [69,70]. Mitomycins do not have binding affinity to various natural and synthetic DNAs unless they are reductively activated. In contrast, 2,7- diaminomitosene (2,7DAM; Figure 4.22), a major end product of the reductive activation of MC, binds to the same series of DNAs [71]. Tomasz determined the structure of

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Figure 4.22 Structure of 2,7-diaminomitosene.

Figure 4.23 Structures of daunorubicin, doxorubicin and product of DOX with G.

its adduct to a 12-mer duplex [72]. The 2,7-DAM molecule is anchored in the major groove of DNA. Sequence selectivity of the alkylation by 2,7-DAM was observed. The selectivity correlated with the sequence specificity of the negative molecular electrostatic potential of the major groove, suggesting that the alkylation selectivity of 2,7-DAM is determined by sequence-specific variation of the reactivity of the DNA. Anthracycline antiobiotics such as daunorubicin (DAU) and doxorubicin (DOX; Figure 4.23), also become intercalating alkylators in the presence of formaldehyde (HCHO). A covalent bond is formed between the 3-NH2 (sugar) of the drug and 2-NH2 of G3 via the CH2 bridge. The covalent Adriamycin–DNA adduct formed under oxido-reductive (Fenton) conditions in Tris buffer is structurally equivalent to that resulting from direct reaction with formaldehyde, and covalent linkage of the drug to one of the DNA strands confers remarkable stability to the duplex [73]. Glyceraldehyde also engenders covalent Adriamycin-DNA complexes, providing a possible relevant biological context for in vivo adduct formation. Incorporation of an alkylating agent into intercalating drugs such as doxorubicin and daunorubicin may enhance their antitumor properties. One example is a natural antitumor antibiotic, hedamycin (Figure 4.24), where a bis-epoxide chain is attached to the chromophore. It forms a covalent bond between its C18 atom and the N7 atom of G. Another example is SN-07. An NMR study [74] on the complex of SN-07 (Figure 4.24), another natural anthracycline antibiotic, with

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Figure 4.24 Structures of hedamycin and SN07.

an unusual DNA triplex establishes the formation of a sequence-specific intercalation complex at a single site in the triplex through covalent adduct formation in the minor groove without disruption of the third strand in the major groove. 4.1.4. Strand-Breaking

The bleomycins (BLMs; Figure 4.25) are a class of glycopeptide antibiotics isolated from streptomyces as copper complexes [75]. The mechanism of cytotoxicity is thought to be related to the ability of BLMs to bind to and degrade duplex

Figure 4.25 Structure of Bleomycin A2.

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DNA in the presence of Fe2+ , O2 , and a reductant as required cofactors [76]. Under in vitro conditions, BLM causes both single-strand and double-strand breaks in DNA, preferentially at the 5 -GpC-3 and 5 -GpT-3 sequences. The ratio of these two cleavage processes is sequence dependent, with double-strand cleavage being relatively minor. A typical bleomycin, BLM-A2 consists of the DNA-binding domain and the metal-binding domain, which are connected by the linker. Coordination of O2 to the vacant axial site leads to the formation of “activated BLM,” which has been identified recently by electrospray mass spectrometry as a ferric hydroperoxide and is the last detectable intermediate prior to DNA strand scission [77]. This intermediate has a half-life of 2 min at 4◦ C. The coordination sphere around the Fe(III) center is a distorted square–planar pyramid formed by the five nitrogen atoms, and the sixth axial position is occupied by the OOH− ligand. This structure is supported by the x-ray crystal structure of a biosynthetic intermediate of BLM complexed to Cu(II) [78]. This short-lived intermediate produces a radical species that extracts the 4 hydrogen atom of the deoxyribose and results in DNA strand scission. Although BLMs can cleave both single- and double-stranded DNA, the less frequent double-stranded DNA cleavage by a single BLM molecule, however, is believed to be responsible for the observed cytotoxicity of BLM because ssDNA cleavage is probably repaired by the human counterparts of prokaryotic DNA repair enzymes in vivo. The subsequent partition ratio depends on the concentration of O2 and the sequence context. Vanderwall [79] carried out molecular modeling studies to account for the mechanism of the double-strand cleavage by BLM-Co(III)-OOH. Starting from the structure of the complex in which the terminal oxygen of the OOH group is close to the H4 of the first cleavage site, they were able to derive a similar structure that brings the OOH group close to the H4 of the second cleavage site on the complementary strand without completely dissociating the ligand from the DNA duplex. Several model compounds of bleomycins have been synthesized based on the two-domain structure model of bleomycins. Lown [80] prepared a series of haemin–acridines (Figure 4.26). Here, acridine acts as the anchor to bring haemin close to the DNA duplex via intercalationas, and haemin produces a radical species in the presence of oxygen and a reducing agent such as 3-mercaptoethanol. Hertzberg [81] synthesized (methidiumpropyl-EDTA)-Fe(II) (Figure 4.26), which causes single- and some double-strand cleavages of DNA in the presence of dithiothreitol and O2 . Methidium is the intercalator and the O2 adduct of Fe(II)(EDTA) is the source of a radical species responsible for cleavage reactions. Enediyne antibiotics such as calicheamicins, esperamycins, and neocarzinostatins can cleave double-strand DNA (Figure 4.27). The molecules contained an enediyne unit embedded within their complex architectural frameworks, and it was proposed (Figure 4.28) that these molecules delivered the enediyne portion within the minor groove of the target DNA and then initiated a series of reactions leading to cycloaromatization of the enediyne and 1,4-benzenoid diradical formation when activated by a reducing agent such as thiol. The

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Figure 4.26 Structures of haemin-acridines and (methidiumpropyl-EDTA)-Fe(II).

Figure 4.27 Structures of calicheamicin1I, esperamycin A1.

Figure 4.28 DNA-cleaving mechanism by calicheamicin.

highly reactive 1,4-benzenoid diradical would then be perfectly positioned to strip hydrogen atoms from the sugar phosphate backbone of adjacent strands of DNA, causing scission of the DNA double helix. These drugs are highly cytotoxic because of the double-stranded DNA cleavage, and their antitumor activities are reported to be more than thousands times stronger than that of

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Adriamycin (doxorubicin), for example. The reaction pathway and mechanism of enediyne antibiotics have been reviewed by Smith and Nicolaou [82]. Calicheamicinγ1I cleaves DNA at pyrimidine-rich sequences. As shown in the DNA-cleaving mechanism by calicheamicins [82] (Figure 4.28), a nucleophile attacks the central sulfur atom of the allylic trisulfide (SSSCH3 ), and forms a thiol that is subsequently added to the adjacent α, β-unsaturated ketone embedded within the framework of the aglycon intramolecularly. This reaction, converting a trigonal bridgehead position to a tetragonal center, causes a significant change in structural geometry, which imposes a great deal of strain on the 10-membered enediyne ring. This strain is completely relieved by the enediyne undergoing the cycloaromatization reaction, generating the highly reactive 1,4benzenoid diradical via Bergman cyclization. The biradical cleaves both strands of DNA simultaneously by abstracting hydrogen atoms from sugar–phosphate backbones. Neocarzinostatin (NCS; Figure 4.29), a natural antitumor antibiotic first reported by Ishida in 1965 [83], is a 1:1 noncovalently associated mixture of a highly labile chromophore (NCS chromophore) and a protein component (NCS apoprotein) that stabilizes the chromophore and acts as a transporter. The DNA-damaging activity of the free NCS chromophore results primarily in single-strand DNA cuts and proceeds via an oxygen-dependent reaction. Thiols and UV radiation greatly enhance the DNA-cleaving properties of the NCS chromophore. Reaction leading to DNA damage is initiated by stereo-specific nucleophilic attack on the enediyne ring by a thiol such as glutathione (glu), the 2,6-biradical species is produced. This biradical species cleave single-stranded DNA with base preference (T>A>C>G) and double-stranded DNA with sequence specificity such as AGCGCT and AGTACT, where the boldfaced bases indicate cleavage sites. The 3-D structure of the glutathione postactivated NCS chrom, namely NCSi-glu bonded to the heptamer duplex [84], showed the

Figure 4.29 Mechanism of Neocarzinostatin and structure of NCSi-gb.

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Figure 4.30 Complex of NCSi-gb with bulge-DNA.

orientation responsible for the double-strand cleavage of DNA duplex by the 2,6-biradical. The complex is also stabilized by the drug contacts with the DNA backbone and minor groove via van der Waals interactions. NCSi-gb, another stable postactivated mimic of NCS chrom, is formed by the general-base(gb)-catalyzed activation of NCS chrom in the absence of DNA under thiol-free conditions. In the complex of NCSi-gb binding with a nucleotide containing two bulged bases [85], the rigidly held NA and THI rings form a molecular wedge penetrating the binding pocket. The NMF moiety attached to the THI ring is located in the center of the major groove. The binding of NCSigb promotes the formation of a bulge binding pocket that was not found in the unbound DNA. Binding of NCSi-gb locks the conformation of the two bulged residues in the major groove with antiglycoside conformation, and the complex is bent by 45◦ around the bulged site (Figure 4.30). NCS-chrom is unique among the enediyne antibiotics in its ability to undergo two different mechanisms of activation to form two different DNA binding and cleaving species. One, NCSi-gb, involves the major groove [85], and the second, NCSi-glu, which is generated by glutathione-induced activation, involves the minor groove [86]. The ability of NCS chrom derivatives to recognize bulged base sites is important [87]. Latter, elucidation of the detailed binding characteristics of the synthetic spirocyclic enantiomers, mimicking the spirocyclic geometry of NCSi-gb, provides a rational basis for the design of stereochemically controlled drugs for bulge binding sites [88]. 4.1.5. Other DNA binding molecules

Bulge structure in nucleic acid has been shown to play a significant biological role in biological processes. Expansion of DNA repeat sequences is associated with many human genetic diseases. Triplet repeat expansion is believed to be caused by the unstable nature of triplet repeats sequence. Bulged DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. Based on the studies on interactions of spirocycle compounds on DNA slippage [89–93], two new binaphthol aminosugars were synthesized as DNA bulge binders to study the triplet repeat expansion due to the wedge-shaped structure of 1,10-bi-2-naphthol (Figure 4.31) [94]. They showed DNA bulge structure binding selectivity with moderate binding affinity. Considering the 3-D

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Figure 4.31 Structure of 1,10-bi-2-naphthol.

structural features of NCSi-gb and compound 2, it seems that the prerequisite for selective motif binding may be the steric similarity between small molecules that interact with DNA motif. G-rich DNA sequences have been identified, cloned, and characterized in the telomeres of many organisms. G-rich sequences can form rigid G-quartet structures first proposed to occur within telomeres. G-quartets arise from the association of four G-bases into a cyclic Hoogsteen bonding arrangement. Each G-base makes two H-bonds with its neighbor G-base. K+ prefers to induce a folded G-quartet structure, and Na+ preferentially forms a linear four-strand Gquartet structure. Telomerase has recently become a target for anticancer drug therapy. Several small molecules were developed using a rational drug design strategy to stabilize G-quartet formation within telomeres, thereby inhibiting the telomerase activity (see review 95,96).

4.2. RNA-BINDING DRUGS

Far from being a passive carrier of genetic code, RNA molecules are the only known molecules that possess the double property of being depository of genetic information, like DNA, and of displaying catalytic activities involved in a wide range of biological processes, like protein enzymes. The vast majority of cellular RNAs form higher-order structures with other cellular components to facilitate the exchange of biochemical information. Successful molecular recognition of RNA often precedes catalytic events that are essential to a wide range of cellular activities. Key events in bacterial and viral reproductive cycles that are mediated by RNA or RNA–protein interactions have therefore become the focus of intense investigation. In addition, RNA serves as the primary genome of most pathogenic viruses. Like proteins, RNAs achieve those biological functions by adopting intricate three-dimensional folds and architectures. As in protein sequences, RNA sequences contain signatures specific for three-dimensional motifs, which participate in recognition and binding. The primary sequence dictates the type of secondary structure formed, which in turn leads to formation of possible tertiary structure via interaction of preformed secondary structures. Formation of RNA secondary structure dominates the free energy of folding, as each base pair

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contributes 1–3 kcal/mol of free energy to the final fold. In regulatory pathways, RNA molecules exist in equilibria between transient structures differentially stabilized by effectors, such as proteins or cofactors. Here, the RNA binding small molecules are classified by the targets to which the small molecules bind. Nature has been taking advantage of RNA–small molecules interactions to regulate important biochemical process at the RNA level. The understanding of RNA recognition by small molecules is limited by the chemical diversity of natural RNA ligands. At the same time, most RNA binding small molecules have lower affinity and target specificity. Designing small molecules with desirable selectivity for their target is the most challenging for new RNA targeting drugs. 4.2.1. rRNA

Antibiotics that target the ribosome differ from compounds used in many other therapeutic areas, as their mechanism of action is dominated by interactions with RNA rather than with target proteins. The importance of RNA-binding small molecules as antibiotics is unquestioned. A large number of antibiotics that bind to ribosomal RNA, and therefore interfere with bacterial protein synthesis, are known, and ribosomal RNA is the only validated RNA target for which drugs are currently available. The high-resolution structures of the bacterial ribosomal subunits and those of their complexes with antibiotics have significantly advanced our understanding of small-molecule interactions with RNA [97]. We updated the list of antibiotics that have been determined in complexes with ribosomes by x-ray crystallography in Table 4.1. There are two hydrophobic crevices on the 50S ribosomal subunit to which many different classes of antibiotics bind. The first is the peptidyl transferase center, and it is the binding site of chloramphenicol, sparsomycin, Blasticidin S, puromycin, linezolid, anisomycin, and clindamycin (Figure 4.32) [98–102]. Basically, PTC binding antibiotics inhibit protein synthesis through hindering peptidyl transfer or tRNA substrate binding. Sparsomycin contacts a P-site-bound substrate with its pseudouracil substituent, but its linear portion interacts with the activesite hydrophobic crevice formed by A2451 and C2452 [98]. Blasticidin S, which is composed of a cytosine linked to a pyranose ring that has an amino acid-like appendage, is a partial mimetic of P-site-bound tRNA [98]. Because the pmethoxyphenyl group of anisomycin completely fills the hydrophobic crevice that normally accepts the amino acid side-chains of A-site bound aminoacyl-tRNAs, anisomycin should compete with them for access to the peptidyl transferase center, consistent with reports that anisomycin interferes with the binding of A-site substrates. The chloramphenicol-binding site in Haloarcula marismortui large ribosomal subunit, is different from the chloramphenicol-binding site observed in the large ribosomal subunit of Deinococcus radiodurans. The chloramphenicolbinding site with higher affinity is at the active-site hydrophobic crevice where chloramphenicol can inhibit peptide bond formation in bacteria at pharmaceutically relevant concentrations. Puromycin minics the 3 terminal of A-site tRNA as the peptidyl transfer accepter and causes the formation of the immature peptidyl product.

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TABLE 4.1 Antibiotics that have been determined in complexes with ribosomes Antibiotics Erythromycin A

PDB ID

3OFR 1YI2 1JZY Erythromycylamine 2O43 CEM-101 3ORB Methymycin 3FWO Carbomycin A 1K8A Josamycin 2O44 Macrolide 2O45 RU-69874 Tylosin 1K9M Rapamycin 1Z58 Spiramycin I 1KD1 Azithromycin 1YHQ, 1M1K 1NWY Telithromycin 1YIJ 1P9X Clarthromycin 1J5A Cethromycin 1NWX (ABT-773) Roxithromycin 1JZZ 13-Deoxytedanolide 2OTJ Paromomycin 2Z4K, 2Z4M 2Z4L, 2Z4N 2WDG, 2WDH, 2WDK, 2WDM 2VQE, 2VQF, 2UXD, 2UXB, 2UXC, 2UUC, 2UU9, 2UUA, 2UUB, 2J00, 2J02, 1XMO, 1XMQ, 1XNQ, 1XNR, 1N32, 1N33, 1IBK, 1IBL, Paromomycin, 1FJG Spectinomycin, Streptomycin Paromomycin, 2WRN, 2WRQ Kirromycin Neomycin, 2QOY, 2QP0 Spectinomycin Neomycin 2QOZ, 2QP1, 2QAM, 2QAO 2QAL, 2QAN Tiamulin 3G4S 1XBP Sparsomycin 1NJM, 1NJN 1VQ8, 1VQ9, 1M90

Subunit

species

50S 50S 50S 50S 50S 50S 50S 50S 50S

E. coli H. marismortui D. radiodurans D. radiodurans E. coli D. radiodurans H. marismortui D. radiodurans D. radiodurans

50S 50S 50S 50S 50S 50S 50S 50S 50S

H. marismortui D. radiodurans H. marismortui H. marismortui D. radiodurans H. marismortui D. radiodurans D. radiodurans D. radiodurans

50S 50S 30S 50S 30S

D. radiodurans H. marismortui E. coli E. coli T. thermophilus

30S

T. thermophilus

30S

T. thermophilus

30S

E. coli

50S 30S 50S 50S 50S 50S

E. coli E. coli H. marismortui D. radiodurans D. radiodurans H. marismortui (continued overleaf )

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TABLE 4.1 (Continued) Antibiotics Linezolid

PDB ID

3DLL 3CPW Hygromycin B 3DF1, 3DF3 1HNZ Troleandomycin 3I56 1OND Gentamicin 2QB9,2QBB,2QBH, 2QBJ 2QBA,2QBC,2QBI, 2QBK Kasugamycin 1 VS5, 1 VS7 2HHH Clindamycin 1YJN 1JZX Chloramphenicol 1NJI 1K01 SLD 3CXC Homoharringtonine 3G6E Bruceantin 3G71 Negamycin 2QEX Nosiheptide 2ZJP Thiostrepton 3CF5 Anisomycin 3CC4, 1K73 Spectinomycin 2QOU,2QOW pleuromutilin 2OGM derivative SB-571519 pleuromutilin 2OGN derivative SB-280080 Retapamulin 2OGO (SB-275833) Girodazole 2OTL Quinupristin 1YJW Quinupristin, 1SM1 Dalfopristin Puromycin 1Q7Y Puromycin-N1FFZ aminophosphonic acid Blasticidin S 1KC8 Virginiamycin M1 1N8R Virginiamycin M1, 1YIT Virginiamycin S1 Lankacidin 3JQ4 Mycalamide A 3I55

Subunit

species

50S 50S 30S 30S 50S 50S 30S 50S 30S 30S 50S 50S 50S 50S 50S 50S 50S 50S 50S 50S 50S 30S 50S

D. radiodurans H. marismortui E. coli T. thermophilus H. marismortui D. radiodurans E. coli E. coli E. coli T. thermophilus H. marismortui D. radiodurans H. marismortui D. radiodurans H. marismortui H. marismortui H. marismortui H. marismortui D. radiodurans D. radiodurans H. marismortui E. coli D. radiodurans

50S

D. radiodurans

50S

D. radiodurans

50S 50S 50S

H. marismortui H. marismortui D. radiodurans

50S 50S

H. marismortui H. marismortui

50S 50S 50S

H. marismortui H. marismortui H. marismortui

50S 50S

D. radiodurans H. marismortui

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TABLE 4.1 (Continued) Antibiotics Fusidic acid Edeine B Tetracycline Pactamycin Viomycin Capreomycin Kirromycin

PDB ID 2WRI, 2WRK 1I95 1I97, 1HNW 1HNX 3KNJ, 3KNH 3KNL, 3KNN 3FIC

Subunit 30S 30S 30S 30S 30S 30S 30S

species T. thermophilus T. thermophilus T. thermophilus T. thermophilus T. thermophilus T. thermophilus T. thermophilus

Figure 4.32 Structures of PTC binding antibiotics.

The other important hydrophobic crevice is near the entrance to the exit tunnel that the nascent polypeptide travels through and is the binding site of macrolides, ketolides, and streptogramin B (Figure 4.33) [98,101–104]. Streptogramin B antibiotics are cyclic depsipeptides that contain 6–7 amino acids. These antibiotics partially overlap the macrolide binding site near the entrance to the peptide exit tunnel in 50S. The location of macrolides, ketolides, and streptogramin B suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. To determine how growing peptides position themselves in the tunnel, Bogdanov generated peptide derivatives (aminooxyacetyl-l-ala-ninel-alanine methyl ester) of tylosin and desmycosin that were modified at the C6 position of the macrocyclic lactone ring, and these analogs inhibited cell-free transcription–translation [105]. The decoding site within bacterial 16S ribosomal RNA is the target for many natural aminoglycoside antibiotics (Figure 4.34), such as neomycin, gentamicin, paromomycin, and kanamycin. The binding of aminoglycoside antibiotics causes disruption of mRNA-decoding fidelity by displacing two adenine residues in

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Figure 4.33 Structures of exit tunnel entrance binding antibiotics.

Figure 4.34 Structures of aminoglycoside antibiotics.

helix 44 of the 16S rRNA and by stabilizing a conformation that normally occurs only when the correct mRNA–tRNA complex is formed [106–110]. The aminoglycosides serve as an excellent template for designing antibiotics because they bind with high affinity to the bacterial decoding site and have properties that allow broad-spectrum antibacterial activity. However, the natural products suffer from low bioavailability and toxicity because of their polycationic character. Over the past decades, numerous attempts have been made to develop improved aminoglycoside analogues. Russell [111] described a strategy in the structure-based design of novel antibiotics that bind to the bacterial A site. The two rings of neamine (a substructure of paromomycin), which itself lacks antibacterial activity, were used as the template in electronic search of other three-dimensional entities that would bind to the A site contiguously to the neamine structure. The best analog 3 (Figure 4.35) has an aminohydroxybutyryl group at N1 and an amino aliphatic substituent at O6 and shows a high degree of translation inhibition. X-ray crystal structures of the complex of structure 3 with a model RNA construct revealed that the amino-terminal group of the N1

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Figure 4.35 Structures of neamine and compound 3.

Figure 4.36 Struture of viomycin.

substituent is in contact with bases G1403, G1498 (via a water), and C1497 and that the hydroxyl group makes a water-mediated contact to U1495 [112]. Translocation, the coupled movement of mRNA and two tRNA in ribosome for one codon, involves not only the movement of tRNA and mRNA, but also the ratcheted rotation of the two subunits. In addition to the three main binding sites of antibiotics, the interface formed between 50S and 30S subunit is also potent for inhibition of protein synthesis because the interface is involved in both the assembly of initiation 70S translation complex and intersubunit rotation of the two subunits in translocation. Viomycin (Figure 4.36), a member of tuberactinomycin family of antibiotics, binds at the interface between helix44 of the small ribosomal subunit and helix69 of the large ribosomal subunit near the binding site for the paromomycin [113]. This class of antibiotics inhibit translocation by stabilizing the tRNA in the A site in the pretranslocation state. As shown in the designing of DNA binding small molecules, conjugation between groups with different functions may be a helpful method to improve binding activities or biological activities. Conjugates of chloramphenicol that had the dichloroacetyl group replaced either by nucleotide groups or by pyrene were generated by researchers in Denmark [114] (Figure 4.37). Molecular modeling

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Figure 4.37 Structures of nucleotide chloramphenicol conjugates and pyrene chloramphenicol conjugates.

Figure 4.38 Design of combination of sparsomycin and linezolid.

with chloramphenicol in the A-site wedge was consistent with additional binding to the P-loop residues G2484 and G2485 when there was nucleotide replacement for the dichloro groups. However, footprinting of the nucleotide conjugates to E. coli 70S did not support binding in the expected region. The pyrene conjugate (Figure 4.37) modeled to stack with nucleotides U2506, G2583 and U2584 appeared to bind as designed. This was shown by its protection of U2506 from modification in footprinting experiments. Another successful example is the combination of sparsomycin and linezolid (Figure 4.38) [115–118]. The program by Rib-X Pharmaceuticals began with a detailed x-ray structural knowledge of the provocative juxtaposition of the linezolid-binding site with that of the nonselective antibiotic sparsomycin within the 50S ribosomal subunit. Both compounds bind within the peptidyl transferase center of the ribosome. Combination of the two molecules leads to a serious of compounds with much higher activities. 4.2.2. Riboswitch

Riboswitches [119,120] are fundamentally different RNA drug targets, in that they have evolved as structured receptors for the purpose of binding low-molecular-weight ligands. As a consequence, riboswitches form ligandreceptor interfaces with a level of structural complexity and selectivity that

RNA-BINDING DRUGS

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Figure 4.39 Structures of FMN and roseoflavin.

approaches that of proteins. Researchers have reported 12 different classes of riboswitches, and members of each class bind to the same metabolite and share a highly conserved sequence and secondary structure. Therefore, riboswitches are another potential bacterial target with some proof of principle [121,122]. Roseoflavin [123] and pyrithiamine pyrophosphate [124] are examples of antibiotics that appear to act by disrupting cellular metabolism through riboswitch-RNA binding. Roseoflavin (Figure 4.39), an antibacterial riboflavin analog, inhibits the growth of several gram-positive bacterial species through a mechanism involving repression of riboflavin biosynthesis. In these species, all the genes involved in riboflavin biosynthesis are expressed in a single operon, regulated by a single FMN-binding riboswitch [125]. A reasonable prediction is that this riboswitch is the cellular target of roseoflavin. Serganov [123] reported the crystal structure of antibiotic roseoflavin with FMN riboswitch. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Pyrithiamine is an analog of thiamine that inhibits the growth of several bacterial and fungal species. Like thiamine, pyrithiamine is readily phosphorylated inside cells to pyrithiamine pyrophosphate (PTPP, Figure 4.40), which differs from TPP only in that the central thiazole ring is replaced by a pyridinium ring. Remarkably, PTPP binds to several TPP riboswitches in vitro with comparable affinity to TPP and represses the expression of a reporter gene fused to a TPP riboswitch inside bacteria. This suggests the possibility that, in its phosphorylated form, pyrithiamine inhibits bacterial or fungal growth by repressing one or more TPP riboswitch-regulated genes in these organisms. Wickiser [126] reported that adenine and 2-aminopurine(2AP) (Figure 4.41) have nearly equal binding affinity, whereas DAP binds with much higher affinity for adenine riboswitch. The association rate constant, however, favors adenine over DAP and 2AP 3- and 10-fold, respectively. Although in vitro studies discovered compound targeting on riboswitch can inhibit bacterial growth, whether these compounds could cure an infection in clinical is still a question. The potent toxicity of riboswitch-targeted antibacterials

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Figure 4.40 Structures of TPP and PTPP.

Figure 4.41 Structures of adenine, 2-aminopurine and DAP.

in humans is a major concern in drug design. It is possible that compounds that target riboswitches in pathogenic bacteria might cause similar effects in human hosts if the host also carries that riboswitch. Perhaps, there are proteins that could be inhibited by a compound that resembles the natural metabolite in mammalian cells. As well, the evolution of bacterial resistance must be taken into consideration when developing any antibacterial drug. 4.2.3. Viral RNA

Regulatory elements located in untranslated regions of viral mRNA often form folded structures that could control gene expression and were among the first structures that could be recognized as targets for small molecule therapeutics. Some of these are signals for binding by viral proteins, such as the HIV transactivation response element (TAR) RNA and the rev response element (RRE). TAR [127–133] and RRE RNA have been the target of efforts to develop small molecules that could disrupt protein binding. The aminoglyciside neomycin B has been shown to bind to the RRE RNA and inhibit the interaction with Rev as well as HIV replication in cells [134]. Tor [135] synthesized and tested a wide range of aminoglycoside derivatives, including dimmers and intercalator conjugates 4 (Figure 4.42), and found that it had an RRE RNA affinity in the submicromolar range and a selectivity of 2000 fold for the RRE RNA versus nonspecific nucleic acids. Guanidinoglycosides obtained by modification of aminoglycosides by guanidinylation of the amino groups have increased specificity, binding affinity, and inhibition activity of HIV replication compared to parental aminoglycosides. Mei [127] described three small molecule inhibitors of the HIV-1 Tat-TAR interaction that target the RNA (Figure 4.43). Each of the three Tat-TAR inhibitors recognizes a different structural feature at the bulge, lower stem, or loop region

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Figure 4.42 Structures of compound 4 and guanidinoglycosides.

Figure 4.43 Structures of compound 5, 6 and 7.

of TAR. Compound 7 has been demonstrated, in cellular environments, to inhibit the TAR-dependent, Tat-activated transcription and the replication of HIV-1 in a latently infectious model. Parolin [128] reported that a 6-aminoquinolone derivative, compound 8 (Figure 4.44), previously shown to exhibit potent activity against replication of HIV-1 in de novo-infected human lymphoblastoid cells, was found to efficiently bind to TAR RNA, with a dissociation constant in the nanomolar range (19 ± 0.6 nM).

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Figure 4.44 Structure of compound 8.

Figure 4.45 Structure of compound 9.

Murchie [129] used NMR and computational methods to model the interaction of a series of synthetic inhibitors of the in vi tro RNA binding activities of a peptide derived from the Tat and found that a series of compounds containing a bi-aryl heterocycle as one of the three substituents on a benzylic scaffold (such as compound 9 in Figure 4.45), induce a novel, inactive TAR conformation by stacking between base pairs at the site of a three-base bulge within TAR. Davidson [132] discovered conformationally constrained cyclic peptide mimetics of Tat that are specific nM inhibitors of the Tat–TAR interaction by using a structure-based approach. The NMR structure of a peptide-RNA complex reveals that these molecules interfere with the recruitment of TAR to Tat by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket. This structure illustrates additional principles in RNA recognition: RNAbinding molecules can achieve specificity by interacting simultaneously with multiple secondary structure elements and by inducing the formation of deep binding pockets in their targets. Tylophorine B (Figure 4.46) was reported with remarkable biological activity to tobacco mosaic virus and can also bind the bulged hairpin oligonucleotide with high affinity [136]. Xi [137] has reported its high affinity for TMV RNA and assembly origin of TMV RNA (oriRNA). It is speculated that tylophorine B likely exerts its virus inhibition by binding to oriRNA and interfering with virus assembly initiation. This work may shed light on the possible molecular inhibition mechanism against TMV by tylophorine B and provide clues in rational design of sequence-specific RNA-binding antivirus drugs.

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Figure 4.46 Stucture of tylophorine B.

Figure 4.47 Structures of enoxacin and two miR-21 inhibitor.

4.2.4. RNAi and miRNA pathway

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CHAPTER 5

G-QUADRUPLEX DNA AND ITS LIGANDS IN ANTICANCER THERAPY ZHI-SHU HUANG, JIA-HENG TAN, TIAN-MIAO OU, DING LI, and LIAN-QUAN GU School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China

The DNA double helix is an important molecular target for many anticancer drugs. The alkylating agents and topoisomerase poisons including cisplatin, doxorubicin, and etoposide, have been used for many years and are still widely used in anticancer therapy. However, these agents have strong side effects to the patients, mainly because of their nonspecific interactions with duplex DNA [1]. Therefore, a new generation of agents that target DNA secondary structures, the G-quadruplexes, are expected to be much more specific and effective [2–4]. Extensive studies have been made by scientists over the past two decades in this area [5–7], and it is time to look back and summarize the results for the evolution of these G-quadruplex ligands.

5.1. G-QUADRUPLEX STRUCTURES

G-quadruplex DNA refers to a series of four-stranded structures formed by the guanine-rich DNA sequences in physiological ions solution. The formation of these structures was first proposed by Davies et al . in 1962, only a few years after Watson and Crick had proposed the double-helix DNA in 1953 [8]. With the rapid development of X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR), and other powerful new technologies, many G-quadruplex structures have been resolved. The G-quartet surface, connecting loop, groove region, and ion channel are four important structural components of G-quadruplexes [9,10] in drug binding studies.


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As shown in Table 5.1, potential G-quadruplex forming sequences have been summarized by Burge et al ., indicating that G-quadruplex structures can be constructed by a single guanine-rich sequence intramolecularly or by the intermolecular association of two (dimeric) or four (tetrameric) separate strands [9,11]. The building blocks of G-quadruplexes are the G-quartets (or G-tetrads), which are derived from the association of four guanines into a cyclic Hoogsteen hydrogenbonding arrangement with two hydrogen bonds between two adjacent guanine bases. G-quartets stack up one on top of another to form the G-quadruplex DNA structures (Figure 5.1) [12–14]. These structures show extensive structural polymorphism compared to duplex DNA [15–18]. Besides the strand stoichiometry, polymorphism arises from the relative arrangement of strand directivity in various ways, all parallel, adjacent parallel, alternating parallel, or three parallel and one antiparallel, resulting in different conformations named as propeller, basket, chair, and hybrid-type G-quadruplexes, respectively (Figure 5.2) [19,20]. Most of the tetrameric G-quadruplexes form the parallel structures, whereas dimeric and intramolecular G-quadruplexes can form both parallel and antiparallel structures (G-quadruplex structures are defined as antiparallel when at least one of the four strands is antiparallel to the others). Meanwhile, such variations in strand directivity affect their appropriate location of the linkers (or loops) between G-rich segments. Accordingly, as shown in Figure 5.2, parallel G-strands need a connecting loop to link the bottom G-quartet with the top G-quartet, leading to propeller-type (double-chain-reversal) loops. Antiparallel G-strands can be linked either by lateral or diagonal (edgewise) loops, depending on whether the strands are adjacent or diagonally opposed [21,22]. The sequence and size of loops usually TABLE 5.1 Guanine-rich sequences possibly form G-quadruplexes Sequence Intramolecular G-quadruplex

Gm Xn Gm Xo Gm Xp Gm

Bimolecular G-quadruplex

Two Xn Gm Xo Gm Xp

Tetramolecular G-quadruplex

Four Xn Gm Xo

Description m represents the number of G residues in each short G-tract. Xn , Xo , and Xp can be any combination of residues, including G, forming the loops. Xn and Xp are any nonguanine nucleotide of length n and p, respectively, Gm is any number of guanines involved in tetrad formation of length m, and Xo is any nucleotide of length o involved in loop formation. Xn and Xo are any nucleotide of length n and o, respectively, and Gm is any number of guanines involved in G-quartet formation of length m.

208


Figure 5.1 Basic structures of duplex DNA and G-quadruplex DNA.

have a dominant role in determining the G-quadruplex topology. The loop residues can form stacking and hydrogen-bonding interactions themselves and further stabilize particular G-quadruplex folds [23–27]. Furthermore, there are four grooves in G-quadruplex structures, defined as the cavities bounded by the guanine phosphodiester backbones [28,29]. The variations in strand directivity also affect the guanine glycosidic torsion angles (Figure 5.3) [30]. Changes in this glycosidic angle will further alter the relative position of the sugar ribose and the groove dimensions. For parallel Gquadruplexes, all the guanine glycosidic torsion angles are characteristic of an equivalent anti conformation, so the four grooves dimensions are also equivalent. When one of the strand orientations runs antiparallel arrangement, guanine glycosidic torsion angles are changed and found to adopt both syn and anti conformations. These changes will further alter the groove dimensions, generating narrow, medium, and wide grooves, as shown in Figure 5.3 [31]. Moreover, G-quadruplexes are characterized by coordination of metal ions, usually K+ and Na+ [32–36]. The hole among G-quartets is suitable for coordinating the right size of cations because the two planes of G-quartets are linked by eight carboxyl oxygen atoms, which have strong negative electrostatic

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Figure 5.2 Topology and loop structure of G-quadruplex DNA.

Figure 5.3 Glycosidic bond and variation in groove width in the G-quadruplexes.

potential to create a channel in the middle of stacking G-quartet (Figure 5.4) [37–41]. Variation of G-quadruplex structure from the same sequence can occur because of different metal ion coordination in a G-quartet. For example, the human telomeric DNA sequence, d[AGGG(TTAGGG)3 ], forms the antiparallel basket-type topology in Na+ solution [42]. On the other hand, the potassium

210


Figure 5.4 Metal ion in the central ion channel.

complex with this sequence has variable and complicated structures, including antiparallel basket-type or chair-type structure [43–45], parallel propeller-type crystal structure [38], and mixed hybrid-type structures [46–49].

5.2. BIOLOGICAL FUNCTION OF G-QUADRUPLEX

As we mentioned earlier, G-quadruplex DNA is a kind of particular DNA secondary structure. Besides the in vitro structural information, bioinformatics studies showed that human genes contained as many as 376,000 potential G-quadruplex forming sequences that could be found in telomeric ends, immunoglobulin switch regions, recombination hot spots, gene regulatory elements, and so on [50–52]. Potential G-quadruplex forming sequences are unevenly distributed in the human genome with a bias toward seven types of regulatory elements, including TSS-proximal regions, nuclease hypersensitive sites, conserved noncoding regulatory sequences, CpG islands, enhancers, insulators, and conserved transcription factor-binding sites. This phenomenon may be progressively favored by natural selection during the gene evolution [53]. However, for the fields of G-quadruplexes studies, what researchers most are concerned about are whether G-quadruplex structures exist in vivo and what their biological functions are. Recent in vivo studies of the potential G-quadruplex DNA have provided evidence that G-rich sequences introduced special properties on DNA [54]. For example, use of single-chain antibodies specific for G-quadruplexes has led to identification that telomeres that end in the macronuclei of Stylonychia lemnae contain G-quadruplex structures [55]. The formation and resolution of this Gquadruplex would further participate in DNA replication and are tightly regulated by the telomerase and telomere end-binding proteins, TEBPα and TEBPβ [56]. Regarding the studies about transcription [57,58], the clearest evidence for a role of G-quadruplex structure in transcriptional regulation has been obtained from the studies of oncogene c-myc [59,60]. The c-myc promoter contains a nuclease hypersensitive element (NHE III1 ) that controls 85–90% of transcriptional activation [61,62]. This element consists of G-rich sequence

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and is proposed to form G-quadruplex structures in vitro [63–65]. It has been observed that gene expression was altered by both a single-base substitution that disrupted the G-quadruplex structure within the c-myc promoter and addition of the G-quadruplex ligands TMPyP4 and SYUIQ-5 to two contrastive cell lines, which provided direct evidence that G-quadruplex structures formed in vivo and controlled transcription [66,67]. Furthermore, recent studies on the role of DNA supercoiling in transcriptional regulation reinforced this in vivo evidence, showing that supercoiling induced by transcription had a dynamic effect on c-myc DNA: it converted duplex DNA to G-quadruplex structures, even at considerable distances from the transcriptional starting site [68]. This information, together with the well-characterized biophysical properties of G-quadruplexes and a number of newly found G-quadruplex structures and their specific proteins [69–71], is consistent with the interpretation that Gquadruplexes exist in vivo and that these extensive structures participate in the regulation of telomere structure, replication, and transcription. Therefore, some of these G-quadruplex structures, including human telomeric, c-myc, and ribosomal DNA, are recognized as promising targets for the design of anticancer drugs. 5.2.1. Telomere DNA

Telomeres are noncoding DNAs located at the termini of chromosomes and can form protective structures at these regions, preventing them from being degraded or fused through DNA repair mechanisms (Figure 5.5) [72]. They range in size from 3 to 15 kb and are composed of tandem repeats of the sequence d(TTAGGG) with a 3 overhang of the G-rich strand, which plays an important structural and functional role [73–76]. Telomere length decreases with each cell division event, and this stringent control of telomere length is important for cell cycle control [77]. Reversion of this degradation through a telomere maintenance mechanism increases cellular replicative capacity, resulting in cellular immortalization, and tumorigenesis [78]. A telomere maintenance mechanism is mainly provided by a specialized enzyme called telomerase, which functions as a reverse transcriptase to add multiple copies of the d(TTAGGG) to the end of the G-rich strand of the telomere [79,80]. Normal human somatic cells exhibit very weak telomerase activity, which is insufficient to maintain a constant telomere length, whereas 80–85% of human tumor cell populations have high telomerase activity for the maintenance of telomere lengths [81–84]. The folding and stabilization of G-quadruplexes in G-rich sequences at the ends of telomeres may influence the protein-DNA recognition and hence regulate telomere length as well as telomere function in the cell cycle [85]. The effects of binding and stabilization of these structures by small ligands have been extensively studied over the past decade [86,87]. For example, many small ligands capable of stabilizing telomeric G-quadruplex are effective in telomerase inhibition because optimal telomerase activity requires an unfolded single strand as substrate (Figure 5.5) [88,89]. Other reported biological effects of ligands

212


Figure 5.5 Structure and biological roles of telomeres and inhibition on telomerase by G-quadruplex ligands.

binding to G-quadruplexes include the shortening of telomere length, induction of senescence, and inhibition of cell growth [90–93]. Moreover, end-to-end fusions of chromosomes have been observed with telomeres in the presence of G-quadruplex ligands [94–96]. Recently, several studies have also shown that some telomeric G-quadruplex ligands inhibit and displace the binding of proteins POT1 (protection of telomeres 1) and TRF2 (telomeric repeat binding factor 2) to the telomeres and hence generate a telomere dysfunction, which results in the activation of DNA damage response and selective inhibition of cell growth [97–100]. As a result, induction and stabilization of the telomeric G-quadruplexes by small molecules have been shown to interfere with telomere biological functions, inhibit telomerase activity, and eventually alter telomere maintenance. Telomere maintenance is crucial for the unlimited proliferative potential of cancer cells; thus the design of drugs targeting the telomeric G-quadruplex is a rational and promising approach for cancer chemotherapy. 5.2.2. Oncogene c-myc DNA

Oncogene c-myc belongs to the Myc gene family and is one of the first identified oncogenes, which is subsequently linked to a wide range of human cancers.

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c-myc functions as a gene-specific transcription factor through its protein product, c-Myc, which is believed to regulate 10–15% of all cellular genes and be involved in cell cycle regulation, apoptosis, metabolism, cellular differentiation, and cell adhesion [101]. Thus, the aberrant overexpression of c-myc is associated with a variety of malignant tumors, including those of colon, breast, small-cell lung, cervix, osteosarcomas, glioblastomas, and myeloid leukemia [102]. The NHE III1 of the c-myc promoter contains a 27-base-pair G-rich strand (5 TGGGGAGGGTGGGGAGGGTGGGGAAGG-3) that has been shown to have the ability to form multiple parallel G-quadruplex structures under conditions of transcriptionally induced superhelicity and may displace transcription factors that induce c-myc expression such as Sp1, CNBP, or hnRNP K, which are known to bind to and modulate the conformation of the c-myc NHE III1 (Figure 5.6) [63,103–105]. Accordingly, c-myc G-quadruplex could function as a silencing element, whose formation can be stabilized by specific G-quadruplex ligands that could potentially be used to specifically repress c-myc expression [66,67]. This on–off switching mechanism may be an effective approach to target cancers that overexpress c-Myc [59,60]. More recently, other promoter G-quadruplex DNA, including c-kit, bcl -2, KRAS and hTERT , were also reported to serve as a silencing element that is analogous to c-myc gene [106–111]. These results have provided the best independent support that promoter G-quadruplexes may be a new and broad therapeutic approach to control gene expression.

Figure 5.6 Structure and biological roles of c-myc. The overexpression of c-myc has been found in a variety of tumors, as its product, the c-Myc protein, is a transcriptional regulator, the target genes of which are involved in cell cycle regulation, apoptosis, metabolism, cell adhesion, and cellular differentiation. The interactions of G-quadruplex DNA in c-myc with ligand may displace transcription factors from NHE III1 and silence the transcription.

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5.2.3. Ribosomal DNA

Human ribosomal DNA (rDNA) is G-rich sequence and contains >400 copies of the rRNA genes, organized in tandem arrays on five different human chromosomes. This G-richness is restricted to the nontemplate strand, and most guanines are within runs that contain three or more consecutive guanines. Hence, this sequence has the potential to form G-quadruplex structures [112,113]. Once these quadruplexes are formed, they can prevent the renaturation of the template DNA, thereby promoting the dense spacing of RNA polymerase I (Pol I) molecules on rRNA genes, which is characteristic of rapid transcribing rDNA (Figure 5.7) [114]. On the other hand, nucleolin, an abundant eukaryotic protein, is associated with rDNA in vivo and is absolutely required for rRNA synthesis, as its knockdown was shown to specifically inhibit Pol I–driven transcription [115–117]. Nucleolin binds tightly to rDNA G-quadruplex structures and thus may further stabilize them to increase the efficiency of Pol I transcription [113]. As the role of excessive rRNA synthesis in tumorigenesis comes to be better understood [118,119], the therapeutic potential of drugs to inhibit this process becomes more evident. Besides, previous studies have showed that treatment of

Figure 5.7 Mechanism of the RNA Polymerase I driven rDNA transcription. G-quadruplex ligand could disrupt the formation of complexes between nucleolin and rDNA G-quadruplexes, and thus inhibiting aberrant RNA Polymerase I transcription in cancer cells.

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yeast with a G-quadruplex ligand (porphyrin) selectively down-regulates rRNA expression, indicating the formation of G-quadruplexes in rDNA in vivo, as well as their potential role in rRNA biogenesis [120]. Thus, targeting nucleolin/rDNA G-quadruplex complexes using specific quadruplex ligands to inhibit aberrant Pol I transcription in cancer cells represents a novel approach to selectively disrupt proliferation of cancer cells (Figure 5.7).

5.3. LIGAND-QUADRUPLEX COMPLEX

G-quadruplex structures represent a new type of molecular targets for selective DNA-interactive compounds in view of the abnormal mechanism of telomere maintenance, overexpression of oncogenes, and excessive rRNA synthesis in most cancer cells but not in normal cells. The design of drugs targeting at the telomere, promoter or rDNA G-quadruplexes is a rational and promising approach for cancer chemotherapy. Therefore, further efforts in drug design are required, especially for controlling the selectivity of ligands to G-quadruplex over duplex DNA because ligand interaction with duplex DNA leads to acute toxic and intolerable side effects on normal tissues [1]. With an increasing number of G-quadruplexes identified in the genome, whether ligand design should consider the selectivity among different types of G-quadruplex species is also required for clarification. Nevertheless, in search for G-quadruplex binding ligands, it is important to understand their binding sites and binding mechanisms with the G-quadruplex. According to crystallographic and NMR studies, the G-quadruplex ligands usually stack over the terminal G-quartets on either end of the G-quadruplex structure. For example, in the crystal complexes of acridine derivatives and G-quadruplexes, the acridine chromophore groups in the ligands stacked directly on the terminal G-quartet [121]. Besides these π-stacking interactions, other segments including nitrogen atom of the acridine ring, side-chain amide nitrogen and hydrogen atoms, and the terminal amine nitrogen atoms would further participate in the water-mediated hydrogen-bonding interactions with loop and groove regions [122,123]. Similarly, the crystal structure of a parallel G-quadruplex and daunomycin complex showed three daunomycin molecules stacking on the 5 -end of the G-quartet, with their amine sugar moieties forming hydrogen-bonding and/or van der Waals interactions with the quadruplex grooves [124]. Moreover, the interactions of fluorinated polycyclic acridinium salt with the G-quadruplex were maintained by stacking two ligand molecules on the G-quartet and also by placing the positively charged ligand above and below the ion channel at the center of G-quartet [125]. In addition, the NMR structure of the complex formed between c-myc G-quadruplex and TMPyP4 showed the π-stacking interactions of one porphyrin molecule onto the external G-quartet. Meanwhile, the positive charges of TMPyP4 were in close contact with several negatively charged phosphates backbone, suggesting the contribution of electrostatic interactions to the stability of complex [126].

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Other than the end-stacking binding mode, several molecules, including TMPyP4 and naphthalene diimide compound, could also stack onto the loop nucleotides [127,128]. More interestingly, distamycin, a noncoplanar DNA duplex minor groove binder, could stack on the two ends of a G-quartet when binding to the tetrameric G-quadruplex d(TAGGGTTA) [129]. However, when the DNA sequence changes to d(TGGGGT), distamycin molecules were found to form dimers and bind simultaneously to two opposite grooves of the G-quadruplex [130]. Removing the positively charged terminal group of distamycin resulted in an unprecedented ligand binding position in which both the groove and the 3 end of the DNA were occupied [131]. It is clear that G-quartet is the basic moiety for ligand-quadruplex interactions, which has a square aromatic surface of G-quartet much larger than that of the Watson-Crick base pairs. The strong and selective binding of the ligand molecule to the G-quadruplex may be attributed to its larger ring system, which allows the molecule to occupy the whole area of the G-quartet. Other binding effects, such as hydrogen bonding, van der Waals and electrostatic interactions with the loop, groove, ion channel, and sugar-phosphate backbone are also important for the enhancement of binding potency and selectivity of ligands toward Gquadruplexes because these binding sites not only differ from duplex DNA but also vary from different G-quadruplex species [132].

5.4. SMALL MOLECULES BINDING TO G-QUADRUPLEX STRUCTURES

Benefiting from a series of biophysical and biological studies, G-quadruplexes have emerged as a significant anticancer drug target. Many groups have searched for small molecules that can bind to G-quadruplexes and hence interfere with telomere maintenance, alter G-quadruplex-related gene expression or inhibit rRNA biogenesis. Although there is still a long way to go in the development of clinical drugs that target G-quadruplexes, some promising small molecules have been discovered through screening and rational structure-based design. In general, most of these compounds can be divided into three categories: fused aromatic system, macrocyclic system, and unfused aromatic system. 5.4.1. Fused Aromatic System 5.4.1.1. Substituted Anthraquinones, Acridines, Isoalloxazines, and Related Analogues Disubstituted anthraquinones (1, Figure 5.8) are one of the earliest and most widely characterized quadruplex-interactive ligands [85]. SAR studies including cytotoxicity, binding potency, and telomerase inhibition activity have been performed for a wide range of diamidoanthraquinones, including 1,4-, 1,5-, 1,8-, 2,6-, and 2,7-isomers with terminal substituent of side chain varying from conventional amine group to special amino acid [133–137]. Some of these compounds have previously been synthesized as cytotoxic

SMALL MOLECULES BINDING TO G-QUADRUPLEX STRUCTURES

217

O 8

1

7

2

R

R 3

6 5

4

O Disubstituted Anthraquinones 1

R = NH-CO-(CH2)n-amine

n = 1,2,3

or CO-NH-(CH2)n-amine or NH-CO-(CH2)n-NH-CO-aminoacid-NH2 or CO-NH-(CH2)n-NH-CO-aminoacid-NH2

Figure 5.8 Structures of disubstituted anthraquinones.

agents with affinity for duplex and triplex DNA. Previous studies have found a significant correlation between in vitro cytotoxic potency and duplex-binding affinity of the diamidoanthraquinones, but no evidence for a significant correlation between telomeric G-quadruplex binding and telomerase inhibition activity [138]. Molecular modeling studies suggested that these compounds bind by a “threading intercalation mode” to telomeric G-quadruplex structures with two terminal amine side chains protruding into grooves, analogous to their behavior with duplex DNA [139]. Interestingly, compounds with AQ-NH-COarrangement exhibited invariantly better stabilization and telomerase inhibition activities than those with AQ-CO-NH- arrangement. Theoretical calculations suggested that planarity of the former compounds are much stronger than the latter series, showing π-stacking interactions contribute positively to these activities [137,140]. Although two amido groups attached to the anthraquinone ˚ it tricyclic system increase the effective length of the system from 7.5 to 12 A, is still not sufficient for the G-quartet. Modification of side-chain terminal group from amine to amino acid improved the ligand’s selectivity toward G-quadruplex DNA, due to the enhanced interactions of side chain and quadruplex groove. Nevertheless, these compounds only showed moderate selectivity [137]. As shown in Figure 5.9, in further searching tricyclic aromatic molecules for G-quadruplex DNA, a series of disubstituted acridines (2) and acridones (3) has been synthesized by the introduction of a potential positive charge in the center of the chromophore to complement the channel of negative electrostatic potential that runs through the center of a quadruplex [141–144]. However, this approach led to faint improvement of the binding ability, selectivity, and telomerase inhibition in comparison with disubstituted anthraquinones, due to the weak alkalinity of the nitrogen atom in the central ring. The incorporation of a third substituent, an anilino group, into the acridine chromophore at the 9-position, which fits into a third groove in this model, enhanced the alkalinity of the nitrogen atom in the central ring [145]. According to SPR (surface plasmon resonance) results, the disubstituted acridines had the similar binding affinities with duplex and quadruplex, whereas the 3,6,9trisubstituted compounds (4–13, Figure 5.9) had much stronger binding affinity to telomeric G-quadruplex DNA (with 10–70 times larger binding constants) than to duplex DNA [146,147]. However, the further elongation of 3- and 6-side chains

218

G-QUADRUPLEX DNA AND ITS LIGANDS IN ANTICANCER THERAPY 9

8

R

1 2

7 6

3

N

5

R = 2,6-disubstituted NH-CO-(CH2)2-amine

R

or 3,6-disubstituted NH-CO-(CH2)2-amine or 4,5-disubstituted CH2-NH-CO-(CH2)n-amine n = 1, 2, 3 or 4,5-disubstituted CH2-NH-(CH2)n-amine n = 2, 3

4

Disubstituted acridines 2 O

8

R

1 2

7 6

3

N H

5

R = 2,6-disubstituted NH-CO-(CH2)2-amine

R

or 2,7-disubstituted NH-CO-(CH2)2-amine or 3,6-disubstituted NH-CO-(CH2)2-amine

4

Disubstituted acridones 3 R 8

N

HN

9

1

7 6

O

O R = N(CH3)2

2 3

O N n

N N N 5 4 n H H Trisubstituted acridines

4

n=2

8

n=2 x=1

5

n=3

9

6

n=4

10

7

n=5

11 12

n=2 x=2 n=2 x=3 n=3 x=2 n=4 x=2

13

n=5 x=2 H N

R 9

8

O N

4 2 3

5

N

H N

1

7 6

N H

N x

R = HN

4

O N H

N

14

R = HN

15

R = HN

O

N 6

O

F

Trisubstituted acridines F

Figure 5.9 Structures of substituted acridines and acridones.

significantly decreased the quadruplex affinity for both groups of compounds (4–7 and 8–13). Among all the trisubstituted acridines, the 3,6-hexanamido derivative 13 displayed the highest quadruplex selectivity. This suggests that the addition of steric hindrance of these ligands at the 3- and 6-positions is unfavorable for quadruplex or duplex binding. Conversely, 8 and 10 exhibited a stronger G-quadruplex binding affinity but a lower selectivity due to a concomitant increase in duplex binding. A balance between G-quadruplex binding affinity and selectivity against duplex binding should be taken into account. Systematic SAR investigation for a number of trisubstituted acridines demonstrated that the 3,6,9-isomers exhibited much better telomerase inhibition activities than 2,6,9- and 2,7,9-isomers. Compound 14 with extended anilino side chain or compound 15 in Figure 5.9 with benzylamino group on 9-position resulted in enhanced telomeric G-quadruplex interaction and superior telomerase


219

inhibitory activity [148]. Among this molecular library, compound 4, also termed as BRACO19, was chosen for a series of in vivo assays because of its potent telomeric G-quadruplex binding affinity (K = 3.1 × 107 M−1 ), high selectivity (31-fold against duplex DNA), and excellent telomerase inhibition activity (tel EC50 = 113 nM) [147]. These studies showed that BRACO19 did not cause nonspecific acute cytotoxicity at similar concentrations to those required to completely inhibit telomerase activity, and there existed a 90-fold difference [149]. Besides, BRACO19 bound to telomeric single-stranded overhang DNA and produced short- and long-term growth arrest in cancer cell lines, but these effects were significantly less potent in a normal cell line [150]. Interestingly, subtoxic concentrations of BRACO19 triggered growth arrest in tumor cells after just 15 days of exposure, before any detectable telomere shortening, showing that this response could not be explained solely by telomerase inhibition [149]. Another observation that BRACO19 caused chromosome end-to-end fusion in combination with the appearance of p16associated senescence led to the proposal that this ligand primarily acted to disrupt the telomere structure [151]. Further in vitro and in vivo data proved this proposal that the anticancer effect of BRACO19 was caused by inhibiting the capping of 3 telomere ends, due to its ability of displacing the human single-stranded protein POT1 from the G-overhang [150]. Thus, we can envisage telomere targeting, in conjunction with telomerase inhibition, may be more practical for the treatment of cancer [89]. Moreover, BRACO19 not only had antitumor activity as a single agent in xenografts model [149,152,153], but also had increased antitumor effect as combination drugs with clinical paclitaxel and flavopiridol [149,154]. Unfortunately, BRACO19 had very poor cell permeability, and recent studies also showed that it might face stability problems during the preparation of dosage forms, their storage, and after application. Further applications will require either chemical modifications or a suitable formulation and delivery strategy to improve BRACO19’s biopharmaceutical properties and reduce stability problems [155,156]. Another example for tricyclic aromatic G-quadruplex ligand is the isoalloxazine derivatives. The design of isoalloxazines as potential G-quadruplex ligands was originated from the finding that oxidized riboflavin binds to an intramolecular G-quadruplex with moderate binding affinity (Kd ) of 1–5 μM [157]. Similar to the design of trisubstituted acridines, three amine side chains were introduced to the isoalloxazine scaffold (16–21, Figure 5.10) for potential interactions with quadruplex grooves, loops, and the negatively charged sugar-phosphate backbone [158]. None of these ligands showed significant binding to the duplex DNA in SPR analysis or any stabilization of duplex DNA in FRET (fluorescence resonance energy transfer) assay. The most remarkable was that compound 16 showed 14-fold G-quadruplex selectivity for c-kit over human telomeric DNA because discrimination between two different G-quadruplexes by a small molecule is a greater challenge than that between quadruplex and duplex. Although structural information of the ligand interactions is still limited, the results have shown that small coplanar tricyclic system with druglike scaffolds had the potential

220


R2

H N

R1

N

N

O N

N

R1

O Trisubstituted isoalloxazines

R1

R2

16

N(CH3)2

N(CH3)2

17

N

N(CH3)2

18

N

19

N(CH3)2

F

20

N

N

21

N

OMe

N(CH3)2

O

Figure 5.10 Structures of isoalloxazines.

O N H

n R R1

O

H N R2

H N

R N H

N H

Ethidium derivatives

Substituted fluorenones

23

22 R

R HN

NH

O

n R = amine

N

N

R1 = H or aromatic group R2 = H or aromatic group R3 = H or aromatic group R4 = H or CH3

R4

R = amine n = 2, 3

O

n

R3

N+

O

O R

n = 2, 3, 4

H N R = (CH2)2-amine

R NH O

Disubstituted phenanthrolines

Benzo(h)quinoline derivatives

24

25

or aromatic group

Figure 5.11 Structures of other G-quadruplex ligands in tricyclic aromatic system.

to discriminate a specific G-quadruplex structure from duplex and other Gquadruplexes. The essential point is that the appropriate number and configuration of substituents are introduced to the right tricyclic rings, hence allowing the molecules to correctly interact with the G-quartet as well as the grooves and loops of G-quadruplex. Besides the typical compounds listed earlier several other tricyclic aromatic analogues have also been designed and synthesized as G-quadruplex ligands, such as substituted fluorenones (22) [159–161], benzo(h)quinoline derivatives (23) [162], disubstituted phenanthrolines (24) [163], and ethidium derivatives (25) as shown in Figure 5.11 [164]. Among these ligands, ethidium


221

derivatives are outstanding for their telomerase inhibition activities, with IC50 values of 18–100 nM. Other compounds exhibit similar binding properties as substituted anthraquinones. 5.4.1.2. Quindoline Derivatives and Triaza-Cyclopenta[b]Phenanthrene Several tetracyclic planar ligands have been synthesized, which aim at extending aromatic rings to fit in the G-quartet dimension. One type of compound has a crescent-shaped scaffold. The two kinds of quindoline derivatives, disubstituted (26, 27) and 11-substituted (28–37), as shown in Figure 5.12, are representatives of this class of molecules [165–167]. All these compounds have shown strong G-quadruplex stabilization and telomerase inhibition potency but only moderate G-quadruplex selectivity (2∼3-fold) over duplex DNA. Further introduction of a positive charge at the 5-N position of the quindoline scaffold (38) not only significantly increases their binding ability and telomerase inhibition activity but also improves the selectivity toward G-quadruplex DNA, due to the better interactions of 5-N electropositive center with cation channel [168]. Moreover, these 11-substituted and 5-N -methylated quindolines have been used as molecular probes to explore the regulatory role of the c-myc and bcl -2 G-quadruplex DNA [67,108]. The results provided direct evidence that G-quadruplex structure formed in c-myc and bcl -2 promoter region could function as a transcriptional repressor element, and G-quadruplex specific ligands could regulate the transcription of c-myc and bcl -2 through stabilization of quadruplex structure. Compound 30, also termed as SYUIQ-5, has been used for a series of anticancer research [93,169,170]. Treatments with SYUIQ-5 inhibited telomerase activity and increased the time of population doublings of cancer cells. At nonacute cytotoxic concentrations, SYUIQ-5 could also induce a marked cessation in cell growth and cellular senescence phenotype after 35 and 18 days, respectively. Growth cessation was accompanied by a shortening of telomere length and induction of p16, p21 and p27 protein expression. Subsequent investigations also demonstrated that SYUIQ-5 could inhibit c-myc gene promoter activity and, therefore, inhibit hTERT, the catalytic subunit of telomerase that was under transcriptional control in part by the c-myc transcription factor. Besides, SYUIQ-5 could trigger potent telomere damage through TRF2 delocalization from telomeres and eventually induce autophagic cell death in cancer cells. These results indicate that the quindoline derivatives are potentially important agents for anticancer treatment. Another tetracyclic planar ligand for G-quadruplex DNA is based on the triazacyclopenta[b]phenanthrene scaffold (39, Figure 5.12) [171]. This compound was first designed to be a minor groove binding agent, but results showed that its binding to G-quadruplex DNA was about 20-fold tighter than to the duplex DNA. Additional experiments aimed at exploring the anticancer activity of 39 and the structure–activity relationships of this new agent are currently underway. 5.4.1.3. Pentacyclic Acridinium Analogues In addition to the development of tetracyclic ligands, researchers have investigated the pentacyclic planar

222


N H N

N N

R

R = amine

N

N

R

Disubstituted quindolines 27

Disubstituted quindoline 26

R

X

R

X

R

28

NH

morphoryl

33

O

morphoryl

29

NH

N(CH3)2

34

O

N(CH3)2

30

NH

CH2N(CH3)2 35

O

CH2N(CH3)2

31

NH

OH

36

O

OH

32

NH

CH2OH

37

O

CH2OH

HN X N 11-subsitituted quindolines

O

R1 HN X

R1 = amine R2

R2 = H or F R3 = H or F

N + I– R3 5-N-Methylated quindolines 38

X = NH or O

N

N

O HN

N H

N NO2 Triaza-cyclopenta[b]phenanthrene 39

Figure 5.12 Structures of G-quadruplex ligands in tetracyclic aromatic system.

system, such as acridinium salts (40, 41) [172], meridine (45), and ascididemin (46) [173], as shown in Figure 5.13. Even without side chains, most of these compounds have exhibited good telomeric quadruplex binding and telomerase inhibition activity, especially some of the acridinium salts had significant telomerase inhibition activity with IC50 value lower than 1 μM. A useful molecular feature for the notable activity of acridinium salts might be their much bigger aromatic scaffold and positive charge at position 13-N of the acridine ring, which acted as a “pseudo” potassium ion positioned above the center of G-quartet in the region of high negative charge density [125]. Compound 41, also termed as RHPS4, showed selectivity for antiparallel telomeric G-quadruplex DNA, implicating favorable interactions with lateral and diagonal loops in vitro. Its IC50 value for telomerase inhibition was 0.33 μM [174]. Similar to other telomeric G-quadruplex ligands as telomerase inhibitors, RHPS4 caused a senescent-like growth arrest in cancer cells and a reduction in telomere length after a long-term

223


R1 2

R2

R3

R4

40 3-Me

Me

Me

MeOSO3

41 3-F

Me

F

MeOSO3

R4

42 3-CH=CHCON(CH2CH2)2O

H

H

I

R2

43 3-CH=CHCON(CH2CH2)2O (CH2)3OAc

H

I

44 2-NHCO(CH2)4CO2Me

H

I

R1 3

+

N

–

R3 N acridinium salts

OH

H

N

N O

N

N

N O

N

Meridine

Ascididemin 46

45

Figure 5.13 Structures of pentacyclic acridinium analogs.

treatment at nonacute cytotoxic concentrations [175]. On the other hand, the short-term biological activity of RHPS4 was not caused by telomere shortening, but was associated with telomere dysfunction, in terms of presence of telomeric fusions, polynucleated cells, and typical images of telophase bridge [94]. Furthermore, treatment of cancer cells with RHPS4 led to the displacement of the hTERT from the nucleus and inhibition of TRF2, which might be corresponding to their telomere dysfunction effects [96,99,100,176]. Moreover, RHPS4 had a good pharmacodynamic profile and could be used as single agent or in combination therapies with taxol, adriamycin, and camptothecin, which all produced a strong antitumoral activity in xenografts model [96,175,177], while the temozolomide and cisplatin antagonized the action of RHPS4 [175]. Expanding the area properly and increasing the positive charge density of aromatic ring does increase the activity of these pentacyclic systems; however, these are not sufficient to gain significant selectivity, as the aromatic ring simply stacks on the G-quartet, and its size is still too small for the dimension of G-quartet. The results from competition mass spectrometry, dialysis assay, and FRET analysis showed that all these pentacyclic compounds have modest Gquadruplex selectivity over other DNA species. Recently, based on the results from trisubstituted acridines, side chains have been introduced to the quinoacridinium pharmacophore in significant positions [178,179]. Some of these new acridinium salts, such as compounds 42, 43, and 44, have shown much higher Gquadruplex selectivity and lower cytotoxicity than the original compounds. These

224


studies suggest that appropriate side chains are also crucial for the G-quadruplex selectivity of this pentacyclic system. 5.4.1.4. Dibenzophenanthroline Derivatives and Related Analogues Besides the pentacyclic acridinium derivatives, a series of pentacyclic dibenzophenanthroline ligands (Figure 5.14) with crescent shaped arrangements and extended amino side chains have been studied [180,181]. The N -methylation of quinacridine and its crescent ring shape in these compounds were considered to be crucial for their G-quadruplex stabilization potency. The global charge created by the side chains had a strong influence on the stabilization because highly cationic species could stabilize the G-quadruplex structure (49 > 48 > 47). Moreover, enhancement in affinity by the introduction of a third side chain might be gained from a synergistic effect between an optimized quadruplex interaction and an increase in the global charge (50 > 47), though the charge increase appeared to reduce the quadruplex selectivity as found with competition dialysis assay or FRET melting method. Compounds 47, 51, 52, and 53 could discriminate quadruplex against duplex DNA, whereas the disubstituted compounds with highly cationic side chains (48 and 49) or the trisubstituted compounds (50) had no such selectivity. Unlike the improved selectivity with the trisubstituted from the disubstituted acridines, the introduction of a third side chain here did not work. The difference might arise from the location or configuration of terminal N

N

H N

H N

2

N

H N

N

H N

N

2

3

48

47 N

N

3

H N

H

NH2

3

2

N

N

H N

N

N

2N

H N 3

H2N

H N

3

2

49 N+

50 N+

H N

N+ Cl

2

H N

N 3

N

N

N

52

53

2

51

Dibenzophenanthroline derivatives

Figure 5.14 Structures of dibenzophenanthroline derivatives.

225


N HN

R = (CH2)3N(CH3)2 N R

N H

N

N O HO

N

N

Disubstituted bispyrimidinoacridine 54

55

HN

n

N H

N

O HO N N N H H Trisubstituted bispyrimidinoacridine

N H

N

N R

N H

N

N

N

R1 = NCH3 or S R2 = OMe or H

R = NO2 or OCH3 n = 2, 3

N H

+

R2

N

N

R2

R1 N Azacyanines

R1

R Substituted benzoindoloquinolines 56

57

Figure 5.15 Structures of other G-quadruplex ligands in pentacyclic aromatic system.

amine of the third side chain being incorporated into different pharmacophores. The side chains have a strong influence on fixation of the ligand in G-quadruplex and duplex DNA. Another class of these pentacyclic compounds is the substituted bispyrimidinoacridines (54 and 55, Figure 5.15), which have two pyrimidine rings incorporated into the acridine core [182]. It is interesting to note that the trisubstituted bispyrimidinoacridine showed strong binding affinity but lower selectivity than the disubstituted form. Other compounds, such as substituted benzoindoloquinolines (56) and azacyanines (57), as shown in Figure 5.15 [183,184], also belong to the pentacyclic G-quadruplex ligands. Azacyanines without any side chains bound poorly to all duplex DNA strands and showed more than 100-fold selectivity for quadruplex over duplex DNA. These SAR are similar to that of dibenzophenanthroline ligands and therefore further confirmed the suggestion that introduction of side chains should consider their interacting objects. 5.4.1.5. Fluoroquinolone Derivatives As shown in Figure 5.16, Fluoroquinolones (58) were proven telomerase inhibitors, probably due to their interaction with G-quadruplex structures [185]. Hence, a series of fluoroquinolone derivatives have been synthesized with an extended aromatic conjugation system. Quinobenzoxazine A-62176 (59) was both a topoisomerase II poison and a catalytic inhibitor, whereas the extended hexacyclic fluoroquinophenoxazine QQ58 (60) was a strong G-quadruplex binder without topoisomerase II poisoning activity, suggesting its high selectivity over duplex DNA [186]. However,

226


O

O F

OH

N O

O

N

O

N

N

O

H CH3

O

F

OH

N

N

N

O

O F

O

H2N

H2N O

Levofloxacin

A-62176

QQ58

58

59

60

O F

O

O OH

N

F

H2N

O N H

N

N O

O H2N

O F

OH

N

N

O

N

N O

N N

FQA-C

FQT-C

Quarfloxin (CX-3543)

61

62

63

Figure 5.16 Structures of fluoroquinolone derivatives.

the increase in aromaticity of the hexacyclic ligands resulted in poor selectivity for the G-quadruplex but higher topoisomerase II poisoning effects of fluoroquinoanthroxazines (61 and 62) [187]. In view of preceding attractive results, a library of fluoroquinolone analogues with various aromatic conjugation systems and side chains has been designed and synthesized by Cylene Pharmaceuticals [114]. The most promising hit in this library was quarfloxin (CX-3543, 63) with pentacyclic system and two specific amino side chains, which was highly selective for c-myc G-quadruplex. These two side chains were chiral substitutes, and their asymmetric feature might allow for the quarfloxin to recognize the groove and loop regions of specific G-quadruplex motifs and gain the selectivity over other G-quadruplex structures or duplex DNA. However, quarfloxin did not show a direct inhibitory effect on c-myc expression in vivo, suggesting an alternative mechanism. It has been shown that quarfloxin concentrated in the nucleoli of cancer cells and selectively disrupted nucleolin/rDNA G-quadruplex complexes in the nucleolus, thereby inhibiting rRNA synthesis by interfering with the elongation step of Pol I-driven transcription and inducing apoptosis in cancer cells. In addition, quarfloxin did not interfere with Topoisomerase I/II function in cells. It is not yet clear whether the mechanism of quarfloxin reflects its preference for

227


rDNA quadruplexes over other quadruplex forms or just the high density of quadruplexes present in rDNA. Now, quarfloxin has entered Phase II clinical trial for treatment of carcinoid/neuroendocrine tumors (C/NET). 5.4.1.6. Perylene Derivatives and Related Analogues Perylene derivatives, such as PIPER (64, Figure 5.17), are classical agents used for interaction with G-quadruplexes. Previous studies suggested a correlation between ligand aggregation and G-quadruplex DNA selectivity, and the derivatives that formed aggregates in a buffer had much higher quadruplex selectivity than the unaggregative species [188,189]. The formation of aggregates as well as the binding selectivity was dependent on the pKa values of their basic side chains. This was explained by the fact that the aggregated ligand molecules became too large to interact with double-stranded DNA by inserting the ligand chromophore between the base pairs. Nevertheless, the self-aggregation of compounds must be carefully considered in the interaction model between different ligands and Gquadruplex DNA because recent results showed that strong drug self-aggregation was related to a minor telomerase inhibition and weaker interactions with the G-quadruplex, suggesting that the higher selectivity for G-quadruplex DNA on aggregation was due to a reduced binding efficiency to duplex and single-stranded DNA, rather than a greater affinity for G-quartets [190]. Besides the cationic basic side chains, several other substituents, including EDTA [191], swallowtailed polyethyleneglycol [192], and anionic groups [193], were also introduced to cleave G-quadruplex DNA or improve water solubility of the derivatives.

O

O

N

N

O

O

N

N PIPER 64 R2

R2 NH

O

O

N

N R1

R1 O

O R1 = amine R2 = amine

O

O

N

N

O

O

R1

R1 HN R2

R1 = amine R2 = amine

R2 Coronene derivatives 65

Naphthalene derivatives 66

Figure 5.17 Structures of perylene derivative and related analogues.

228


Further modifications on the perylene ring led to a series of coronene (65) and naphthalene (66) derivatives [194–196], showing increasing and decreasing aromaticity, respectively. Interestingly, all these compounds had four side chains aimed at four grooves of G-quadruplex, and some of them did exhibit good selectivity toward G-quadruplex in comparison with perylene derivatives. Whether this selectivity is related to the abundant side chains on the chromophore is still under investigation. 5.4.2. Macrocyclic System

Porphyrins are 5.4.2.1. Porphyrin Derivatives and Related Analogues well-known binding agents for duplex DNA that have been investigated for more than three decades. The planar arrangement of the aromatic rings in porphyrins provides the potential for binding to G-quadruplexes by stacking on the Gquartets. The pioneer compound 5,10,15,20-tetra-(N -methyl-4-pyridyl) porphine (TMPyP4, 67, Figure 5.18) could stabilize different G-quadruplexes [197–199], and also bind nonspecifically to all structural forms of DNA, including singlestranded, duplex, triplex, and quadruplex according to competitive equilibrium dialysis [200].

N H N

HOOCH2C

H N +

N

N

H3C

N

N+

4Cl–

C2H5

H3C

N+

+N

CH3 N

NH

C2H5

N

HOOCH2C

TMPyP4

CH3 NMM 68

67 OH N+

N +

NH

N

N OH

HO N

N

HN I

– +

N

Mn N N

N +

I

+

O

O N+ – I

N

H N

H N

–

I

–

O

O +

N

N H

–

5Cl

N H

OH TQMP

Pentacationic manganese(III) porphyrin

69

70

Figure 5.18 Structures of porphyrin derivatives.

+

N


229

TMPyP4 was initially found to be an effective telomeric G-quadruplex ligand, showing telomerase inhibition activity in cancer cells [201]. Interestingly, TMPyP4 suppressed the proliferation of ALT-positive cells and telomerasepositive cells within several weeks at noncytotoxic concentrations [202]. Subsequent investigation also demonstrated that TMPyP4 could bind to the c-myc G-quadruplex DNA and therefore down-regulate c-myc gene expression [66,203]. The hTERT gene, which encodes the catalytic subunit of telomerase, is transcriptionally regulated by c-myc. Accordingly, the down-regulation of the hTERT gene might be another mechanism for the effect of TMPyP4 on telomerase activity. Besides, TMPyP4 has also been used as a molecular probe to explore the in vitro and in vivo functions of other promoter G-quadruplex DNA, such as VEGF [204,205], bcl -2 [107], RET [206], and so on [111,207]. This molecule seemed to be a versatile quadruplex ligand, and cDNA microarray analysis revealed TMPyP4 could up-regulate 33 genes and down-regulate 54 genes in leukemic cells [208]. On the other hand, another closely related porphyrin N -methyl mesoporphyrin IX (NMM, 68), which is assumed to be anionic rather than cationic in an aqueous solution at physiological pH, has shown specific binding affinity to G-quadruplex structures with no apparent affinity to any other form of DNA [200]. Treatment of Saccharomyces cerevisiae with NMM caused down-regulation of loci connected with the function of the rDNA [120]. All these promising results from the early studies have motivated further research efforts for synthesizing new porphyrin derivatives to improve their binding affinity toward G-quadruplex DNA and to generate much better molecular probes with special functions. The works mainly focused on the synthesis of metal-porphyrin complexes and modification of substituent group on N -methylpyridyl positions. The most typical molecules in this series are 5,10,15,20-tetra[4hydroxy-3-(trimethylammonium)methyl-phenyl]porphyrin (TQMP, 69) and pentacationic manganese(III) porphyrin (70), as shown in Figure 5.18 [209,210]. TQMP is a nonpyridinium cationic porphyrin with a phenol quaternary ammonium and has been shown by SPR assay to bind 30-fold more strongly to telomeric G-quadruplex than to duplex DNA. TQMP might be more flexible than the rigid porphyrin TMPyP4 due to a higher steric effect. The introduction of a hydroxyl group might also be an important factor for increasing the interaction with Gquadruplexes by hydrogen bonding. These properties might favor its binding to G-quadruplexes in the grooves and increase its selectivity. Compared with TQMP, pentacationic manganese(III) porphyrin is even more promising with 10,000-fold selectivity for telomeric G-quadruplex over duplex DNA. This porphyrin contains a central aromatic core and four flexible cationic arms. The bulky cationic substituents surrounding the aromatic core, which prevent a close interaction with the duplex DNA, might be responsible for its low affinity for duplex DNA. Its high affinity for the G-quadruplex DNA might be attributed to the interactions between the G-quartet and the porphyrin core and between the grooves or loops and the flexible cationic arms.

230


Moreover, several modifications have also been made to the porphine core to generate a group of porphyrin analogues, such as tetramethylpyridiniumporphyrazines (TMPyPz), their zinc complex (3,4-TMPyPz zinc(II), 71), and octacationic quaternary ammonium zinc phthalocyanine (ZnPc, 72) [211,212]. As shown in Figure 5.19, their cores are of higher aromaticity than the porphyrins. In addition, methylation of the pyridyl groups of tetrapyridinoporphyrazines or introduction of highly cationic side chains on phthalocyanine improved their Gquadruplex selectivity over duplex (>30-fold by 71 and >6-fold by 72) due to electrostatic interactions with grooves or loops. On the basis of these studies, several guanidinium-modified phthalocyanines and their metal complexes were synthesized [213]. One of the molecules, Zn-DIGP (73), exhibiting good solubility in water, is the first example of a high-affinity G-quadruplex ligand showing both in vivo “turn-on” luminescence and the ability to knock down RNA expression. Besides the introduction of cationic groups, an anionic copper phthalocyanine (Cu-APC, 74) has been reported to selectively bind to and stabilize telomeric G-quadruplex DNA and inhibit telomerase activity efficiently, even in the presence of excess duplex DNA. The high selectivity is consistent with studies of NMM. However, binding affinity of Cu-APC to the telomeric G-quadruplex DNA was modest, and further improvement of these anionic macrocyclic molecules is required [214]. Other typical porphyrin analogues also included the corrole derivatives (75) following similar modification ideas as porphyrins. SPR analysis showed that several of these compounds exhibited more than 100-fold selectivity for c-myc G-quadruplex DNA against duplex DNA [215,216]. 5.4.2.2. Telomestatin and Related Analogues Telomestatin (76, Figure 5.20) is the first natural telomerase inhibitor with high potency (IC50 = 5 nmol/L) due to its ability to facilitate the formation of G-quadruplex structures or to bind to and stabilize the G-quadruplex structures [217]. It consists of one thiazoline ring and seven oxazole rings. Telomestatin appeared to interact preferably with basket-type intramolecular G-quadruplexes rather than intermolecular quadruplexes [202,218]. Moreover, the specificity of telomestatin binding to telomeric G-quadruplex structures was 70-fold over duplex according to polymerase stop assay [202]. The results from electrospray mass spectrometry and competitive FRET assay also indicated ineffective binding or stabilization of single-strand or duplex DNA by telomestatin [219,220]. On the basis of these results, detailed investigations in vivo for telomestatin have been performed. Treatment of telomestatin in cancer cells led to inhibition of telomerase activity, reduction in telomere length, induction of senescence, and cell apoptosis [221]. Further competition experiments indicated that telomestatin strongly bound in vitro and in vivo to the telomeric overhang and impaired its single-stranded conformation. Long-term treatment of cells with telomestatin greatly reduced the G-overhang size, supporting the hypothesis that the telomeric G-overhang was an intracellular target for the action of telomestatin [222]. Besides, the treatment of HT1080 tumor cells with telomestatin induced

231


N+ I–

N+

+

N –

+

Cl N

+ –

+

N

N N

N N

N

N +N Cl–

I–

+

H N

N

– HN +

+

I – +N

NH Cl–

O– O O S

O S –O O

N N

N

HN N H

N N

N

N N H

N Cu

N

N N

NH Cl

ZnPc

N+ I–

N Zn

N

– +I

N

N

HN

N

N

O

O

72

H N

NH

N N

N

71

Cl

N Zn

N

3, 4-TMPyPz zinc(II)

+ – HN

N I–

N

N

N Cl– + N

O

O

Cl–

N Zn

N

I

I–

NH +

–

Cl

O O S –O

Zn-DIGP

Cu-APC

73

74

O O– S O

R NH

N R

R NH HN

R = N-methylpyridyl or N, N, N-trimethylbenzenaminium group

Corrole derivatives 75

Figure 5.19 Structures of porphyrin analogs.

prompt cell death and a rapid decrease of the telomeric G-overhang and of the doubles-stranded telomeric repeats [98]. Telomestatin treatment also provoked a complete dissociation of POT1 and TRF2 from telomere, suggesting that the ligand triggered the uncapping of the telomere ends. The effect of the ligand was associated with an increase of the γ-H2AX foci, one part of them colocalizing at telomeres, thus indicating the occurrence of a DNA damage response at

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the telomere [97,98,223]. Another study on the DNA damage pathway also demonstrated that such telomere dysfunction induced by telomestatin activated the ATM-dependent DNA damage response [224]. Interestingly, telomestatin was shown to completely dissociate TRF2 from telomere in cancer cells but not in normal or immortalized cells. It rapidly dissociated TRF2 from telomeres in cancer cells, when given at a concentration that did not cause normal cells to die. In addition, telomestatin also induced anaphase bridge formation in cancer cells. These effects of telomestatin were similar to those of dominant negative TRF2 [223]. Accordingly, all results together suggested that telomestatin exerted its anticancer effect not only through inhibiting telomere elongation, but also by disrupting telomere maintenance and ultimately resulting in telomere dysfunction by rapidly disrupting the capping function at the ends of telomeres. Unlike the TMPyP4 as versatile molecule, telomestatin seemed to be a purer telomeric G-quadruplex ligand. Gene expression profile of myeloma cells after 1 and 7 days of telomestatin treatment revealed >2-fold change in only 6 (0.027%) and 51 (0.23%) of 33,000 genes surveyed, respectively. There were no changes observed in expression of genes involved in cell cycle, apoptosis, DNA repair, or recombination [221]. Furthermore, effect of telomestatin on the growth of U937 cells in xenograft mouse model was evaluated. Administration of telomestatin in U937 xenografts decreased tumor telomerase levels and reduced tumor volumes. Tumor tissue from telomestatin-treated animals exhibited apparent apoptosis. None of the mice treated with telomestatin displayed any signs of toxicity [225]. Moreover, enhanced chemosensitivity toward imatinib, daunorubicin and cytosine-arabinoside was also observed in cell models [224,226,227]. Inspired by the promising anticancer activity of telomestatin, several related analogues have been designed and synthesized. Because telomestatin has no convenient functional groups, such as hydroxy and/or amine groups, its further structural development is difficult. Therefore new modifications are all based on new scaffold. The most typical molecules in these compounds are a series of oxazole-containing macrocycles (77–81, Figure 5.20), most of which contain one or two amine side chains to enhance compounds’ water solubility and binding potency to G-quadruplex DNA [228–234]. Compound 78, also termed HXDV, is one of the pioneer molecules of this series, showing no impact on the thermal stabilities of the duplex and triplex DNA. Furthermore, HXDV has been found to bind to the telomeric G-quadruplex DNA through end-stacking binding mode and inhibited cell cycle progression causing M-phase cell cycle arrest, with a mode of action dependent on its G-quadruplex binding activity [235–237]. The lysinyl derivative of HXDV, HXDL, also termed L2H2-6OTD (79), has been synthesized by two independent groups, showing improved binding potency toward telomeric G-quadruplex DNA [229,231]. On the other hand, hybrid compounds of telomestatin and TMPyP4, the selenium-substituted expanded porphyrin Se2SAP (82) and cyclo[n]pyrroles (83–87) have been synthesized (Figure 5.20). Atom-by-atom superimposition and the electrostatic field-fit alignment studies showed significant similarity

233


between the oxazole ring of telomestatin and the bipyrrole ring of Se2SAP. Accordingly, Se2SAP should overlay very well with the entire G-quartet like telomestatin, and its charged N -methyl-4-pyridyl groups might also recognize the grooves/loops of different G-quadruplexes. SPR experiments have confirmed the selectivity of Se2SAP for the c-myc G-quadruplex (∼50-fold) over the duplex DNA. Se2SAP also showed a fairly high selectivity of binding to G-quadruplexes with a single lateral loop and the syn-anti -anti -anti arrangement of guanines in the G-quartets [238]. For the cyclo[n]pyrroles, the order of their solution binding affinities towards telomeric G-quadruplex DNA was 84 > 83 > 85 > 86 87, showing an optimized combination of size and positive charge resulted in the highest level of binding affinity. Smaller porphyrinoid systems

N O

CH3

O

S

N

O

N

N

N

N

O

O

CH3

N

O

N

3

N H

O

O O N

N

N N

O

O

O

N H

N

N

H N

O

R O

O

N

N

O

N H

N H

+

O

N N+

N Pyridyl polyoxazole 81

NH HN NH

–

4Cl

Se2SAP 82 R R N NH H HN

R R

HN

R R

+HN

NH+

NH HN

R n

R NH H HN N

R R

Octaethylporphyrin 83

O

+

N H Se

N Se

R

N HN

N

N

N +

N

R NH N

O N

78 HXDV, R = CH(CH3)2 79 HXDL(L2H2-6OTD), R = CH2(CH2)3NH2

O

N

R

N O

N

O

L1H1-7OTD 80

N

O

N

N

O

N

O

O N

O HXPV 77

NH2 O N

N

N

O

O O Telomestatin 76

O

N H

O

N

O

N

N

O

O

84 Cyclo[6]pyrrole, n = 1 85 Cyclo[7]pyrrole, n = 2

R

R

R R

86 Cyclo[8]pyrrole, R = CH3 87 Cyclo[8]pyrrole, n = CH2CH3

Figure 5.20 Structures of telomestatin and related analogs.

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tended to bind more strongly, as long as the positive charge on the macrocycle was retained [239]. 5.4.2.3. Peptide-Based Macrocyclic Compounds The unique selectivity observed in the macrocyclic system made it a favorable molecular model for the design of other G-quadruplex ligands, including a series of peptide-based macrocyclic compounds (88–94, Figure 5.21). These compounds should have good interaction to G-quadruplexes by overlaying the entire G-quartet, which are similar to that of telomestatin. Accordingly, the transformation of oligoamides from macrocylic (88) to helical structure (89) resulted in loss of binding affinity and selectivity [240]. Another example for these compounds is oxazole-based peptide relatives (90–93), which contain three stereo amine side chains in the macrocyclic system. These side chains are protonated at physiological pH and might be involved in stabilizing interactions with the grooves and loops of the quadruplex as well as the negatively charged phosphate backbone. SPR data indicated that inversion of all three stereocenters would not affect the recognition of homo chiral quadruplex structure (90 and 91), but inversion of only one stereo center side chain on both faces of the macrocycle (92) led to a four- to sixfold drop in binding affinity for c-kit and human telomeric quadruplexes. Moreover, for compounds 90 and 91, it is noteworthy that introduction of side chains improved the c-kit selectivity threefold over human telomeric G-quadruplex. With shorter methylamine side chains (93), macrocycle showed a lower binding affinity for human telomeric G-quadruplex (fourfold), but a similar equilibrium binding to c-kit (sevenfold) [241]. The highly selective binding of macrocyclic ligands to G-quadruplex over duplex DNA is mostly attributed to their larger ring system. Results from the furan-based cyclic oligopeptides (94) also supported this point [242]. The macrocyclic scaffold would overlap completely with the four guanines in the G-quartet, allowing the ligand molecule to occupy the whole area of the quartet region. However, targeting the G-quartet in the ligand design is insufficient to gain the selectivity among different quadruplex species because this binding site is common to all quadruplexes. Findings from the study on oxazole-based peptide derivatives (90–93) suggested that appropriate side chains are also crucial for designing macrocyclic ligands with selectivity for different G-quadruplex structures. 5.4.3. Unfused Aromatic System

The investigation of inter5.4.3.1. Distamycin and Related Analogues actions between G-quadruplex and unfused aromatic compounds began with studies on the carbocyanine dye and traditional groove binder distamycin (95, Figure 5.22) [200,243]. Unlike its binding to the minor groove of duplex DNA, distamycin could either stack on both ends of a G-quadruplex or bind simultaneously to two opposite grooves of the quadruplex in a 4:1 binding mode forming two antiparallel dimers [129,130]. Recent studies also showed that the

235


R

R O

O

N HN O R

N

NH N HN

O

N

O N * H N HN

R * NH N

O O R = O(CH2)3NH3+CF3CO2–

R

R

Macrocyclic oligoamides 88

O

* R

(CH2)4NH2

S, S, S

91

(CH2)4NH2

R, R, R

92

(CH2)4NH2

S, S, R

93

CH2NH2

S, S, S

O

oxazole-based peptide R

NH N OH R

N

O NO2

O

O O

n

NH O

O

HN

N H

O O

NH

H O N

HN

O O

O

R R = O(CH2)3NH3+CF3CO2– R = NH2 or NHCOCH2CH2NH2 n = 1 or 3 Furan-based cyclic oligopeptides Helical oligoamides 89

R

O

R O

Stereochemistry

90

O

H N O

94

Figure 5.21 Structures of peptide-base macrocyclic compounds.

positively charged terminal group of distamycin was important for its groove binding mode [131]. To enhance the selectivity of distamycin, several derivatives (96) have been designed and synthesized. However, their quadruplex selectivity was still modest even though the number of pyrrole rings was increased to five and a much longer side chain was introduced, showing such arrangement and orientation of polyamide were easily fit for the duplex grooves [244]. Other quadruplex groove binders include a series of heterocyclic diamidines, such as DB832 (97, Figure 5.22). Its adjacent furan rings are the key moiety that forms hydrogen bonds with G-NH2 amino group hydrogen in the quadruplex grooves. Addition of a phenyl ring between these two furan rings would diminish the compound’s groove binding mode [245]. Similar to the distamycin, DB832 showed no selective binding toward different DNA species [246]. On balance, arrangement of these five member rings is important for the future ligand design because the grooves of G-quadruplex DNA have different groove geometries and patterns of donor–acceptor sites from those of duplex DNA. Recently, a series of biaryl polyamides (98), which seemed to be hybrid compounds of distamycin and DB832, were synthesized. The rearrangement of these rings gave high selective compounds showing inability to bind to duplex DNA. However, the selectivity arises from their planar U-shaped structures that match the surface area dimensions of a terminal G-quartet in quadruplex structures rather than the grooves of duplex DNA [247]. Thus, design of pure quadruplex groove binder with high selectivity on the basis of polyamides is still a challenge.

236


O

O

NH

O

H N

N

N

N H

NH HN

O

N HN

H N

N O

N

O

R

n

O

O H

NH2

H N

R=

N H

N

n = 1–5

Distamycin

Distamycin derivatives

95

96 N

O

NH

O H2N

HN

NH O

H N N

O

O O

HN

NH2

N DB832

Biaryl polyamide

97

98

N

Figure 5.22 Structures of distamycin and related analogues.

5.4.3.2. Triazine derivatives and Related Analogues Triazine derivatives are other pioneer molecules of unfused aromatic compounds with much higher aromaticity than the distamycin and its related analogues. These compounds tend to stack on the G-quartet surface because of their steric bulky conformation. In this series, 12459 (99, Figure 5.23) is the most selective Gquadruplex interactive compound, which showed a 25-fold telomerase inhibition over the Taq polymerase inhibition [90,248]. Other structures are analogues with the similar butterfly-shape (Figure 5.23), such as 1,4-triazoles (100) and biarylpyrimidine derivatives (101) [220,249,250], all having high selectivity for G-quadruplexes. The high selectivity of these ligands was attributable to their adaptive structural feature arising from the rotatable bonds, which allowed the ligands to adopt different conformations to fit the shape of groove and loop regions of G-quadruplex while maintaining the rigidity for G-quartet. Another consequence for the high selectivity was the steric requirements of these compounds, which were difficult to allow intercalative binding or groove binding to duplex DNA to take place. In view of this feature, and enlightened by the tempting G-quadruplex selectivity exhibited by 1,4-triazoles and biarylpyrimidine derivatives, more and more butterfly-shape compounds were designed and synthesized, such as bis-indole or bis-benzimidazole carboxamides [251,252], triarylpyridines [253], diarylethynyl amides (102) [254], substituted diarylureas [255], and 2,6-diphenylthiazolo[3,2-b][1,2,4]triazoles [256]. More recently, the third substituted group was also incorporated into the center body and generated several trisubstituted unfused aromatic compounds, including compound 103

237


NH2

N+

N N H

+

N N H

N

N H

N

N

O R2N

1, 4-triazoles

99

100

O H N R = aliphatic group

R NR2

NH2

O

Diarylethynyl amides 102

NH2

O

O

HN

N

R2N

O

N NH

N H

R

X = N or CH R = (CH2)3N(CH3)2

O O

H2N

H2N

101

O

NR2

O

X

N H

Biarylpyrimidine derivatives

O

O N H

N

H N O

N

R = aliphatic group

12549

N

R2N

N

N

N

N

N H

N

H N

N O

NR2

NH2

N Isaindigotone derivatives 103

104

Figure 5.23 Structures of triazine derivative and related analogues.

and symmetric phenyl derivatives [257,258]. It is noteworthy that diarylethynyl amides (102) showed very good binding affinity and excellent quadruplex over duplex selectivity. NMR results also indicated that the recognition of the parallel G-quadruplex by these ligands was achieved through groove binding, showing the first paradigm for the selective quadruplex groove binders. Besides aforementioned symmetrical molecules, other unsymmetrical compounds, such as berberine derivatives and isaindigotone derivatives (104, Figure 5.23), also belong to these high selective unfused aromatic G-quadruplex ligands [259–263]. 5.4.4. Other G-Quadruplex Ligands

Besides the molecules we introduced in an early section of the chapter, several other types of compounds have also been reported as G-quadruplex ligands, such as numerous metal complexes [264], hybrid and conjugation molecules [265–270], steroids [271], and so on. Although these compounds may not fit in any of the structural categories reviewed earlier, they share the similar design principles for interaction with G-quadruplex. We can envisage more and more G-quadruplex ligands would be discovered because of the introduction of virtual screening and high-throughput screening in this area.

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5.5. SUMMARY AND OUTLOOK

Over the past decade, DNA quadruplex research has rapidly moved from fundamental to clinical studies. The biological significance of G-quadruplexes has been recognized by numerous research efforts. With the advent of x-ray crystallography and NMR studies in this field, the structure and topology of G-quadruplexes and their ligand complexes have become clearer than ever before. This structural information is particularly important for the design of druglike molecules specific for G-quadruplexes. The G-quadruplex is recognized as a significant drug target for cancer, and extensive efforts have been directed toward the discovery of promising lead compounds capable of binding and stabilizing G-quadruplexes. In this chapter, we presented a large collection of G-quadruplex ligands. Their binding affinity and selectivity depend on properties of both the chromophores and the substituted groups, particularly the shape and electron density of the chromophore, the size, number and location of substituted groups. Besides the most preferred amine side chains, other substituted groups have also shown notable selectivity, including peptides, aminoglycosides, anilino groups, quinolinium moieties, and so on. According to the complexes of G-quadruplex and ligand, the target binding sites for G-quadruplex ligands include the G-quartet surface, grooves and loops, and the possible modes of interaction include stacking (onto the planar ends), electrostatic attraction, hydrogen bonding, and other molecular forces. Substituted groups are crucial for the ligands that belong to fused aromatic system to achieve G-quadruplex selectivity over duplex DNA. An emerging trend is toward the enhancement of grooves and loops recognition by the introduction of additional substitutes on the coplanar chromophore. Although the individual side chains may have different affinities for a quadruplex, their synergistic effect is also important for differential interaction with different G-quadruplex structures. An important characteristic of the substituted groups is their selective interaction with the structurally more complex loop or groove regions in addition to π–π stacking on the G-quartet. Most of the G-quadruplex ligands have extended planar chromophores facilitating their stacking onto the planar ends of a G-quadruplex. An elegant exemplification for this design principle is provided by the macrocyclic compounds. Furthermore, several unfused aromatic molecules may be more promising candidates in the search for high-selective Gquadruplex ligands with druglike scaffolds. Clarification of their binding modes with G-quadruplex is warranted for better understanding of their selectivity. Today, we are glad to see that some of the G-quadruplex ligands have shown excellent affinity and selectivity for G-quadruplex and significant telomerase inhibition or suppression of the transcription activity of oncogenes, and a few have entered clinical trials for cancer therapy. These achievements have enlightened the promising prospects of G-quadruplex ligands as anticancer agents with reduced side effect and toxicity. With increased knowledge of ligand–quadruplex interactions, we can anticipate that more effective G-quadruplex ligands can be developed for cancer therapy in the near future.

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CHAPTER 6

MOLECULAR MODELING IN NUCLEIC ACID-TARGETED DRUG DESIGN LIDAN SUN, HONGWEI JIN, LIANGREN ZHANG, and LIHE ZHANG State Key Laboratory of Natural & Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China

6.1. INTRODUCTION

Following the publication of the human genome, we have made great progress in understanding the structure and biological functions of nucleic acid. Even though the concept of gene targeting could not be explicitly formulated until much later, there were early realizations that nucleic acid could provide a promising target for drugs [1]. Growing knowledge about the detailed information has prompted major research efforts aiming at the design of potential drug molecules that are able to interact and alter the nucleic-acid structure [2]. A large percentage of the chemotherapeutic agents to treat cancer currently fall into the category of DNA-targeting drugs. DNA was and continues to be one of the primary targets in the development of anticancer, antiviral, and antiphrastic drugs [3]. At the same time, the key role of RNA to facilitate the essential biological activities such as information storage, signal transduction, and replication also made RNA the potential drug target [4]. Functional RNA complex includes ribozymes, riboswitches, and ribosomal and transfer RNAs. The development of molecules that can selectively interfere with undesired RNA activity has become a promising new direction for drug development. For example, aminoglycoside antibiotics and their analogs for targeting RNA have been widely synthesized and studied [5,6]. Although the value of DNA and RNA as a target for selective drug action had been evident from the outset, it also quickly became apparent that few Medicinal Chemistry of Nucleic Acids, First Edition. Edited by L.H. Zhang, Z. Xi and J. Chattopadhyaya. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

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known drugs showed much, if any, selectivity for binding to particular nucleotide sequences. The selective recognition of single- and double-stranded nucleic acids, as well as their secondary and tertiary structures, constitutes a great challenge to chemists and biologists. Eventually it was realized that one might need to recognize a sequence composed of a sufficient number of base pairs to locate a specific site in the human genome [7,8], which is really difficult for small-molecule drugs to achieve. On the side of nucleic acid system, a few challenges also need to be considered and solved [9]. First, the highly charged characteristic of DNA/RNA molecules results in a strong solvation effect as well as tight association with ionic molecules. The second challenge is rising from the highly flexible nature of nucleic acid. These long-chain molecules can undergo large but restrained conformational changes to achieve their diversed functions in biological environment. Therefore, understanding the native conformation and mechanisms by which drug/ligand molecules can interact with nucleic acid have been a focus of interest for many years [10–15]. Although a wide range of experimental methods have been applied to the nucleic acid field to study in detail the relationship between their structure and function, the information provided by experimental assay so far was quite limited. To compensate the technique limitations in the “wet” experiments, tremendous efforts has been made to theoretically model the structure of nucleic acid, the best two of which are molecular dynamics (MD) simulation [16] and molecular docking [17]. The principle of MD simulation is based on solving Newton’s laws of motion for all atoms in one system and to act as a useful tool that can be used not only for developing a static model for the structure of nucleic acid, but also for providing dynamic information that cannot be obtained through current experimental methods [18,19]. The most widely used software for nucleic-acid MD simulation is AMBER and CHARMM software packages [20]. Through the years, we have witnessed tremendous improvement in the reliability of MD simulation applied to the field of nucleic acids. For example, refining of force fields, simulation algebras, and protocols, as well as optimized computational power, have all contributed to achieve nanosecond-scale simulations of both DNA and RNA commonplace [21,22]. Several reviews have been published to provide practical examples and brief guidelines for the setup of various programs and procedures for running MD simulations [23,24]. Regarding molecular docking, on the other hand, most of the methods and programs that are based on proteins are not adapted to nucleic acid [25–27]. The increasing structural knowledge of DNA and RNA molecules and the awareness of their essential roles in biological processes emphasize the necessity for developing effective docking methods to apply on nucleic acid [28]. Docking a ligand into a receptor binding site involves the cooperation of a sampling algorithm and a scoring function to assess which conformation the molecule is most likely to adopt. The modeled interactions include electrostatic interaction, van der Waals (VDW) interaction, hydrophobic interaction, hydrogen bonding (H-bond), and solvent effects. However, because most of the protein-docking programs cannot effectively consider the high flexibility and high charge of the

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TABLE 6.1 Molecular-Modeling Software of Nucleic Acid Drug Design Model-building

MD simulation

Molecular docking

Insight II Discovery studio modeling Sybyl

AMBER CHARMM NAMD

DOCK AutoDock Surflex Dock

nucleic acid, precisely predicting the binding mode and sequence selectivity is still quite challenging by using the conventional docking software. To solve this problem, several specific docking programs and scoring functions, such as DrugScore, DrugScoreCSD, DrugScoreRNA [29], and Kscore [30], were developed for nucleic acid systems. Due to the massive amount of work for nucleic acid–targeted drug design, a review to cover all the studies appearing in the literature would be beyond the scope of this chapter. Here we will only briefly browse the fundamental methodology and a few classical applications of molecular modeling in the field of nucleic acid–targeted drug design (Table 6.1).

6.2. TARGETING DNA 6.2.1. MD Simulations of DNA

During the past decades, drug design for targeting DNA structures have attracted great attention and became a subject of intensive experimental and theoretical research. Most studies on DNA have been focused on two aspects: structure-based function prediction and DNA–drug/ligand complex interaction. The MD simulation method provides a lot of detailed information that helps to elucidate the structure, thermodynamics, and kinetics of the native, modified, and complexed nucleic acid for rational drug design [31–33]. The successful applications have been seen in simulation of DNA single strand [34], duplexes [35,36], triplexes [37,38], quadruplexes [39–41], DNA catenanes [42], modified backbones [43,44], modified bases [45,46], antisense oligonucleotides [47,48], and damaged DNA [49,50], as well as DNA/RNA hybrid [51]. 6.2.1.1. Native DNA Sequence-dependent changes in the native 3D structure of DNA play a major role in the recognition process. It has been suggested that the base sequence influences the conformation of the nucleic acid chain in solution. However, few experimental methods are currently available that are able to detect the real-time DNA conformation at the single-base or base-pair level. For these reasons, it is attractive to use MD simulations to make an attempt to analyze the impact of primary sequence on the DNA structure, and many works have reported on this topic [52–54]. A method based on atomistic MD simulation was developed for estimating the complete set of sequence-dependent shape, stiffness, and mass parameters of a

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261

DNA oligomer [55]. This method defined two models, namely, rigid base model and base-pair model, each of which was evaluated by MD trajectories analysis. The sequence-dependent variability analysis suggested that MD simulation could be used to estimate complete parameter sets for a local rigid base model but not for the rigid base-pair model of B-form DNA. Models in which bases or base pairs were modeled as rigid form offered a promising approach to understand various structural features at these scales, such as sequence-dependent curvature and flexibility. Meanwhile, Lavery and his colleagues selected 39 double-stranded BDNA oligomers to study the nearest-neighbor effects on base-pair oriented conformations and fluctuations in B-DNA based on a systematic simulation approach [56]. The results indicated that the sequence-dependent effects on B-DNA structure at the base or base-pair level were relatively small, whereas the next-nearest-neighbor effects were increased at the tri- or tetra-nucleotide level. As a conclusion of these observations, the sequence dependence of DNA structure and dynamics certainly requires the consideration of next-nearest-neighbor interactions. 6.2.1.2. Artificially Modified Oligonucleotide Over the past decades, a large number of artificial oligonucleotides were synthesized with modifications occurring in the phosphodiester, sugar, or nucleobase moiety, which were shown to be resistant to chemical degradation and were able to selectively hybridize to target nucleic acids with strong affinity [57,58]. They were proved to be capable of hybridizing with the complementary DNA or RNA sequence with substantial increased thermal stabilities compared to the parent DNA: DNA or DNA: RNA duplexes, which made themselves promising candidates for clinical applications [59]. These nucleotide analogs, such as phosphoramidates [60], PNA [61], LNA [62], and INA [63], provide a convenient method to design stable antisense oligonucleotide by incorporating these modified nucleotides into the native sequence. Due to the high clinical potential, these hybrid complexes have been studied systematically by MD simulation. For instance, isonucleosides (INA) in which the nucleobase is linked to various positions of ribose other than C1 (Figure 6.1) (1–5)), belong to a class of nucleoside analogues that is capable of facilitating the design of stable antisense oligonucleotide as mentioned earlier. As a proof of principle, unrestrained MD simulations were performed in aqueous solution on the 18-mer of an antisense sequence 5 -AACATCTCCTGAGGGAAC-3 duplexes with isonucleosides incorporated at the 3 -end or at the center of single strand. The MD trajectories were analyzed to extract structure parameters. The results indicated that all structures remained in the B-form during the simulation process. Within the modified duplexes, the observed changes that occurred in the interbase pair parameters and backbone torsion angles were primarily localized at the isonucleoside-incorporated region. The modeling results were in perfect agreement with the experimental results [64]. Locked nucleic acids (LNA), another modified oligonucleotide mentioned earlier in the chapter, carry a modified sugar fragment that is restrained to the

262


O

B

O

B

O

HO

B O

O

O HO

O

O 1

O

2

O

B

3

O

B

O OH

O 4

O –O

5

B

O

O

O

O P

O O 6

Figure 6.1 The structure of isonucleosides (1–5) and locked nucleic acids (6).

C3 -endo conformation (Figure 6.1) (6). This modification entails a more rigid structure of the phosphate backbone but does not prevent the hybridization of LNA strand with its complementary DNA or RNA [62]. Ivanova et al . reported the unrestrained MD simulations [65] on a series of LNA-incorporated DNA 9mer duplexes whose structural information was available via NMR experiments. The MD results indicate that the effect conferred by a single LNA incorporation was fairly limited. Accompanied with increasing degree of LNA modification, a “gradual” transition from B-DNA to A-DNA conformation was observed. As an extreme case, the duplex with one fully locked chain displayed a complete A-DNA-like conformation. This is exactly what was indicated in the NMR experiments. Therefore, MD simulations can generate valuable structural information on LNA-incorporated DNA duplexes and appeared to be an effective way to predict the dynamic conformation of more complicated LNA:DNA systems. 6.2.1.3. DNA-Drug/Ligand Complex In recent years, there have been an increasing number of drug molecules being designed to target specific DNA sequences for treating diseases. Using MD simulations was proved to be a reliable way of understanding the static and dynamic properties of these DNA–drug complexes in solution [66–68]. Figure 6.2 shows two classical types of interaction between small molecule drugs and the duplex DNA. The first systematic analysis by MD simulation of the structure, energy, and dynamics of noncovalent complexes of DNA bound with mGB (minor groove binders) in the gas phase was reported by Shaikh et al . [69]. Later, a case study of 25 DNA–mGB complexes was investigated by Shaikh et al . [70] who applied the method to a series of DNA sequences. These MD simulations allowed the prediction of the structure properties of the complex, as well as whether a particular structural change would have an impact on ligand binding. A derived approach from MD simulations, called “in silico footprinting” (ISF), was proposed to predict the sequence selectivity for DNA-interacting

TARGETING DNA

(a)

263

(b)

Figure 6.2 Two interaction models of small molecules bound to DNA duplex (a) molecules (blue color) found within the minor groove. (b) Molecules (yellow color) intercalulating between the base pairs. (Full color version of the figure appears in the color plate section.)

molecules [71]. Specifically, the DNA backbone was viewed as rigid, and the inspected ligand would move along the minor groove of DNA using MD simulation from the starting base pair until the end. Thousands of binding modes would be generated and evaluated, and the putative binding sites were identified according to the potential energy plot. This method may offer a rapid prescreen approach for locating where the ligands may preferentially bind, although whether it is generally adaptive or not is still to be evaluated. 6.2.2. Molecular Docking on DNA

Unfortunately, a reliable program based on molecular docking method for the prediction of drug–DNA binding mode is still under development. The conventional docking programs used on protein system, such as DOCK, AutoDock, and Surflex, have been tried on docking certain small molecules to the minor groove of B-DNA [72,73]. The results suggested that the docking accuracy and pose ranking of these programs were far from satisfactory. Why would that happen? The major reason is, the sequence diversity of DNA allows for the large conformation change on ligand binding and cannot be treated as a rigid backbone. Therefore, the receptor flexibility has to be included in the DNA-ligand binding calculation. Monte Carlo (MC) algorithm seems to be a good choice to handle this issue because it is able to consider the receptor and ligand flexibility [74]. The relative movements of the ligand/drug and the DNA, as well as the internal flexibility of the two molecules, can be described and defined by MC variables. This method has been successfully used to reproduce the binding mode of methylene blue (MB)–DNA complex.

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6.3. TARGETING RNA 6.3.1. MD Simulations of RNA

Recently, numerous results have been reported to discuss the applications of MD simulation on RNA. The modeling subjects included isolated RNA molecules as well as complexes bound with drugs or ligands [75,76]. For instance, a series of MD simulations were performed to elucidate the thermodynamic basis for the relative stabilities of hairpin, duplex, and single-stranded forms of the 5 -CGC (UUUU) GCG-3 oligonucleotide [77]. Another example came from the study of antibiotics, where aminoglycoside and their analogs are known to exert their antimicrobial effects by targeting the decoding A-site of rRNA and therefore interfere with the protein synthesis. MD simulations were carried out on the complex of aminoglycoside paromomycin (gentamicin C) bound with a eubacterial ribosomal decoding A-site oligonucleotide [78]. It was revealed that the inspected compound interacted with the RNA major groove as shown in Figure 6.3c. In addition to predicting the RNA–ligand binding mode, MD simulation was also utilized to study the dynamic features of RNA secondary and tertiary structure. It was well noted that RNA chains can fold into complex structures, which frequently exhibit minimum free energy. Based on this property, many energybased simulation strategies for analyzing and predicting RNA structures were developed. A new application called discrete molecular dynamics (DMD) [79], for example, was designed to be able to rapidly generate tertiary conformation ˚ deviations from the for 150 structurally diverse RNA sequences within 4 A experimental results. Hajdin and his colleagues improved this method and used so-called replica exchange DMD to generate RNA-like folds for sequences up to 161 nucleotides that have complicated tertiary structure [80]. These methods can be used as convenient tools for RNA structural and functional analyses. 6.3.2. Molecular Docking on RNA

Just like DNA, researchers have also tried the conventional docking method on the RNA systems [81,82]. For example, a series of synthesized compounds, including β-carboline–nucleoside, neamine–nucleoside conjugates [83,84], neamine-dinucleosides, and neamine-PNA conjugates [85,86], were investigated for their interactions with the corresponding RNA targets. AutoDock 3.0 program was tried at that time, although the updated DOCK 6 program [87] seems to have the optimized parameters and more suitable scoring functions for RNA docking. Compared to DNA target, molecular docking on RNA has more challenges and limitations, mostly because a single RNA molecule is more flexible in solution. At the same time, extensive efforts have been devoted to developing flexible docking methods for RNA targets. One program [88] called MORDOR (Molecular Recognition with a Driven dynamics OptimizeR) appeared as one of the most promising tools, which allows induced-fit binding of small-molecule ligand

265

(b)

(c)

Figure 6.3 The structure of (a) ribozyme (PDB:299D), (b) tRNA (PDB:1FIR), and (c) the complex of RNA bound with gentamicin C (highlighted as cyan color) in the major groove (PDB:12BYJ). (Full color version of the figure appears in the color plate section.)

(a)

266


with RNA via flexible docking. The flexible ligand can probe the surface of the receptor (RNA in this case) and explore a low-energy path at the surface of the receptor by carrying out energy minimization with root-mean-square-distance constraints. This strategy was applied on 57 RNA complexes and successfully ˚ for reproduced experimental conformations within an atomic RMSD of 2.5 A 74% of tested complexes.

6.4. SUMMARY

Over the years, targeting nucleic acid such as DNA and RNA for drug design has been one of the major focuses in the pharmaceutical research field. Although we have witnessed the substantial improvements on the reliability of molecular simulation and modeling techniques applied to nucleic acids, the current available tools are still far from mature, and a great deal of issues and problems need to be resolved. MD simulation and molecular docking, which are the two major “flows” in this field, will still coexist and compensate each other for some time. Meanwhile, we are anxiously expecting more efforts directed into this field to better help researchers model and design nucleic acid–targeted drugs.

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CHAPTER 7

STRUCTURE OF 10–23 DNAzyme IN COMPLEX WITH THE TARGET RNA IN SILICO—A PROGRESS REPORT ON THE MECHANISM OF RNA CLEAVAGE BY DNA ENZYME OLEKSANDR PLASHKEVYCH and JYOTI CHATTOPADHYAYA Bioorganic Chemistry Program, Department of Cell & Molecular Biology, Biomedical Center, Uppsala University, Uppsala, Sweden

7.1. INTRODUCTION

Unlike RNA enzymes, which are widely present in living organisms, all known catalytically active deoxyribonucleic acids were isolated using artificial in vivo selection procedure [1] from a large pool of random oligonucleotides in combinatorial DNA libraries [2–4]. DNA enzymes have been shown to be capable of catalyze cleavaging and forming phosphodiester bonds, oxidative cleavage of DNA [5,6] and RNA [3,7], and porphyrin metallation (for review see Ref. 8). The first reported RNA-cleaving deoxyribozyme, which was found to cleave almost any target RNA molecule in a highly sequence-specific manner, is called “10–23 DNA enzyme” (DNAzyme) [3,4]. DNAzyme consists of 15 nucleotides catalytic core (Figure 7.1, Panel B) and two flanking arms complementary to the target RNA. Most of the nucleotides constituting the catalytic core are highly conserved (especially G1 to G6 as well as C13 and G14 ); however, nonessential nucleotides (C7 to A12 ) could be exchanged by some other nucleotides without complete loss of the enzymatic activity [9]. The Mg2+ -dependent 10–23 DNAzyme promoted catalytic cleavage of a particular phosphodiester bond occurs between an unpaired single mismatched purine and Medicinal Chemistry of Nucleic Acids, First Edition. Edited by L.H. Zhang, Z. Xi and J. Chattopadhyaya. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

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paired pyrimidine residues in the target RNA, as shown in Figure 7.1, Panel A. The transesterification reaction yields two cleavage products, one terminated with 2 ,3 -cyclic phosphate and the other with a 5 -hydroxyl [3]. Despite numerous applications in suppressing target genes in vivo, in vitro, and in animal models and intensive studies of the sequence requirements in the catalytic core of the DNAzyme [9,2,3,9], only very little is known about why this specific DNA sequence shows catalytic activity and how the molecular structure of the active conformation of the 10–23 DNAzyme orchestrates this process. Earlier attempts [10,11] to resolve the molecular structure by x-rays demonstrated that upon crystallization the DNAzyme takes advantages of its palindromic sequence in the catalytic core and crystallizes as a complex containing two strands of the RNA and two strands of DNA forming a four-way junction of two different

RNA 1 5′-G G | | 3′-C C 52 DNA

3 5 7 9 12 14 U GA GA C CG UC A A C | | | | | | | | | | | | A C T C T G G A G T TG 50 48 46 A G 25 42 30 G G C C 40 32 A T A A 34 38C G A 36T C

16 19 A A G A-3′ | | | | T T C T-5′ 23 20

(a)

16

p G 2p 2G 3 14p14 p G 3C 4 13 13 p p C 12 4 T A 12p 5 5 11 A p 11 A 6 p10 6 G p C 10p 9 8 7 7p A 9 T8 C p p A

1

(b) ′5

′5

O

O

B1

O O P O

S –

O

O

B2

O

B1

O O– P O S

O

3′

3′ Sp

B2

O

O (c)

1

p

15 15 p

Rp

Figure 7.1 (a) Sequence of the RNA cleaving 10–23 DNAzyme (T20 to C52) and its target RNA (G1 to A19). (n) The 15 nucleotides of the catalytic core (G29–A43) as well as the corresponding phosphates are renumbered in the 5 → 3 direction (becoming G1 –A15 ) for easy comparison with previous works (these residue names are formatted using italic style in the text). Single G to A mutation has been introduced in the catalytic core at the positions 2 , 6 , and 14 (marked in red), respectively, which in every single case resulted in the loss of the catalytic activity of the DNAzyme. Another mutation, T to C at the position 8 (marked in blue), led to modified deoxyribozyme with the catalytic activity preserved. (c) Definition of Sp and Rp configuration of stereodefined phosphorothioates.

274

STRUCTURE OF 10–23 DNAZYME IN COMPLEX WITH THE TARGET RNA

types: (a) “stacked-X” conformation of an 82 nucleotide RNA–DNA complex characterized by Nowakowski et al . [10] and (b) the 135◦ -rotated “crossed” conformation from their later study of a 108 nucleotide RNA–DNA complex [11]. It has, however, been proven that these complex RNA–DNAzyme crystal structures do not reflect active conformation of the DNAzyme in solution [10,11]. Proposed [4] mechanism of the DNAzyme promoted target RNA cleavage suggests internal phosphoester transfer involving the 2 -hydroxyl group, assisted by two metal cations, which requires (1) an in-line attack on the phosphorous center of the scissile phosphate of the RNA target by the neighboring 2 -oxyanion, via (2) dianionic phoshorane intermediate complex, followed by (3) the degradation of this pentacoordinate structure to yield the cleavage products. However, the kinetic characteristics of the cleavage reaction were found [12] to be inconsistent with exclusive use of this catalytic scheme [12]. Thus, 10–23 DNAzyme appeared to reach saturation with high concentrations of Mg2+ , but the rate constant for this enzyme shows no indication of plateau even at pH 9 [12]. Under suboptimal reaction conditions (30 mM Mg2+ ), the deoxyribozyme achieves a rate constant (∼5 min−1 ) that is in excess of the speed limit predicted by this kinetic model [12] (see also Santoro and Joyce [4]). In addition, the 10–23 deoxyribozyme does not exhibit the thio effect [13], familiar for the hammerhead ribozyme, indicating that this enzyme achieves its sizeable rate enhancement most likely without using metal coordination to a nonbridging phosphate oxygen [12]. To get an insight into the molecular structure of the active 10–23 DNAzyme and to investigate mechanism of the DNAzyme-promoted transesterification reaction, we have performed theoretical modeling of the active and nonactive 10–23 DNAzyme complexes with complementary 19mer RNA target of the nucleotide sequences studied experimentally by Zaborowska et al . [9]. Our selection of the mutation positions in the 10–23 DNAzyme for the present simulation studies has been based on the experimental kinetic data [9] of the target RNA cleavage, measured employing systematic substitution of nucleotides in the catalytic core by naturally occurring nucleotides [9]. Molecular structure, conformations, and internal motions of six selected native and mutated 10–23-type deoxyribozymetarget RNA complexes have been studied in silico using molecular mechanics (MM) and dynamics (MD) techniques in an aqueous medium in presence of either Mg2+ or K+ cations. Two of these complexes, involving 10–23 DNAzyme and its mutant having residue T8 (T36) substituted by cytosine (Figure 7.1), represent an enzymatically favorable situation, which in biochemical experiments in vivo [9] led to selective and effective cleavage of the target RNA in presence of Mg2+ cations. Other three investigated complexes represent negative controls and involve DNAzyme mutants having guanosine residues in the positions 2 , 6 , and 14 of the catalytic core (G30, G34, G42) substituted by adenosines (Figure 7.1). In the experimental conditions [9] these three mutated deoxyribozymes have shown no cleavage of the target RNA, even in presence of Mg2+ ions in high concentrations. Another negative control we have selected to use is the original 10–23 DNAzyme in complex with the target RNA and in presence

RESULTS AND DISCUSSION

275

of monovalent K+ cations, which have experimentally been shown to suppress the catalytic activity of the DNAzyme [3,4]. Recently Nawrot et al . [14] have performed RNA cleavage studies with the modified 10–23 DNAzymes, in which the phosphates in the catalytic core were systematically substituted with P-stereorandom phosphorothioates (PS). They demonstrated that the cleavage rates were dependent on the position of the thiosubstitution in the 10–23 DNAzyme. They also showed that the thio-substitution at P5 phosphate (Figure 7.1) resulted in complete loss of the enzymatic activity [14]. We have therefore selected two stereoselective P5-substituted DNAzyme phosphorothioates for the modeling to understand how they are structurally different from the native counterpart. Selected examples of the active deoxyribozymes in different ionic conditions, as well as those DNAzyme mutants lacking the enzymatic activity (Figure 7.1), have been expected to systematically highlight structural and conformational differences between the original and mutated DNAzymes. This should allow identification of structural components and internal motions that led to or suppress the ability of the DNAzyme to cleave the target RNA. Restrictions of the employed MD technique does not allow step-by-step follow-up of the cleavage reaction due to the fact that parameters of the force field remain constant during the whole simulation, so the sequence of events during the transesterification reaction taking place in presence of the DNAzyme cannot be restored. However, the obtained molecular structures represent equilibrated initial state just before the transesterification reaction. This, in combination with information about timedependent behavior and dynamics of the simulated DNAzyme–RNA complexes, as well as about spatial distribution and time-dependent dynamics of the Mg2+ cations, provide important clues about the initial conditions of the DNAzyme promoted transesterification reaction and about its plausible mechanism.

7.2. RESULTS AND DISCUSSION

All molecular structures of the original and mutated 10–23 DNAzyme complexes with the target RNA (Figure 7.1) have been optimized and evaluated in the time-dependent manner during six 2 ns MD simulations based on Cheathan–Kollman’s [15] procedure employing modified version of Amber 1994 force field implemented in amber 7 program package [16] using explicit water solvent and K+ or Mg2+ counter-ions to mimic the experimental conditions (see details of the Calculations section). 7.2.1. General structure of the 10–23 DNAzyme 7.2.1.1. Original 10–23 DNAzyme in the monovalent and bivalent metal ion environment The presence of divalent cations, especially Mg2+ , was experimentally found to be vital for the 10–23 DNAzyme promoted cleavage of target RNA to occur. To investigate the exact influence of the divalent versus

276


DNAzyme with Mg2+(active)

(a)

DNAzyme with K+ (not active)

(b)

DNAzyme T8 C8 with Mg2+ (not active) (c)


(d)


(e)

DNAzyme G14 A14 with Mg2+ (not active) (f)

Figure 7.2 Structure of the catalytic pocket formed by folding of the catalytic loop in the catalytically active (A, C, with Mg2+ ) and inactive (B, with K+ ; D–F, with Mg2+ ) 10–23 DNA enzyme and its mutants (single nucleobase mutation G to A in positions 2 , 6 and 14 or T to C in position 8 in the catalytic core) in a complex with the target RNA. Enlargements show the location of cations near the scissile phosphate and key distances ˚ from Mg1 and Mg2 to pro-Rp oxygen atom of the scissile phosphate and from Mg3 (A) to O5 of the leaving rU11. For stereo views of these compounds see Figures S9–S14 in SI, Appendix I. (Full-color version of the figure appears in the color plate section.)

monovalent cations on the molecular structure of the 10–23 DNAzyme we have simulated behavior of the DNAzyme complex with the target RNA in presence of Mg2+ and in the presence of K+ (deoxyribozyme-RNA complexes A–B, Figure 7.2) in explicit water solvent. 7.2.1.1.1. DNAzyme structure in presence of potassium cations In presence of K+ ions (i.e., inactive catalytically), the DNAzyme-RNA complex is found to be a good example of a typical B-type hybrid DNA–RNA duplex [15] created by flanking arms (T20-A28, G44-C52) with the catalytic core “hanging”


277

from the side of the duplex (Figure 7.2, deoxyribozyme-RNA complex B). The sugar pucker in the RNA strand of the duplex is varying between North-type (with exception of rC15 all pyrimidine residues: rU3, rC8, rC9, rU11, and rC12, for details see Table S1, and Figure S3 in SI, Appendix I) and South-type (all purines: rG1, rG2, rG4-rA7, rA13-rA19, for details see Table S1, Figure S3 in SI, Appendix I) conformation. The residues of the DNA strand, including those of the catalytic core, have the sugar moieties in the South-type conformation (for details see Table S1, Figure S3 in SI, Appendix I). Although the average structure of the duplex remained over time largely unchanged (RMSd between snapshots ˚ Figure S5 in SI, Appendix I), the nucleotides of the last 500 ps are up to 1.7 A, of both the catalytic loop and the duplex show significant internal dynamics with the pseudorotational phase angle (P) fluctuating within ±16–30◦ of its average value. Average values of the pseudorotational phase angle are provided in Table S1 and Figure S3 in SI, Appendix I. 7.2.1.1.2. DNAzyme structure in presence of magnesium cations Presence of Mg2+ cations, which in experiments makes the DNAzyme catalytically active [2,7,9], lead in our simulation to a significantly different molecular structure of the DNAzyme–RNA complex (Figure 7.2, deoxyribozyme complex A), as compared to that formed in presence of monovalent K+ cations (Figure 7.2, deoxyribozyme complex B). The overall folding of the catalytic loop has been found significantly changed, being no longer folded away from the duplex, as observed in presence of K+ (Figure 7.2, complex B; Figure S1 in SI, Appendix I, complex B, ˚ between the scissile phosphate and the closest C13 (C41)—G14 (G42) ∼15.3 A phosphate; compare with complexes A, C–F in Figure S1 in SI, Appendix I), but being bent toward the duplex. It appears being wrapped about the stem of the duplex so that a stretch of the catalytic loop relocates close to the scissile phosphate (Figure 7.2, complex A; Figure S1 in SI, Appendix I, complex A, P–P distance between the C13 (C41)–G14 (G42) phosphate and the scissile ˚ phosphate is ∼6.5 A). While the flanking arms of the DNAzyme are still forming a stable DNA:RNA heteroduplex with the target RNA, the sugar conformation of the residues in the DNA strand in the presence of Mg2+ cations is found to be predominantly North-type (Table S1 in SI, Appendix I), which is not like in a typical DNA:RNA heteroduplex [15]. The interaction of Mg2+ cations with negatively charged phosphates leads to a shift of the South –North pseudorotational equilibrium of the sugar moieties in the DNA strand toward North-type conformation in T22, A28, G31 (G1 ), T36 (T8 ), C41 (C13 ), T46, C47, and T49 residues (Table S1, Figure S3 in SI, Appendix I). The divalent cations also direct folding of the catalytic loop bridging in some instances (phosphates in positions P3, P6, P9 in the native DNAzyme, Table S3 in SI, Appendix I) together the phosphates in the catalytic loop and phosphates in the backbone of the target RNA and/or of the recognition arms of DNAzyme (Table S3 in SI, Appendix I). As the result, the catalytic loop of the 10–23 DNAzyme folds in a particular way (Figure 7.2, Figure S1 in SI, Appendix I),

278


bringing together stretches of backbones of the target RNA and the catalytic loop (Figures 7.2 and 7.5). This creates an electrostatic pocket, which attracts, ˚ of the scissile phosphate lines—up, and holds three Mg2+ cations within 6 A (Figure 5), notably in cases of the deoxyribozymes (shown in Figure 7.2, insets A and C), which have shown enzymatic activity [9]. Our simulations have shown that mutations of the key residues in the catalytic pocket, stereoselective thio-substitution, and monovalent cations environment are changing folding of the catalytic loop, thus disrupting/rearranging the structure of the active site of the DNAzyme. As a result of these structural rearrangements, the number of magnesium cations strategically positioned close to the scissile phosphate decreases to none to one cation (Figure 7.2, deoxyribozymes in Insets B, D–F, Figure S4 in SI, Appendix I), which apparently is inadequate to promote and/or assist cleavage of the scissile phosphate. The Mg2+ cations are also found to influence the conformations of some phosphates, changing the β (P-O5 -C5 C4 ) torsion at catalytic loop’s residues A5 (A33), A9 (A37), A15 (A43) (Table S2 in SI, Appendix I) to unusual syn conformation. Other key backbone torsions α (O3 (i-1)-P-O5 -C5 ) and γ (O5 -C5 -C4 -C3 ; Figure S3, Tables S1, S3 in SI, Appendix I) are found in mostly —sc and +sc conformations, respectively, as in presence of K+ cations (Figure S3 in SI, Appendix I). Similarly, the residues at the ends the catalytic core and their neighbors (A28-G29, A43-G44; Figure S3, Tables S1, S3 in SI, Appendix I) show unusual backbone conformation switching α and γ torsions to ±ap. Relative higher flexibility of the sugar moiety of these key residues mirrors the behavior of the local helical and base step parameters directly attributed to these residues (Figure S6 in SI, Appendix I). 7.2.1.2. Influence of the mutations in the catalytic core Systematic substitution of nucleotides in the catalytic core by other naturally occurring nucleotides revealed [9] that the nucleotide sequence of the catalytic core is highly conserved and the substitution at any but T8 (T36) position results in partial or complete loss of catalytic activity. We have also investigated four singlepoint mutations introduced into the catalytic core of 10–23 DNAzyme, namely T 8 → C8 (deoxyribozyme complex C, Figure 7.2), G2 → A2, G6 → A6 and G14 → A14 (Figure 7.2, deoxyribozyme complexes D–F, respectively) in presence of Mg2+ cations in aqueous environment (see Experimental section for details). 7.2.1.2.1. Pyrimidine mutation at position 8 of the catalytic loop The T to C mutation at position 8 of the catalytic core (Figure 7.1, Inset B, complex C in Figure 7.2) has resulted in the molecular structure that has a catalytic loop ˚ loop folded similar to that of the original DNAzyme (RMSd of duplex ∼3 A, ˚ ˚ ∼2.2 A, sugar ∼3 A). Structural adjustments due to the mutation are probably arising from the difference between the C7 -C8 -A9 stacking motive in the T8 → C8 mutated DNAzyme and the C7 -T8 -A9 stacking pattern in the original 10–23 DNAzyme (Figure S2 in SI, Appendix I). The folding of the catalytic loop of the T8 → >C8 mutated DNAzyme (Figure 7.2) is actually positioning the backbone


279

˚ closer to the scissile phosphate compared to that of the catalytic loop ∼1.2 A ˚ compared to 6.5 A ˚ for the P11–P41 distance, of the original DNAzyme (5.3 A Figure S1 in SI, Appendix I, Insets C and A, respectively). However, although the position of Mg2 at the active center of the T8 → C8 mutated DNAzyme remains virtually the same as in the native counterpart (Mg2 distance to pro-Rp oxygen of ˚ and 4.1 A, ˚ in the native and the T8 → C8 mutant, the scissile phosphate is 4.4 A respectively, Figure 7.2, Insets A and C), the position of the key Mg1 cation ˚ further from the scissile phosphate (distance is found localized about ∼1.3 A ˚ Figure 7.2, from Mg1 to pro-Rp oxygen of the scissile phosphate is 4.2 A, Insets A and C) than that in case of the original DNAzyme (Mg1 to pro-Rp ˚ Figure 7.2, complex A). This oxygen of the scissile phosphate distance is 2.0 A, suggests that Mg1 in the T8 → C8 mutated DNAzyme is interacting with the scissile phosphate not directly, but via a water molecule. This results in weaker electrostatic interaction of the Mg1 with the scissile phosphate, which tunes somewhat down the reactivity of the phosphate. This probably explains why the closer proximity of the catalytic loop and the scissile phosphate (Figure 7.2, deoxyribozyme A vs. C) does not result in increase of the relative catalytic potency of the T8 → C8 mutant [9]. 7.2.1.2.2. Purine mutations at positions 2, 6, and 14 of the catalytic loop Change of folding of the catalytic core has also been found as the main consequence of the G to A mutations at positions 2 , 6 and 14 (Figure 7.1, complexes D, E and F in Figure 7.2, respectively). These mutations seem to introduce local perturbations (modified nucleotide and its neighbors), which affect the overall ˚ longer distance between the folding of the catalytic loop, resulting in ∼1.5 A ˚ compared to ∼6.5 A ˚ in C13 -G14 phosphate and the scissile phosphate (∼7.8 A the original DNAzyme, Figure S1 in SI, Appendix I, Panels D–E vs. A). The ability of the active center to hold key Mg2+ cations and to direct/arrange them properly to assist the transesterification reaction has been found reduced (see the positioning of cations in the catalytic pocket shown in Figure 7.2, Panels D–F vs. A). As in the case of the T8 → C8 mutation, the main structural perturbations have probably been caused by different stacking patterns of the G1 -A2 -C3 , A5 -A6 -C7 and C13-A14-A15 triads as compared to that of the original 10–23 DNAzyme (Figure S2 in SI, Appendix I). 7.2.1.3. Influence of the thio-phosphate substitution in the catalytic core Systematic substitution of phosphates in the catalytic core of 10–23 DNAzyme with P-stereo-random phosphorothioates by Nawrot et al . [14] have shown variation of the relative cleavage rates depending on the position of thio-substitution (PS). Thus, the rates decreased by up to 50% in cases of the substitution at P2, P4, P9–P13 positions while they remained largely unchanged (P3, P6, P7, P14) or even increased (P1, P8, P15) [14] in other cases (for phosphates numbering see Figure 7.1, inset C). The authors [14] suggested that in the cases showing cleavage rates reduced by one half, only one of the nonbridging phosphate oxygens (pro-Rp or pro-Sp ) is directly involved in coordination with

280


divalent metal ion(s), whereas phosphorothioates at positions P3, P6, P7, and P14–16 seem to have no functional relevance in the deoxyribozyme-mediated catalysis. 7.2.1.3.1. Stereoselectivity of the phosphates in the catalytic core (P2, P9–P12 phosphates) Folding of the catalytic loop positions pro-Sp and proRp atoms of the catalytic loop phosphates in a different chemical environment which depends on the distance and orientation toward the DNAzyme duplex with the target RNA. Thus, pro-Sp atoms of P1–P6 phosphates have been found buried inside the major grove of the duplex, whereas pro-Rp atoms of P9–P16 phosphates have been found located in the positions where indirect (via cation) interactions with the target RNA backbone are possible (for details, see Table S13 in SI, Appendix I). This effectively makes interactions of the pro-Sp and pro-Rp atoms in the catalytic loop phosphates stereoselective. Changes in the cleavage rates [14] observed as the result of thio-substitution in the catalytic core [14] apparently reflect this stereoselectivity. Thus, in the cases of thio-phosphate substitution at P2, P9–P12 positions, the cleavage rates [14] have been shown to be reduced by ≈50% . This was interpreted [14] that only one stereoisomer is enzymatically active. The results of our calculations support this notion because only pro-Rp of P2 and pro-Sp of P9–P12 positions are found to be exposed to the solvent and ionic environment (Table S13 in SI, Appendix I). 7.2.1.3.2. Bridging and interaction with other phosphates in the folded conformation of the active DNAzyme (P3 and P6 phosphates) Only pro-Rp oxygen atom of the P3 phosphate has found to be associated with one Mg2+ cation while pro-Sp atom remains inaccessible for the Mg2+ cations during the whole 2 ns simulation of the native DNAzyme-RNA complex (Figure 7.2, deoxyribozyme complex A). This may suggest that on a stereo-random thio-substitution at P3, the cleavage rate will be reduced by ∼50%. The experimental cleavage rate was, however, found to be 0.89 of that of the native [14]. The probable cause of this discrepancy is the location of the P3 phosphate (phosphorotioate) at the beginning of the catalytic loop, which allows the associated Mg2+ cation to bridge pro-Rp oxygen at P3 with pro-Rp oxygen in the backbone of the target RNA (rG27-rA28 phosphate) and thus to stabilize and direct the folding of the catalytic loop. We expect this kind of metal-assisted bridging remain even on thio-substitution, which preserves folding of the catalytic loop into active conformation reducing the anticipated thio effect. Similar reduction of the thio effect in the P6 thio-substituted DNAzyme-RNA complex is probably due to the fact that P6 microenvironment consists of one Mg2+ atom complexed with pro-Sp oxygen bridging to pro-Rp of A5-G6 phosphate (Figure S1 in SI, Appendix I). The suggested scenario has, for example, been evident from our simulations of the P5 thio-substituted DNAzyme (Figure S7 in SI, Appendix I), where the Mg2+ atom associated with pro-Sp oxygen of P7 bridges to pro-Rp oxygen of G44-G45 phosphate (Table S13, Figure S3 in SI, Appendix I) on pro-Sp PS substitution at P5.


281

7.2.1.3.3. Catalytic loop conformational changes on sulfur substitution at P5 To get an insight into the most intriguing 16-fold reduction of cleavage activity [14] in the case of the thio-deoxyribozyme substituted at P5 [14], we performed two 2 ns MD simulations of the DNAzyme pro-Sp and pro-Rp Ssubstituted at P5. Comparison of the molecular structures of the native 10–23 DNAzyme and its P5 pro-Sp and pro-Rp counterparts (Figure 7.2 deoxyribozyme complex A, Figure 7.4, Figures S1, S7 in SI, Appendix I) suggests that on phosphorothioate substitution at P5 position, the Mg2+ cations associated with either sulfur atom at P5 pro-Sp position or with sulfur atom at P5 pro-Sp position (P5 pro-Sp or pro-Rp substitution, respectively) bridge the catalytic loop and the backbone of the target RNA between C3 -T4 -A5 stretch of the catalytic loop and the rG2-rU3 phosphate (Figure S3 in SI, Appendix I). This changes folding of the catalytic loop and, consequently, disrupts the active site arrangements (Figure S7 in SI, Appendix I, Insets A and B) pushing pro-Rp associated Mg2+ at P14 in ˚ (Figure S7 in SI, the active site (Mg2 position, Figure 7.4) by more then 2.5 A Appendix I, Insets A and B) away from the scissile phosphate, which apparently hinders the enzymatic activity [14] by the P5 substituted thio-deoxyribozymes. 7.2.2. Structure of the catalytic site

The original 10–23 DNAzyme and all the considered mutants in presence of Mg2+ cations (deoxyribozyme complexes A, C–F, Figure 7.2) have shown con˚ in the sistently near identical DNA–RNA duplexes (backbone RMSd < 2.6 A) recognition arms region and similar overall folding of the catalytic loop resulting in creation of the electrostatic pocket between backbones of the RNA strand and that of the catalytic loop (Figure 7.2, insets A, C–F). The configuration of this pocket and its ability to attract, hold, and direct metal cations toward scissile phosphate have, however, been found to vary drastically (see scissile phosphate P11—Mg2+ distances, Figure S4 in SI, Appendix I) between the catalytically active [9] and nonactive [9] 10–23 DNAzyme-type deoxyribozymes (Figure 7.2). The folded catalytic core is found to be able to come close enough to the scissile phosphate only under very strict experimental conditions [3,4,9], which makes it not surprising to find a single nucleotide mutation in the catalytic loop having profound effect on the overall catalytic activity [9]. Local conformational changes initiated by the single nucleotide mutation lead in the single-stranded catalytic core to small adjustments along the strand—a “domino effect”—resulting in overall different folding of the catalytic core and, more important, rearranging the structure of active site. The catalytic loop of the active original 10–23 DNAzyme [3,4] and the T8 → C8 mutated analog [9] (A and C in Figure 7.2) has been found to be folded so that it has created a catalytic pocket between active loop and target RNA backbones. Functionally, this active site has been found to attract and ˚ of the scissile phosphate and 5 -O of the position three Mg2+ cations within 6 A leaving rU11 (Figure 7.3 for 10–23 DNAzyme), which have also been found to be distinctively associated with the scissile, C13 -G14 and A12-C13 phosphates and remained effectively immobilized in their respective positions for the whole

282


Cleavage products H N

O

O

N

O

H2O

N

O

O

P

O Mg1 O

5′

Target RNA

+

5′

O

O

NH

N

O

O

O

3′

O

3′

O 3′

O

N

N

O

O

NH2

O O

O OH

O–

P

NH2

O

C13

DNA enzyme

N

O

HO

O

N

O NH

N

N

O O

P O

rG10

O O

NH2 N

G14

O Mg2 – O H2O

rU11

NH N

3′ O

H N

5′

OH

Mg3

NH2

rG10

O

N

HN

O

rU11

O

O

O

–

P

O

5′

O

O

N

O

N N

N

C13

N NH2

H2N

G14 N H

O

Distances: Mg1 to O atom of the scissile phosphate: 2.0 Å Mg2 to O atom of the scissile phosphate: 4.3 Å Mg2 to O5′ of rU11: 5.4 Å Mg3 to O atom of the scissile phosphate: 6.0 Å Mg3 to 2′-O of rG10: 5.9 Å Mg3 to O5′ of rU11: 4.9 Å

Figure 7.3 Structure of the catalytic site in the 10–23 DNAzyme and plausible mechanism of the 10–23 DNAzyme action as originally proposed in the “two metal ion” mechanism of cleavage by ribozymes (for hammerhead ribozymes see review in Ref. 17). See the text for discussion. (Full-color version of the figure appears in the color plate section.)

length of the 2 ns MD simulation (see scissile P11—Mg2+ distances, Figure S4 in SI, Appendix I). The folding of the catalytic loop in the G2 → A2, G6 → A6 and G14 → A14 mutated deoxyribozymes (D–F in Figure 7.2) has been found ˚ wider gap between the RNA and catalytic loop backbones, to create about 1.5 A thus making the electrostatic pocket less effective in attracting Mg2+ cations. As a result, only one (G2 → A2) or two (G6 → A6, G14 → A14) cations has been ˚ of the scissile phosphate (see scissile P11—Mg2+ found positioned within 6 A distances, Figure S4 in SI, Appendix I). The folding of the catalytic loop apparently makes access of Mg2+ cations to the scissile phosphate stereoselective. In our simulations the Mg2+ cation in position Mg1 (Figure 7.3) has always been found located only from the side of the catalytic pocket, thus effectively associating with the nearest nonbridging oxygen atom of the scissile phosphate (pro-Rp atom) only.


10-23 DNAzyme + Mg2+

283

DNAzyme pro-S sulfur DNAzyme pro-Rpsulfur substituted at P5 + Mg2+ substituted at P5 + Mg2+

B

D

Figure 7.4 Comparison between molecular structure of active 10–23 DNAzyme and those of the inactive DNAzymes having stereodefined thio-phosphates (pro-Sp and proRp ) substitution at P5 position of the catalytic loop. Insets B and D expand region around scissile phosphate (Inset B) and around loop end of the catalytic loop (Inset D). Sulfur substitution at distant P5 phosphate in both pro-Sp and pro-Rp conformations is found to facilitate additional interactions with rG4-rA5 phosphate of the target RNA (see Inset D). The net result of this interactions manifests as change in the folding of the catalytic loop leading to structural and electrostatic rearrangements at the catalytic site (see Inset B in Figure S7 in SI, Appendix I), which leads to a shift of magnesium cation in key ˚ Mg2 position further from pro-Sp oxygen atom of the scissile phosphate (from ∼4.3 A ˚ (Full-color version of the figure appears in the color plate section.) to 5.8–6.0 A).

7.2.3. Plausible Mechanism of the 10–23 DNAzyme action

Similarity between final products of the RNA cleavage catalyzed by 10–23 DNAzyme and hammerhead ribozyme (5 -hydroxyl and 2 , 3 -cyclic phosphate esters), together with the divalent metal cations’ dependence of these enzymes has been used as an evidence [4] to support a general acid–general base mechanism involving metal-assisted deprotonation of 2 -hydroxide adjacent to the cleavage site, followed by SN 2 attack of the scissile phosphorus by the generated nucleophilic 2 -oxyanion yielding observed cleavage products. It was suggested [4] that the metal ion may participate either as a metal hydroxide that functions as a general base to assist in deprotonation of the 2 -hydroxyl or as a Lewis acid that coordinates directly to the 2 -hydroxyl and enhances its acidity. Furthermore, a general “two metal ion” mechanism of action, hypothesized for the hammerhead ribozymes [17] (for review see Takagi et al . [18]), suggests that metal cations are also stabilizing the transition state during in-line attack by the 2 -oxyanion as well as stabilizing leaving 5 -hydroxyl product. The metal might also be playing a purely structural role, helping to organize the enzyme into its active conformation [4]. Our calculations suggest that the aforementioned mechanism of transesterification reaction promoted by 10–23 DNAzyme[4,19,17,18] is similar to that utilized by the hammerhead ribozymes [17,18]. However, the hammerhead ribozyme was

284


found [20] to be able to utilize alternative routes of catalysis, which involve functional groups of ribozyme instead of the metal cations. In contrast, the catalytic activity of DNAzyme is only metal dependent, which probably is originated from the in vitro selection procedure employed [4] to isolate the catalytically active DNA sequences. There also are inconsistencies between the overall similar solution [21–23] and crystal [24,25] structures and the biochemical data identifying essential functional groups. Thus, the crystal structures of the hammerhead ribozyme [25–28] have revealed the scissile phosphate being located not in a position aligned for the SN 2 attack, and no functional group has been found located strategically in position to carry out the proton transfer or to assist as electrostatic catalysts. The solution structures of the hammerhead ribozyme studied by NMR, FRET [21] hydroxyl radical protection, and transient electric birefringence methods [22,23] were found to be quite similar to molecular structure emerged from x-ray studies [25,24] with little differences attributed to conditions of the crystallographic packing on crystallization. It had also been shown [19,25] that the ribozyme can cleave in the crystal, suggesting that slight conformational changes would allow for hydrolysis. On the other hand, later studies [29,30] have suggested that the ribozyme undergoes significant conformational ˚ from changes, which involve large-scale movement of domain 2 by 10–15 A the position seen in the crystal structure to position allowing the two nucleosides flanking the cleavage site to stack on two particular guanosines, G8 and G12, in a transient conformation. Summarizing the large number of inconsistencies between the available structures and the biochemical data identifying essential functional groups, Blount and Uhlenbeck [26] concluded that the hammerhead ribozyme must undergo a conformational isomerization prior to the transition state, which is likely to be promoted/stabilized by interactions between essential functional groups in domains 1 and 2 within the core. The isomerization must also include changes in the cleavage site to position the scissile phosphate for the in-line attack, whereas one or more nucleotide functional groups are most likely positioned near the scissile phosphate to act as a general acid or base catalyst or to stabilize the transition state [26], the role of which may also be attributed to divalent ions, most notably Mg2+ , at the cleavage site. It is likely [26] that hammerhead ribozyme must undergo a conformational isomerization prior to the transition state, which should also include changes in the cleavage site to position the scissile phosphate for the in-line attack. Despite these uncertainties and the fact that the number of Mg2+ ions involved in catalysis by hammerhead ribozymes remains obscure, a general double-metal-ion mechanism seems to be well suited to give general explanation of catalysis by 10–23 DNAzyme, hammerhead ribozymes or by protein enzymes, such as polymerases and alkaline phosphatases [17,31]. 7.2.4. Two-metal or three-metal ion mechanism of 10–23 DNAzyme action?

The significant difference that distinguishes the catalytically active original 10–23 DNAzyme and its T8 → C8 mutant and their inactive counterparts, as found in


285

our simulations, is the ability of the former to immobilize three Mg2+ cations at the active site for a sufficiently long time (in our simulation 100% of 2 ns MD), long enough to initiate and assist the transesterification reaction. Notably, the G14 → A14 mutated deoxyribozyme was able to recruit three Mg2+ cations at the active site only for ∼10% of the simulation time (∼0.2 ns), which in conjunction with the experimental results [9] (i.e., catalytically inactive [9]) clearly suggests that this retention time of Mg2+ cations by the active site is not sufficient to promote the transesterification reaction. 7.2.4.1. Cyclic phosphate formation To form the 2 , 3 -cyclic phosphate, -O− (2 -oxyanion) nucleophile at rU11 is required, a general base action by 2 which can possibly be generated by metal-coordinated hydroxide ion acting as a general base, abstracting the proton from the 2 -OH or, alternatively, by a metal ion acting as a Lewis acid to coordinate with the 2 -oxygen lone-pair to accelerate the deprotonation of 2 -OH [31]. The inversion of configuration of the scissile phosphate after cleavage suggests a direct in-line attack with development of a pentacoordinate transition state or an intermediate, which requires the 2 -oxygen nucleophile to be in line with the 5 -oxygen of the scissile phosphate for cleavage to occur [32,26]. The positions of the three Mg2+ cations closest to the scissile phosphate (sites Mg1, Mg2, Mg3 in Figures 7.2 and 7.3) observed in our 10–23 DNAzyme MD simulation (A in Figure 7.2) suggest that Mg1 and Mg2 cations associated with the scissile phosphate are likely to be involved in formation and stabilization of the transitional active site configuration (Figure 7.3). The closest Mg1 ion is apparently directly coordinated to the nonbridging oxygen of the scissile phosphate rendering the phosphorus center more susceptible to nucleophilic attack. The Mg2 ion, which is associated with the C13 -G14 phosphate ˚ from O2P of the scissile phosphate of the catalytic core, is located within 4.3 A (Figure 7.3), which makes it a good candidate for participation in the cleavage by stabilizing the charged trigonal-bipyramidal intermediate (or transition state) through hydrogen bonding between a metal-bound water molecule and the nonbridging oxygen. 7.2.4.2. Is there any evidence of the 2 -OH activation by Mg2+ ? The third magnesium ion, Mg3, present in the active site of the 10–23 DNAzyme (Figure 7.2, complex A) has been found located at a position that is relatively close to the 2 -OH of rG10 (Mg3, distance to oxygen atom of the 2 -OH is ˚ Figure 7.2, Panel A). Assisted by two metal cations [4], this 2 -hydroxyl 5.9 A, group was suggested [4] to participate in the internal phosphoester transfer and in the in-line attack on the phosphorous center of the scissile phosphate. Thus, the Mg3 may have some role as a general base catalyst to initiate the abstraction of proton from 2 -OH of rG10 to form a 2 -oxyanion participating in the in-line attack. However, the relatively long distance to the oxygen of 2 -OH of rG10 ˚ suggests otherwise, and without relocation of the Mg3 from its current (5.9 A) ˚ this seems to position to a position closer to the 2 -O of rG10 by at least 2 A, be an unlikely scenario, even taking into account possible interaction via water

286


molecule. Because the 2 -OH itself is found not in the position for a direct inline attack on the scissile phosphate, a precleavage conformational change at the catalytic center might be needed to facilitate the in-line attack in the general base catalysis scenario, which might also bring the Mg3 in place to promote this attack. 7.2.4.3. Stabilization of the developing negative charge on the 5 oxygen as a leaving group The developing negative charge on the 5 oxygen leaving group can possibly be stabilized by a proton that is provided by a water molecule from a bulk of solvent or by an Mg2+ -bound water molecule as a general acid catalyst or, alternatively, by direct coordination of an Mg2+ ion that acts as a Lewis acid catalyst. Judging from the distance (Mg3 to O5 of ˚ Figure 7.2, Panel A), the Mg3 cation may also participate in stabirU11: 4.9 A, lizing 5 -OH leaving group of rU11 via Mg2+ bound water molecule or hydroxyl. For this role the Mg3 cation is competing with the Mg2 (Figure 7.3), which is ˚ from O5 of rU11. localized within 5.4 A Due to the limits of the employed theoretical (molecular mechanics) model, which does not have tools of quantum mechanics to follow the reaction path, the obtained molecular structures should be considered representing initial configurations of the “10–23”-type DNAzymes that must undergo a conformational change to allow in-line attack in the transesterification reaction.

7.3. CONCLUSION

Our in silico simulations have shown considerable insight into the molecular structure of the active conformation of the 10–23 DNA enzyme, which has been unknown to date. Simulations and comparison of the molecular structure of the original 10–23 DNAzyme with those of the catalytically active and inactive single nucleotide substituted mutants have provided important mechanistic clues for understanding of the catalytic function of the DNAzyme. This study has also shed light on the role of Mg2+ cations in the 10–23 DNAzyme promoted cleavage of the target RNA. Correlation between the molecular structure of the DNAzyme and enzymatic data suggest that the Mg2+ cations play a dual role in the 10–23 DNAzyme promoted cleavage (1) inducing proper folding of the catalytic loop of the DNAzyme to transform to an active conformation and (2) participating in the transesterification reaction. (A) Comparison of the structures of the native and mutated DNAzymes in complex with the target RNA with the reported enzymatic activity [9] suggests that the metal-dependent DNAzyme cleavage reaction (Figure 7.3) is assisted by three Mg2+ cations immobilized within the active center as follows: 1. The closest to the scissile phosphate Mg1 cation is interacting directly (native 10–23 DNAzyme, Figure 7.2, complex A) or indirectly via

CONCLUSION

(B)

(C)

(D)

(E)

287

water molecule (T8 → C8 mutants, Figure 7.2, complex C) with the nonbridging pro-Rp oxygen of the scissile phosphate, rendering the phosphorus center more susceptible to nucleophilic attack. 2. The Mg2 cation associated with C13 -G14 phosphate is located within ˚ from the nearest oxygen of the scissile phosphate (Figure 7.2, 4.4 A complex A) possibly stabilizing the charged trigonal-bipyramidal intermediate (or transition state) through hydrogen bonding between a metal-bound water molecule and the non-bridging oxygen. 3. The developing negative charge on the 5 -oxygen atom as a leaving group of rU11 is probably stabilized by Mg3 cation, which is located ˚ of O5 of rU11 (Figure 7.2, complex A). For this role, within 4.9 A Mg3 may be competing with Mg2 cation, which is localized within ˚ from O5 of rU11. 5.4 A The presence of Mg2+ cations is found to have a profound effect on the molecular structure of the DNAzyme, directing folding of the singlestranded 15-membered catalytic loop. Their presence also has a stabilizing effect on a relatively flexible DNA:RNA duplex formed by the flanking arms of DNAzyme strand and the target RNA (average RMSd fluctuations ˚ which is much in the respective simulations with Mg2+ were ∼0.6 A, ˚ fluctuations observed in presence of monovalent K+ lower then ∼2.5 A cations, Figure S5 in SI, Appendix I). Folding of the catalytic loop has been found to result in formation of an electrostatic pocket between the backbones of the target RNA and the catalytic loop. The active site has been shown to be capable of attracting and holding up to three Mg2+ cations (in case of the catalytically active “10–23”-type deoxyribozymes). The rationale behind the conserved nucleotide sequence in the catalytic loop has been found to be due to the need to fulfill strict folding requirements to form an electrostatic pocket close to the scissile phosphate, which is dictated by the stacking interactions within the loop and by the adjustments of the backbone controlled by ionic environment. Any small changes in the stacking pattern at the 5 -end (G1 -G7 ) of the catalytic loop have produced profound effect on the DNAzyme catalytic function, which, as we expect, would also take place with other artificial nucleotides because of change of electrostatics. This, for example, is evident from the fact that the pKa modulation within a single-stranded RNA is known to take place depending on the sequence context [33,34]. Small conformational changes at the 5 -end of the catalytic loop leads in our simulations to repositioning of the 3 -end residues of the catalytic loop, which perturbs the geometry of the active site. This is consistent with the fact that any nucleobase substitution at the positions 1 to 7 of the catalytic core of 10–23-type deoxyribozymes leads to hindering of the catalytic activity [9]. Because nucleotides positions 13 and 14 of the catalytic loop are functional in the catalysis (Figure 7.3), their chemical modification (as well

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as of their neighbors) is expected to be the most promising strategy in order to tune directly the catalytic activity of 10–23 DNAzyme. (F) Simulation results reported in this chapter constitute our first attempt to understand the mechanism of 10–23 DNAzyme enzymatic activity. Due to technical constraints the reported simulations have been limited to 2 ns, which, although being reasoned by stable energy and low RMSd values along the trajectories, may still prove to be inadequate to describe a relatively slow process of nucleic acids folding. Increase of the simulation length will most certainly bring about the need for deploying a newer force field instead of the Amber 94 force field currently used in our simulations. The limitations of Amber 94 have been shown to lead to the deformation of DNA backbone due to artificial noncanonical values of α and γ backbone dihedrals originated from either insufficient sampling along the trajectory or ergodic problems in the potential energy surface [35]. However in sub-10 ns simulations these force field limitations are shown to contribute insignificantly [36] while clearly manifesting in longer simulations (see [36] and references therein). A recent modification of backbone parameters of the Amber force field by Pérez et al . [36] offers the way to prevent α/γ ration from sliding away from the gauche− , gauche+ state toward the gauche+ , trans geometry, and we are presently testing them in our ongoing 20 ns simulations of the native and modified 10–23 DNAzymes. 7.3.1. Details of calculations

The molecular structure of the native 10–23 DNAzyme has been optimized using Amber force field molecular mechanics employing explicit water solvent and Mg2+ cations and Cl− counter-ions to mimic the experimental setup. The initial structure of the native 10–23 DNAzyme has been obtained as the result of molecular mechanics optimization of 3DNA [37] generated structure, followed by 2 ns production run of unconstrained molecular dynamics (MD) simulation. The minimal energy DNAzyme structure from the last 500 ps of this MD simulation has been used to build up the initial structures of all mutated DNAzymes, as well as the starting structure of the DNAzyme MD simulation in presence of K+ cations instead of Mg2+ . The protocol of the MD simulations is based on Cheathan-Kollman’s [15] procedure employing modified version of Amber 1994 force field as it is implemented in the AMBER 7 program package [16]. Periodic boxes containing 15760 TIP3P [38] water molecules to model explicit solvent around the deoxyribozyme-RNA complexes (complexes containing phosphorothioates were surrounded by 16580 TIP3P [38] water molecules) has been ˚ from these molecules in three dimengenerated using xleap extending 12.0 A sions. High ionic content has been simulated by addition of 100 Mg2+ and 150 Cl− ions to simulate effects of magnesium and by 100 K+ and 50 Cl− ions to examine the effect of monovalent cations on example of native DNAzyme in presence of potassium cations.

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Structural analysis of the DNAzyme-RNA complexes has been performed using tools of AMBER 7 [16] and 3DNA [37] program packages. Because the complexes contain both the duplex and single-stranded fragments, helical parameters have been measured separately for the duplex part and for the DNA and RNA strands of the of the DNAzyme–RNA complexes. Visualization and analysis of the molecular structures have been performed using VMD [39] and Jmol [40] molecular visualization programs. The appendix for this chapter is available here: ftp://ftp.wiley.com/public/ sci_tech_med/medicinal_chemistry.

ACKNOWLEDGMENTS

Generous financial support from the Swedish Natural Science Research Council (Vetenskapsr˚adet), the Swedish Foundation for Strategic Research (Stiftelsen för Strategisk Forskning), and the EU-FP6 funded RIGHT project (Project no. LSHBCT-2004-005276), as well as computational time at the National Supercomputer Center (Linköping, Sweden) awarded by the Swedish National Allocation Committee for the High Performance Computing, are gratefully acknowledged. REFERENCES 1. Breaker, R.R. (1997). In vitro selection of catalytic polynucleotides. Chemical Reviews, 97 , 371–390. 2. Breaker, R.R. and Joyce, G.F. (1994). A DNA enzyme that cleaves RNA. Chemistry & Biology, 1 , 223–229. 3. Santoro, S.W. and Joyce, G.F. (1997). A general purpose RNA-cleaving DNA enzyme. Proceedings of the National Academy of Sciences of the USA, 94 , 4262–4266. 4. Santoro, S.W. and Joyce, G.F. (1998). Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry, 37 , 13330–13342. 5. Carmi, N. and Breaker, R.R. (2001). Characterization of a DNA-cleaving deoxyribozyme. Bioorganic & Medicinal Chemistry, 9 , 2589–2600. 6. Carmi, N., Balkhi, S.R. and Breaker, R.R. (1998). Cleaving DNA with DNA. Proceedings of the National Academy of Sciences of the USA, 95 , 2233–2237. 7. Breaker, R.R. and Joyce, G.F. (1995). A DNA enzyme with Mg(2+)-dependent RNA phosphoesterase activity. Chemistry & Biology, 2 , 655–660. 8. Breaker, R.R. (1997). DNA aptamers and DNA enzymes. Curr. Opin. Chemistry & Biology, 1 , 26–31. 9. Zaborowska, Z., Furste, J.P., Erdmann, V.A. and Kurreck, J. (2002). Sequence requirements in the catalytic core of the “10–23” DNA enzyme. Journal of Biological Chemistry, 277 , 40617–40622. 10. Nowakowski, J., Shim, P.J., Prasad, G.S., Stout, C.D. and Joyce, G.F. (1999). Crystal structure of an 82-nucleotide RNA-DNA complex formed by the 10–23 DNA enzyme. Nature Structural & Molecular Biology, 6 , 151–156.

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11. Nowakowski, J., Shim, P.J., Stout, C.D. and Joyce, G.F. (2000). Alternative conformations of a nucleic acid four-way junction. Journal of Molecular Biology, 300 , 93–102. 12. Breaker, R.R., Emilsson, G.M., Lazarev, D., Nakamura, S., Puskarz, I.J., Roth, A. and Sudarsan, N. (2003). A common speed limit for RNA-cleaving ribozymes and deoxyribozymes. RNA, 9 , 949–957. 13. Scott, E.C. and Uhlenbeck, O.C. (1999). A re-investigation of the thio effect at the hammerhead cleavage site. Nucleic Acids Research, 27 , 479–484. 14. Nawrot, B., Widera, K., Wojcik, M., Rebowska, B., Nowak, G. and Stec, W.J. (2007). Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10–23. FEBS Journal , 274 , 1062–1072. 15. Cheatham, T.E., III and Kollman, P.A. (1997). Molecular dynamics simulations highlight the structural differences among DNA:DNA, RNA:RNA, and DNA:RNA hybrid duplexes. Journal of the American Chemical Society, 119 , 4805–4825. 16. Case, D.A., Pearlman, D.A., Caldwell, J.W., Cheatham III, T.E., Wang, J., Ross, W. S., Simmerling, C.L., Darden, T.A., Merz, K.M., Stanton, R.V., Cheng, A.L., Vincent, J.J., Crow-ley, M., Tsui, V., Gohlke, H., Radmer, R.J., Duan, Y., Pitera, J., Massova, I., Seibel, G.L., Singh, U.C., Weiner, P.K. and Kollman, P.A., AMBER 7 . (University of California, San Francisco, 2002. 17. Steitz, T.A. and Steitz, J.A. (1993). A general two-metal-ion mechanism for catalytic RNA. Proceedings of the National Academy of Sciences of the USA, 90 , 6498–6502. 18. Takagi, Y., Warashina, M., Stec, W.J., Yoshinari, K. and Taira, K. (2001). Recent advances in the elucidation of the mechanisms of action of ribozymes. Nucleic Acids Research, 29 , 1815–1834. 19. Scott, W.G., Murray, J.B., Arnold, J.R.P., Stoddard, B.L. and Klug, A. (1996). Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science, 274 , 2065–2069. 20. Canny, M.D., Jucker, F.M., Kellogg, E., Khvorova, A., Jayasena, S.D. and Pardi, A. (2004). Fast cleavage kinetics of a natural hammerhead ribozyme. Journal of the American Chemical Society, 126 , 10848–10849. 21. Tuschl, T., Gohlke, C., Jovin, T.M., Westhof, E. and Eckstein, F. (1994). A threedimensional model for the hammerhead ribozyme based on fluorescence measurements. Science, 266 , 785–789. 22. Amiri, K.M. and Hagerman, P.J. (1994). Global conformation of a self-cleaving hammerhead RNA. Biochemistry, 33 , 13172–13177. 23. Amiri, K.M. and Hagerman, P.J. (1996). The global conformation of an active hammerhead RNA during the process of self-cleavage. Journal of Molecular Biology, 261 , 125–134. 24. Pley, H.W., Flaherty, K.M. and McKay, D.B. (1994). Three-dimensional structure of a hammerhead ribozyme. Nature, 372 , 68–74. 25. Scott, W., Finch, J. and Klug, A. (1995). The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell , 81 , 991–1002. 26. Blount, K.F. and Uhlenbeck, O.C. (2005). The structure-function delemma of the hammerhead ribozyme. Annual Review of Biophysics and Biomolecular Structure, 34 , 415–440.

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CHAPTER 8

LABELING OLIGONUCLEOTIDES TOWARD THE BIOMEDICAL PROBE IL JOON LEE and BYEANG HYEAN KIM Department of Chemistry, Pohang University of Science and Technology, Pohang, South Korea

8.1. INTRODUCTION

Development of highly sensitive and selective probes to recognize biologically important analytes such as peptides, proteins, nucleic acids, carbohydrates, or even whole cells has long been a focus of biomedical research. Probes are known as devices that respond to physical or chemical stimuli and give detectable signals. A probe contains two essential components, namely, target recognition and signal transduction. The target recognition element can be any chemical or biological entity. On the other hand, the signal transduction part is responsible for converting target recognition events to physically detectable signals such as color, fluorescence, nuclear magnetic resonance, and electrochemical signals. To find out efficient signal transduction elements, many labeling methods have been developed [1]. In this chapter, we discuss various types of oligonucleotide labeling, which may have a huge potential toward oligonucleotide-based biomedical probes. Oligonucleotide is one of the most important analytes because it is a carrier of genetic information. Needless to say, genetic analysis methods and protocols have been developed tremendously so far, and they still continue to draw attention of many research groups. Single nucleotide polymorphism (SNP) probing system is one of the good examples showing the current situation. A lot of methods for detecting genetic mutations at single nucleotide level such as SNPs have been developed, but it is still necessary to improve the present techniques or to develop Medicinal Chemistry of Nucleic Acids, First Edition. Edited by L.H. Zhang, Z. Xi and J. Chattopadhyaya. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

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new techniques for simultaneous detection of multiple SNPs mapping. For future development of oligonucleotide-based biomedical probes, the research activities should be aimed at devising cost-effective, high throughput, fast, and highly accurate analytical probes with the help of suitable oligonucleotide labeling [2]. This chapter is therefore devoted to the importance and usefulness of oligonucleotide labeling for the development of biomedical probes. We will describe representative labeling methods and their potential applications toward biomedical research. For any practical use, oligonucleotide labeling should fulfill the following criteria: detection with high sensitivity and selectivity, no interference in the original function after labeling, good thermodynamic stability, costeffectiveness, and easy to use. With these requirements in mind, we chose five attractive methods of oligonucleotide labeling as the topics of this chapter. 8.2. RADIOACTIVE LABELING

The insertion of 13 C and 15 N labeling into oligonucleotides has recently contributed to DNA and RNA structural researches using nuclear magnetic resonance (NMR) [3]. Synthesis of 13 C and 15 N containing RNA by using enzymatic method [4] is a general method that permits other types of overlapped 1 H resonances in heteronuclear multidimensional NMR studies [5]. The consequent increment in precise 1 H assignments and the constraint of merged 1 H-NOE (Nuclear Overhauser Effect) provide the correctness of the consequent structure models [6]. For three-dimensional nucleic acid molecules, specific labels may be needed. Two classes of enzymatic syntheses have been developed. One is selective isotopic enrichment [7], in which oligonucleotide is tagged by residual type, another is segmental isotopic labeling [8], in which only selected regions of oligonucleotides are labeled, and then unlabeled segments are ligated. Despite developments of these types of labeling methods, the best method to assign protons of large nucleic acid structure may be the chemical modification using specifically labeled monomers, especially nonhelical regions [9]. In addition to precise analysis for determination of nucleic acid structure, structure-independent insights can be supported by 15 N-labeling of functional nitrogen atoms for protonation, hydrogen bonding, stacking, hydration, and ligand interactions. The information for these interactions is directly obtainable from changes in 15 N chemical shifts, yet complementary to whole structures determined from 1 H-NOE constraints. One definitive advantage is to increase the meaningful information from a single experiment of NMR, whether selective labeling is utilized for 1 H assignments or 15 N chemical shift data. If many 15 N containing monomers are efficiently incorporated into nucleic acid, the structure of nucleic acid can be precisely determined. The first published synthetic schemes for 15 N labeling of nucleosides showed singly labeled products [10]. 15 N multilabeled nucleosides have been synthesized such as [1,3-NH2 -15 N3 ]-adenosine, [1,7-NH2 -15 N3 ]-adenosine, [1,3-NH2 -15 N3 ]-guanosine, and [1,7-NH2 -15 N3 ]-guanosine, including their 2 deoxy form [11]. In the case of incorporating multilabeled nucleoside into an

294


oligonucleotide fragment, however, the 15 N labels cannot confirm the identity. Incorporation of 13 C labels adjacent to significant 15 N labels in a nucleoside was reported as a solution for this limitation. This route permits induction of two 15 N multi-labeled nucleosides into an oligonucleotide fragment with suitable 13 C labels [3]. Typical examples are [8-13 C-1,7-NH2 -15 N3 ]-guanosine and [2-13 C-1,7-NH2 -15 N3 ]-guanosine, even though there are many other possibilities (Figure 8.1) [12]. Resonances between the 13 C and 15 N labels can be definitively discriminated from the uncoupled spectra.

8.3. ELECTROACTIVE LABELING

Electroactive labeling is generally utilized in the detection of biological and chemical changes of target recognition element at the solid surface of electrodes. The synergistic effect between electrochemical responses and delicate biomolecules will make sensitive and selective recognizing techniques, which are also novel and rapid. Electrochemical probes consist of one electroactive species and the recognition partners, which can be labeled or tagged with the electroactive species. Such labeling may vary the original properties of a main biomolecule, and the consequent changes may not be directly realized, even though electroactive labeling allows the highly sensitive analysis of a single biomolecule. Moreover, the labeling stage induces intangible changes to chemical interactions such as thermodynamic stability, kinetic energy, affinity, and conformational changes. Recently, some results in the field of solid surface electrode have shown that the essential functions of many biomolecules are altered from what is shown before the labeling [13]. Labeling methods have some problems. Nevertheless, they are better approaches than label-free methods because many electroactive labeling probes are available for highly sensitive and selective transduction [14]. In electrochemical oligonucleotide probe, the labeling is usually performed by redox active materials [15,16], which can also intercalate between the base pairs of the double-stranded DNA, although they don’t significantly interact with any other DNA structures such as single-stranded DNA, loop position in hairpin structure, or bulge region. There have been reported various techniques when the labeling materials are enzymes [17], nanoparticles [18], or liposomes [19]. The DNA hybridization probe using electrochemical materials, Co(bpy)3 3+ and Co(Phen)3 3+ , specifically bound to the minor groove of duplex DNA was first reported by Mikkelsen and coworkers [15]. Recent research results in this field showed that the newly synthesized groove binder made by cobalt ion [20] for the detection of HIV DNA sequences displayed a low limit of detection, ∼27 pM. The research work of Ozsoz and coworkers using methylene blue (MB), the famous redox active materials for probing the hepatitis B virus–related sequences [21], is a well-developed system of the sequence specific binding method. The distinguishable aspect of the approach of the Mikkelsen group is that the MB has a higher affinity for the single-stranded DNA rather than for

295

Figure 8.1 Preparation of

13 C

or

15 N

labeled nucleoside (Ada: Adenosine deaminase) [12].

296


double-stranded DNA. MB is performed as a DNA intercalator, especially it has a high affinity for extruded guanine bases. When the position of MB is far from the guanine bases, the signal induced by MB decreases after hybridization. The relationship between the amount of current charge and the number of guanine bases in the fixed probe is linear because the charge can be determined from the number of fixed probes if the number of guanines per probe is known [22]. The electrochemical signal of detecting DNA hybridization mentioned so far is dependent on a variation in the amount of DNA at the external surface of the electrode. In probing systems more recently developed, the formation of a double-stranded DNA is directly affected by electrochemical signals of the probes. This system can be accomplished by using electroactive molecules to probe the variation in electron transfer properties when the flexibility of duplex DNA is changed by hybridization. By introducing a ferrocene tag to the terminal position of the immobilized probes, electrochemical sensing systems can use the structural changes in DNA from the flexible single-stranded DNA to the rigid double-stranded DNA [23]. The ferrocene moiety can be close to the electrode surface due to the flexibility of single-stranded DNA. When the distance from the electrode to the ferrocene moiety is increased by hybridization, recognizable electron transfer can be decreased. Heeger and coworkers [24,25] reformed these concepts to confirm that the ferrocene moiety was located near the surface of the electrochemical probe before DNA hybridization. A ferrocene molecule dangles to a hairpin DNA structure that has stem and loop position, and the hairpin DNA is immobilized on a gold surface (Figure 8.2). Before hybridization, the hairpin DNA structure is constructed; therefore, the ferrocene molecule is placed near the gold surface and shows a fine electrochemical signal. When the target sequence is appeared and hybridized with the loop position of the probe, the distance from the ferrocene moiety to the gold surface is increased, and the electrochemical signal is decreased. Although

Figure 8.2 Schematic diagram of the hairpin DNA electrochemical probe immobilized on Au surface (Fc: ferrocene tag, eT: electron transfer) [25].

CHEMILUMINESCENT LABELING

297

this system indicates a signal turn off method, in which it is easy to make false positives, the limit of detection (10 pM) is very good. Later, the Heeger group has developed the “turn on” sensing probe by using strand displacement strategy [26]. Using the difference in ability of charge transfer between a double-stranded and a single-stranded DNA is also good strategy to signal DNA hybridization. Barton and coworkers are pioneers of the long-range charge transfer, which can transduce the formation of DNA duplex. Using intercalators such as daunomycin or methylene blue (MB), they developed highly selective and simply operating probes [27]. This system is related to the property of double-stranded DNA to ˚ distances or more [28]. Therefunction as a wire for charge transfer about 40 A fore, the daunomycin or MB intercalates into the base pairs of DNA duplex and can be oxidized through charge transfer when a DNA duplex is built up. If the probe didn’t hybridize with the target DNA sequence, then a highly decreased current was detected. This probing method can discriminate single-base mismatched sequences without any washing. (In most other systems, a washing step is needed to separate between perfectly matched and mismatched sequences.) This character makes this system very simple to operate as an electrochemical DNA probe. Representative examples in this direction were reported by Wang and coworkers, who developed multistep probing systems utilizing nanoparticles [18]. This method is utilizing two types of biotinylated DNAs, which can be hybridized by each other. One DNA is immobilized onto a streptavidin-coated magnetic bead, and another DNA is linked with streptavidin-modified gold nanoparticle. Hybridization can be realized by dissolving the gold tags with HBr and performing potentiometric stripping analysis. The same types of detection methods were developed by the Wang group [18]. For probing the hybridization, a label-free system based on the inosine methods has also been reported. Before denaturing the hybridized target sequences the nonhybridized sequences are magnetically collected, and then a signal is detected due to oxidation of guanine, which is amplified by the copper addition. These multistep methods show good limit of detection with the copper amplification (0.8 nM).

8.4. CHEMILUMINESCENT LABELING

Chemiluminescent labeling has been used as a basic tool of research application and clinical assay [29]. The chemiluminescent nonseparated hybridization protection assay (HPA) for probing DNA or RNA sequences was the first example of chemiluminescence use in nucleic acid assay (Figure 8.3) [30]. This method uses an acridinium ester tagged DNA probe, which is easily hydrolyzed to nonluminescent products. When this probe meets the target sequence, then it is hydrolyzed with a chemiluminescent signal (Chugai-Gen Probe). This class of chemiluminescent assay is broadly utilized to analyze for infectious agents. The branched DNA assay (Quantiplex, Bayer) utilized the concept of the chemiluminescent endpoint for probing HCV (hepatitis C virus) RNA [31]. In this

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Figure 8.3 Mechanism of acridinium ester reaction, and principle of the hybridization protection assay (HPA) [30].

system, the probe binds to both immobilized capture probe and target sequence, and then branched DNA reporter probes are hybridized to the bound probe. In last step, alkaline phosphatase-labeled probes react with each branch of the DNA reporter probe, and this step induces a high level of target amplification into the assay. An AMPPD (1,2-dioxetane substrate) is detecting the bound enzyme labels. Using this type of assay, other methods are developed for HIV-1 RNA and hepatitis B virus DNA. Hybrid Capture assay (Digene Corporation) utilizes a dioxetane substrate that has chemiluminescent property for probing alkaline phosphatase [32]. In the procedure of this assay, there are two types of hybridization. One is DNA target with capture RNA probe immobilized antibodies, another is RNA target with DNA probe at the surface of a microwell plate. The hybridized sequences react with an antibody-labeled alkaline phosphatase, and then enzyme labels are attached to the reacted sequences. Probing human papillomavirus (HPV) DNA, Neisseria gonorrhea and Chlamydia trachomatis DNA, and human cytomegalovirus DNA is main purpose of this assay. Enzymes such as horseradish peroxidase (HRP) and hemin can also catalyze chemiluminescent reactions, which can be detected by potentials between reference and working electrode [33]. Luminol firstly reported by Albrecht is frequently utilized for electrochemiluminescent and chemiluminescent reactions [34]. Oxidation of luminol makes an aminophthalate ion in an excited state, and then the aminophthalate ion emits luminescence and returns to the ground state. Luminols are mainly oxidized by peroxidase for detecting analytes such as target DNA and RNA [35].

CHEMILUMINESCENT LABELING

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For detecting oxidative damages of DNA base, Salles and coworkers developed a chemiluminescence microplate assay with FPG (formamidopyrimidine DNA glycosylase) [36]. Willner and coworkers also utilized luminol and HRP derivatives, which contain the rotating system of oligonucleotideconjugated magnetic particles and naphthoquinone-linked magnetic particles for increasing chemiluminescence [37]. By using the rotating system, the chemiluminescence reactions are amplified, and the sensitivity of viral DNA is increased (limit of detection: 1 × 10−14 M). The Willner group called this system a magnetically amplified DNA assay and detected viral (M13 phage) DNA and single-base mismatched DNA. Polymerase-promoted labeling, which can be controlled by temperature and the rotating system, makes low limit of detection such as 8.3 × 10−18 M of M13 phage DNA (Figure 8.4) [38]. Zhou and coworkers produced a cytochrome P450 1A1 (CYP1A1) polymorphisms probing system using differences in the thermal stability between matched and mismatched sequences [39]. Roda and coworkers proposed a probing system for human papillomavirus (HPV) using polymerase-chain reaction (PCR) chemiluminescence enzyme immunoassay [40]. Biotin-conjugated DNA probes immobilized on the streptavidin-coated 384-well plate was hybridized with digoxigenin-linked PCR product, and then antidigoxigenin antibody-labeled HRP quantified the amount of products. This system provided semiquantitative (a) Taq Polymerase dNTPs Biotin-dUTP

Avidin-HRP conjugate

55°C 94°C 72°C 90 Cycles

1 modified magnetic nanoparticle

Biotin Label

1 : 5′–SH–(CH2)6–CCCCCACGTTGTAAAQACGACGGCCAGT–3′

Avidin-HRP

Light detector

(b) O2

H2O2

hν Luminol 3-Aminophthalate

Au electrode S

N Magnet

Rotation

Figure 8.4 (a) DNA-conjugated magnetic particles labeled with the HRP derivatives, (b) Diagram of rotating system of the functionalized magnetic particles and luminol for increasing chemiluminescence [38].

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function on the viral load, with a detection limit of 10–50 DNA copies for human papillomavirus. Danielsson and coworkers [41] reported a sensitive assay for detecting the amount of 2,4-dichlorophenoxyacetic acid utilizing chemiluminescence and ELISA (enzyme-linked immunosorbent assay) based on disposable glass capillaries. The glass capillaries contain the monoclonal antibodies onto the inner side, using the activated carboxymethyl dextran with different sizes. HRP derivatives were applied for labeling, and the detection limit of 2,4-dichlorophenoxyacetic acid is 2 pg/ml in a competitive detection method. Wang and coworkers also applied this method for detecting Fumonisin B1 (FB1) in food samples [42]. Roda and coworkers proposed a system for probing Cry1Ab protein in a maize sample employing chemiluminescence and sandwich-type ELISA method [43]. HRP derivatives were utilized for labeling, and the detection limit is 3 and 5 pg/ml using 96-well and 384-well plates, respectively. Goddes and coworkers introduced low-powered microwaves for triggering metal-enhanced chemiluminescence [44], and developed a probing system of protein with a detection limit of 150 fmol in less than 1 min using this technique [45]. Vo-Dinh and coworkers proposed a biochip system composed of an intensifier for signal amplification and an integrated circuit photosensing array for the detection of Bacillus globigii spores [46]. The enzymatic amplification was also used for detecting low concentrations of the spores with the intensified biochip device. This combined probing system has the detection limit of approximately 1 × 105 B. globigii spores.

8.5. NANOPARTICLE LABELING

The combination of nanoparticles and biomolecules such as nucleic acids and proteins has some synergistic effect for development of probing system because of their unique and applicable properties. Nanoparticles include several types of metal and semiconducting materials that show useful character such as fluorescence and magnetic property. Special features and applicable research fields of some notable nanoparticles are listed in Table 8.1 [47]. The nanoparticle size can be regulated from 1 nm to more than 10 nm, depending on the nanomaterial, offering an appropriate platform for the interactions between oligonucleotides and other analytes [48]. There are two types of conjugations between nanoparticles and nucleic acids; one approach is the covalently linking method, and another is based on the utilization of noncovalent interactions such as electrostatic, hydrophobic, and hydrogen bonding interactions (Figure 8.5) [49]. Noncovalent methods offer simple preparation of functionalized nanoparticles and facility of modifications. Functionalized nanoparticles can be influenced by charge repulsion, groove binding, stacking, intercalation, and target oligonucleotide hybridization [50]. Useful receptors for oligonucleotides can be presented by nanoparticles, which have the characteristic resemblance to nucleic acid–protein interactions [51]. The introduction of cationic moieties

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TABLE 8.1 Special Features of Some Notable Nanoparticles Nanoparticles

Special features

Linker

Gold

Colorimetric change, fluorescence quenching, stability

Thiol, disulfide, phosphine

Silver

Surface-enhanced fluorescence Photoluminescence, photostability Magnetic property

Thiol

Quantum dot Iron oxide

Thiol, phosphine, pyridine Diol, dopamine derivatives, amine

Applicable fields Biomolecular recognition, delivery, sensing Sensing Imaging, sensing MRI agent and biomolecule purification

(a)

(b)

Figure 8.5 Introduction of nucleic acids into nanoparticles. (a) Utilization of noncovalent interactions [52]. (b) Covalently linking method [54].

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on the surface of nanoparticle offers an electrostatic binding with the phosphate backbone of oligonucleotides. Rotello and coworkers developed the mixed monolayer protected gold clusters (MMPCs), which have tetraalkylammonium ligands to recognize a 37-mer duplex DNA (Figure 8.5a) [52]. Because of the high affinity of the nanoparticles–oligonucleotides complex, transcription of T7 RNA polymerase is inhibited by the complex. This system shows that the nanoparticles–oligonucleotides complex can regulate gene transcription. Murray and coworkers also demonstrated that intercalation offers another effect for DNA binding [53]. Covalently linking method for conjugation provides the highly selective and strong DNA–DNA interactions (Figure 8.5b) [54]. In this section, we will introduce several examples of the combination between nucleic acid and diverse nanoparticles, such as gold nanoparticle, quantum dot nanoparticle, and magnetic nanoparticle. 8.5.1. Gold Nanoparticle

Both the Mirkin and Schultz groups independently reported about oligonucleotides covalently linked with gold nanoparticles (AuNPs) [54]. AuNPs have a unique property of colorimetric change from red to blue color when they are aggregated. The AuNPs functionalized single-stranded DNAs were prepared and aggregated by adding the complementary sequences, and then the color of the AuNPs was changed from red to blue. This system can recognize the target oligonucleotides at the level of subpicomole with single-base mismatched sequence analysis by regulating the temperature [55]. Using this method, oligonuceotides were detected at subpicomolar level without the assistance of PCR [56]. This type of probing system was also developed for the colorimetric sensing of duplex DNA binders [57] and triplex DNA binders [58]. Aptamers also can be utilized for recognizing many other analytes, such as metal ions, small molecules, and proteins because they can specifically bind to these analytes with high affinity. Hence, various types of probing systems can be manufactured by using the binding property of aptamers. Chang and coworkers introduced two aptamers of platelet-derived growth factor (PDGF) into AuNPs for sensing PDGF selectively [59]. Functionalized AuNPs were aggregated on addition of PDGF (Figure 8.6a) [59]. This method, which requires more than two aptamers, cannot easily apply to other analytes because most of aptamers have only one binding site for their analytes. Liu and Lu proposed a sensor platform based on the disorganization of gathered AuNPs. They utilized structure switching of the aptamer from duplex DNA made by two types of single-stranded DNA [60]. The two types of single-stranded DNA were bound to two different bundles of AuNPs, and a middle DNA including the aptamer sequence was used as a cross-linker of these two single-stranded DNAs (Figure 8.6b). When the target molecule is introduced for inducing the aptamer structure, one single-stranded DNA part shortly hybridized with the aptamer is disaggregated from another single-stranded DNA, and then the color of the probe is changed from blue to red (Figure 8.6b). This sensing platform was also applied to recognize small


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(a)

(b)

(c)

(d)

Figure 8.6 Schematic diagrams of colorimetric systems based on assembly or disassembly of AuNPs. (a) Assembly system of aptamer-modified AuNPs by target PDGFs [59]. (b) Disassembly system of AuNPs functionalized by adenosine aptamers [60]. (c) Assembly system of AuNPs induced by target adenosine [62]. (d) Disassembly system aptamerlinked AuNPs to bind target adenosine [63].

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molecules such as adenosine and cocaine, potassium ions, and multianalytes [61]. More simple system based on disorganization of AuNP aggregates was developed by Li and coworkers [62]. This system is similar with the previous system mentioned; however, one single-stranded DNA is not immobilized to AuNPs. The structure switching of the aptamer from duplex DNA is induced by the target molecule (adenosine), and the AuNPs are disaggregated each other (Figure 8.6c). This system had a detection limit of 10 μM for target adenosine. It was later found that folding of the aptamer tethered on AuNPs could stabilize dispersed AuNPs. The Li group [63] and Chang group [64] independently discovered that targetbound aptamers immobilized on AuNPs prefer to be dispersed in buffer solution (Figure 8.6d). Mirkin and coworkers proposed an AuNP sensor for Hg2+ detection based on coordination chemistry of thymine-Hg2+ -thymine [65]. Mercury ion was well known to specifically bind with two thymine bases; therefore many studies about T-T mismatched DNA duplex using Hg2+ were reported [66]. When T-T mismatched two DNA strands are individually linked to different AuNPs, mercury ion can be detected by the inter-particle DNA hybridization. At that time AuNP aggregation is induced, and melting temperature is increased by the presence of the mercury ion. Using this system, they determined a detection limit of 100 nM [65]. Liu and coworkers proposed a simple detecting probe that can be operated at the room temperature, with a detection limit of 1 μM [67]. Rothberg and Li developed a label-free colorimetric system that did not require chemical modification. They used citrate coated AuNPs for easy aggregation with short single-stranded DNA, while double-stranded DNA could not bind to AuNPs and thus induced assembly of the AuNPs [68]. Based on this probing system, they reported hybridization assays to recognize target DNAs [69]. Aptamers are also applicable in the label-free colorimetric system. Unstructured aptamer without target and folded aptamer bound to target have different interactions with AuNPs. Fan and coworkers reported the label-free colorimetric detection of potassium [70] and adenosine triphosphate (ATP) [71], and the Dong group used this system for thrombin detection [72]. Other label-free colorimetric systems for specifically detecting Hg2+ (Figure 8.7a) [73] and Pb2+ (Figure 8.7b) [74] were also developed. 8.5.2. Quantum Dot Nanoparticle

Quantum dots are semiconducting nanomaterials that have unique optical properties such as narrow emission and broad absorption of photoluminescence. The maximum wavelength of emitted photoluminescence can be regulated by the composition, shape, and size of the quantum dot. Therefore, multiplex emissions of photoluminescence can be produced by excitation of a single wavelength photon. Quantum dots also show less photobleaching effect than general organic fluorophores. Based on these unique properties, quantum dots were widely applied to various probing systems [75]. For instance, quantum dots were introduced into the quantitative recognition of diverse analytes such as protease


(a)

305

(b)

Figure 8.7 Label-free colorimetric system using (a) aptamer [73], (b) DNAzyme [74]. (Full-color version of the figure appears in the color plate section.)

[76], maltose [77], and target DNA [78] utilizing fluorescence resonance energy transfer (FRET) system [79]. Ellington and coworkers first developed the combination system of aptamer and quantum dot for FRET detection of protein (Figure 8.8a) [80]. They prepared aptamer-linked quantum dots and fluorescent DNAs that could be hybridized with aptamer sequences. In the absence of analytes, duplex formation induced FRET event because the fluorophore was placed at close to quantum dots. When the target protein was bound to the aptamers, the fluorescent DNA receded from the quantum dots and produced fluorescence emission. Charge transfer between quantum dots and electron donating molecules was also used to construct probing systems. Benson and coworkers proposed a maltose binding protein (MBP)–based quantum dot probe by regulating the interactions between quantum dots and a ruthenium moiety-conjugated MBP [81]. The ruthenium moiety functioned as an electron donor that could quench the photoluminescence of quantum dots. The attachment of target maltose to the MBP could induce its conformational change, and the ruthenium compound was extruded to the quantum dot surface, resulting in an increased photoluminescence emission. Strano and coworkers introduced thrombin binding aptamer (TBA) into this type of probing system. They produced TBA-functionalized PbS quantum dots, which could be selectively quenched on binding to target thrombin (Figure 8.8b) [82]. They proposed a “turn off” system quenched by charge transfer from thrombin and reported the limit of detection to be 1 nM. 8.5.3. Magnetic Nanoparticle

The ferromagnetic materials in bulk type have permanently magnetized properties in the absence of an external magnetic field. In the case of nanometer scale, the particles show the magnetized property only in the presence of an external magnetic field because of fast randomization of the magnetic dipoles [83]. Generally speaking, these types of nanoparticles are called superparamagnetic nanoparticles (shortly, magnetic nanoparticles) [84]. Based on their unique properties, labeling of magnetic nanoparticles has found wide applications such

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(a)

(b)

Figure 8.8 Schematic diagrams of (a) aptamer sensor based on quantum dot FRET (turnon system) [80], (b) aptamer sensor based on quantum dot FRET and charge transfer (turn-off system) [82]. (Full-color version of the figure appears in the color plate section.)

as drug delivery, cancer therapy, separation of biological samples, and magnetic resonance imaging (MRI) [85]. Weissleder and coworkers manufactured magnetic nanoparticles as magnetic relaxation switches (MRS) of MRI. The aggregationdisaggregation of magnetic nanoparticles influences the magnetic relaxation [86]. Lu and coworkers developed aptamer-linked iron oxide magnetic nanoparticles for MRI as contrast agents (Figure 8.9a) [87]. For probing target thrombin, they designed a “turn-off” contrasting system utilizing two types of thrombin-binding aptamer-functionalized nanoparticles that could be aggregated by the addition of thrombin. The aggregations induced a weaker contrasting image, indicating a shorter spin-spin relaxation time. They also proposed a “turn-on” system that target adenosine promoted the disaggregation of two types of DNA-linked magnetic nanoparticles cross-linked by adenosine aptamer, resulting in a brighter contrasting image and larger spin-spin relaxation time (Figure 8.9b) [88].

8.6. FLUORESCENT DYE LABELING

In the initial stage of DNA technology there were no readily recognizable methods for nucleic acids because fluorescent detection was also dependent on fluorescent dyes that can intercalate or bind to the oligonucleotides by means of noncovalent interactions. In postgenomic era, nucleoside monomers covalently linked with diverse fluorophores were utilized increasingly. Especially, sequence-specific hybridization probes composed of fluorescent synthetic oligonucleotides have been developed for genetic analysis in the postgenomic era. Currently, such fluorescent oligonucleotides play a decisive role in

FLUORESCENT DYE LABELING

307

(a)

(b)

Figure 8.9 (a) Schematic diagrams of MRI “turn-off’”contrasting system of thrombin [87]. (b) MRI “turn-on” contrasting system of adenosine [88]. (Full-color version of the figure appears in the color plate section.)

analysis of the genetic information and SNP typing. The simple chemistry for synthesizing oligonucleotide has led to diverse applications of modified nucleic acids using commercially available phosphoramidite monomers of fluorophore and quencher (Figure 8.10). Many researchers have also reported their own labeling methods to meet their purposes [89]. 8.6.1. Hybridization System

Labeling method of a synthetic oligonucleotide with a fluorescent molecule is cost effective to make and easy to use. When the fluorescent probe and the target DNA are hybridized, fluorescence polarization is increased by the changes in the molecular volume of a fluorophore [90]. Simple fluorescent probes can show detectable fluorescence on hybridization to target DNA that is potential methods in genetic analysis. However, they have unpredictable changes in fluorescence due to the influence of the base sequence. HyBeacons developed by French and coworkers overcame this problem, as they recognized patterns of dye-conjugation that authentically induced fluorescence enhancement on hybridization (Figure 8.11a) [91]. Fluorescein is placed in the major or minor groove by incorporation to

308

Figure 8.10 Commercially available phosphoramidite monomers of fluorophore and quencher.


309

(a)

(b)

(c)

Figure 8.11 Schematic diagrams of (a) HyBeacons [91], (b) fluorescein inherent quenching probes [95], and (c) fluorescein dequenching probes [96].

either the base [91] or the 2 -position of ribose [92]. HyBeacons have been used to analyze polymorphic targets directly amplified from saliva [93]. It is a well-known phenomenon that fluorescence is effectively quenched by nucleobases such as guanine bases due to their good electron-donating properties [94]. This phenomenon was used in two fluorescent probing systems that achieve definite quenching or dequenching on hybridization. Crockett and Wittwer proposed fluorescein inherent quenching probes that could be accomplished by linking the fluorophore with two continuous cytidines in the probe sequence. When the probe is hybridized with the target sequence containing two guanosines, the fluorescein is placed in close to the guanosines and leads to a decrease in fluorescence. This system has been utilized in real-time PCR by amplification curves and melting temperature (Tm ) analysis (Figure 8.11b) [95]. This method can be applicable to most sequences because target strands with only one guanosine in the 5 -end position also display detectable quenching. Fluorescein dequenching probes reported by Elenitoba-Johnson could also be achieved by positioning two continuous guanosines next to the fluorescein in the probe strands. Internal quenching is inhibited on hybridization, leading to signal amplification. Using this system, melting temperature analysis was used to discriminate various SNPs (Figure 8.11c) [96]. FRET (fluorescence resonance energy transfer) can be utilized as a signaling system in hybridization probes. For effective energy transfer, the donor fluorophore must be placed close to the acceptor on hybridization [97]. This type

310


of hybridization system has been proposed using a pair of fluorescently labeled oligonucleotides. One probe is labeled with a donor fluorophore at its 3 -end position, and another is linked with an acceptor fluorophore at its 5 -end. When they are hybridized with target sequence, two fluorophores are collected at adjacent sites and conveniently positioned for FRET to occur (Figure 8.12). Organic dyes such as fluorescein and LC (Light Cycler) Red 640 have been utilized [98], and the applications of luminescent europium [99,100] or terbium [101] complexes as LRET (luminescence resonance energy transfer) donors have also been reported. Intercalators that have fluorescence quenching properties can also be used in combination with conventional fluorophores as a FRET system. DNA probe with a fluorescein (the donor fluorophore) and acridine (the acceptor quencher) pair is a good example proposed by Brown and coworkers (Figure 8.13a) [102]. On hybridization, the fluorescence of fluorescein is increased because the acridine

Figure 8.12 Schematic diagram of hybridization probes utilizing FRET phenomenon [98].

(a)

(b)

Figure 8.13 Schematic diagrams of (a) fluorophore-intercalator combination probe [102], and (b) MagiProbe [103].


311

moiety intercalating into DNA duplex becomes less efficient in energy transfer. Yamane developed a similar probing system (MagiProbe) utilizing the interaction between fluorescein and pyrene quencher (Figure 8.13b) [103]. 8.6.2. Molecular Beacon

Molecular beacons (MBs) are fluorescent-labeled synthetic oligonucleotides composed of three parts [104]: (1) loop position that generally has 15–30 nucleotides and specifically bind with target sequence; (2) stem position that contains 5–8 base pairs; (3) signaling position that includes fluorophore connected to 5 end and quencher in 3 -end. Diagrams in Figure 8.14 represent a chemical structure and proposed mechanism of the MBs. In this conventional MBs, the quencher is made by Dabcyl (4-((4-(dimethylamino)phenyl)azo)-benzoyl) group, and the fluorophore by Texas red or the luciferase (5-(2-aminethylamino)-1naphthalenesulfonic acid, EDANS). According to FRET theory, the efficiency of energy transfer is inversely proportional with 6th power of the distance between the donor and the acceptor [105]. Therefore, amplification of fluorescence is dependent on the distance between the fluorophore and the quencher. In the normal condition, MBs are maintained as a hairpin; thereby fluorophore and quencher are in close proximity (about 7–10 nm). At the point FRET occurs, the emitted fluorescence is entirely absorbed by the quencher and is transferred

(a)

(b)

Figure 8.14 (a) Chemical structure of conventional Molecular Beacon, and (b) proposed mechanism of conventional Molecular Beacon [104].

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to the heat energy. When the target sequences are complementarily hybridized with the loop position of MBs, the fluorophore and the quencher in each end of MB strands will be separated. After hybridization, the fluorescence of MBs is amplified. Dabsyl (4-dimethylaminoazobenzene-4 -sulfonyl) group, which is generally used as the fluorescence quenching molecule in MBs, has an excellent quenching effect to some fluorophores, especially fluorescein [106]. Dubertret and coworkers introduced 1.4 nm diameter gold nanoparticle into fluorescence quenching group, they checked quenching efficiency of various fluorophores such as fluorescein, rhodamine 6G, Texas red, and cyanine-5 [107]. The gold nanoparticle quencher induced a higher sensitivity enhanced up 100-fold. Demidov reported no stem MB structures using DNA or PNA [108]. As phosphate (DNA) or polyamide (PNA) backbone has good flexibility, the MB structures can be stabilized by hydrophobic interaction between fluorophore and quencher in the absence of the target. When the MBs are hybridized with the target sequence, the probe and target will make a rigid double-stranded DNA, and the fluorophore and quencher will be separated. Compared to conventional MBs, no stem MBs are simple and cost effective in designing and synthesizing. Moreover, no stem MBs responded faster and have higher sensitivity and selectivity. The efficiency of MBs is influenced by many factors. These factors often induce false-positive and false-negative signals. The distance between fluorophore and quencher is the most important factor, as mentioned earlier [104]. According to probing systems, the fluorescence signal is generally increased 10-fold to 100-fold on hybridization. Thermal stability also plays an important role because the main working mechanism of MB is dependent on the difference in stability between hairpin structure of MB and double-stranded MB, which binds with target DNA. The melting temperature of the MB probe is affected by the chain length of the stem, GC contents ratio, and the ionic concentration of the buffer. Kramer and coworkers studied thermodynamic analysis of the MB transitions [109]. Their study showed that enhanced specificity is a general feature of conformationally constrained probes. Environmental pH value also affects the MBs’ function [104]. Too high or low pH will also break down the stem position. The MBs degenerate and lead to a false-positive result. MBs are usually functionalized by a single fluorescent group. In a homogeneous system, the fluorescence signal changes demonstrate that the probes detect the only one target sequence. To analyze various target sequences at the same time, various fluorophores can be introduced into different MB sequences. Tyagi and coworkers used four types of MBs for allele detection [110]. They found that hairpin structure enables the utilization of differently colored fluorophores. Using diverse MBs designed to recognize different target sequences they demonstrated that multiple targets can be discriminated in the same solution, even if they differ from one another by as little as a single nucleotide. The MB fully matched with the target sequence can specifically bind and emit fluorescence at corresponding maximum wavelengths. The thermal stability of perfectly matched sequence was higher than that of single-base mismatched sequences.


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Tyagi and coworkers also reported wavelength-shifting MBs, which are oligonucleotide hybridization probes that emit various colored fluorescence, yet are excited by a common monochromatic light source [111]. Their schematic diagrams are shown in Figure 8.15. Dabcyl group was used for quenching fluorescence in the 3 -end of the MB probes and luciferase for harvesting fluorescence at the internal position in 5 strand, and Texas red was linked with the distal end of the 5 strand as a fluorescence emission group. In the state of hairpin structure, the speed of energy transfer from the internal harvesting fluorescence molecule to the quenching group was much faster than that of transfer to the final fluorescence emission group. The wavelength-shifting MBs have good fluorescence efficiency and high signal-to-noise ratio. This system can be introduced into different colored MB probes excited at the same wavelength, and thus multigene can be analyzed by simultaneously detecting of these fluorescence intensities utilizing one excitation wavelength. Recently fluorescent hairpin oligonucleotides that can function as MB even without additional quencher moiety have been proposed, and such probing systems can be called quencher-free molecular beacons (QF-MBs). 8.6.3. Quencher-Free Molecular Beacon

The QF-MBs can be defined as hairpin type fluorescent oligonucleotides containing one or multiple fluorophores attached at any part of the sequence without additional quencher. The QF-MBs mentioned in this part are yet another extension of conventional MBs (Figure 8.16) [2]. A hairpin structure and a fluorescent part are the essential building blocks for the construction of QF-MB systems. In most QF-MBs the whole or part of loop is hybridized with the target sequence. Based on the signaling systems, the QF-MBs can be broadly classified into two different types: (1) QF-MBs containing only one fluorophore at the middle or end position of the oligonucleotide (Figures 8.16a and 8.16b) and (2) dual-labeled MBs at the stem or at the ends of the hairpin structure (Figures 8.16c and 8.16d). In the case of mono-labeled QF-MBs, fluorescence is controlled by neighboring DNA bases, and thus structural change of oligonucleotide is sensitively affected to the fluorescence. In dual-labeled QF-MBs (Figure 8.16c) different kinds of two

Figure 8.15 Wavelength-shifting molecular beacons [111].

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(a)

(b)

(c)

(d)

Figure 8.16 Different types of QF-MBs [2].

fluorophores are attached to a hairpin oligonucleotide. One fluorophore is linked at the 5 -end and another fluorophore at the 3 -end. In the case of Figure 8.16d two fluorophores of same kind are attached at the ends. The natural purine and pyrimidine nucleobases have poor fluorescent properties, and thus they cannot be introduced into sequence probing systems. Recently, there have been diverse efforts to design modified nucleosides containing expanded nucleobases and conjugated nucleobase analogues [112]. Kim and coworkers proposed the QF-MB system containing fluorescent nucleoside Fl U at the middle loop of the hairpin DNA (Figure 8.17) [113]. In the fluorescent nucleoside building block (Fl U), fluorene is attached to the base C-5 position of deoxyuridine through rigid conjugated ethynyl linker. Fluorene moiety has relatively excellent quantum yield (about 0.54 in ethanol), and it is less massive as compared to the commonly used fluorophores such as coumarin, pyrene, fluorescein, rhodamine, and cyanide derivatives. The modified nucleoside thus incorporated should not affect the natural hydrogen-bonding properties of DNA. In general, a substitution at the base C-5 position of the

Figure 8.17 Schematic diagram of a loop-modified QF-MB and structure of Fl U nucleoside [113].


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pyrimidine ring and the base C-8 position of the purine ring or the base C-7 position of the 7-deazapurines does not interfere with the ability to form a base pair in the resulting DNA duplex [114]. The fluorene unit was covalently attached to the C-5 position of deoxyuridine using Sonogashira coupling, and the fluorescent deoxyuridine (Fl U) was subsequently introduced into the oligodeoxyribonucleotides (ODNs) using the phosphoramidite chemistry. Similar to the conventional MB, the QF-MBs are fluorescent hairpin DNA, but the fluorophores in QF-MBs are anchored at the loop, rather than at the stem end. It means that another functional group can be introduced into the end position of the QF-MBs. The fluorescent hairpin ODNs shown in Figure 8.18 are examples of the mono-labeled QF-MB systems containing fluorophore at the stem end or middle position. The fluorophore can be attached either through a linker

(a)

(b)

(c)

Figure 8.18 QF-MB systems with fluorophore at the stem end (a, b) [115,116], or middle position (c) [117].

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(Figure 8.18a) or directly incorporated in the form of a modified fluorescent nucleoside (Figure 8.18b) using the phosphoramidite chemistry. The QF-MBs in Figure 8.18a, known as smart probes, were developed by Sauer and coworkers [115]. In the smart probes an oxazine (MR121 in Figure 8.18a) or rhodamine (R6G in Figure 8.18a) derivative is attached as fluorophore at the 5 -end of the guanosine-rich hairpin ODNs through an alkyl linker (C6) using classical N -hydroxysuccinimidyl ester (NHS-ester) chemistry. The full or major part of the loop is complementary to the target sequence. On the other hand, the hairpin ODNs in Figure 8.18b contain a pyrene modified deoxyadenosine (Py A) unit and the pyrene modified deoxyuridine (Py U) at the 5 -overhang position [116]. In the modified nucleoside analogues, Py A and Py U, the pyrene is covalently linked to the nucleobase through a rigid ethynyl linker using Sonogashira coupling. It may be noted that in all the ODNs, the fluorophore-containing nucleosides lack the corresponding nucleosides in the opposite strand to form a nucleobase-pair. Rashtchian and coworkers have reported a series of mono-labeled hairpin ODNs containing different types of dyes [117]. In a few of these ODNs, the dye is directly attached through corresponding dye-phosphoramidite derivatives, at the 3 - or 5 -end. In few other ODNs a nucleoside analogue tagged with the dye (for example, C5-fluorescein-dT) is incorporated through the phosphoramidite method, at any position of the blunt-ended stem (Figure 8.18c) or as an overhang unit. The dye labeling of the ODNs (to the nucleobase) could also be done postsynthetically. In blunt-ended hairpin in Figure 8.18c, also known as self-quenched probe, the fluorescein modified thymidine (T-FAM) has been incorporated at the 3 -end as penultimate nucleoside unit. In T-FAM, thymine is tagged with a fluorophore at its C5-position through a C6 spacer. Residual fluorescence (incomplete quenching) and poor sensitivity are some of the major drawbacks of conventional MB. In an effort to eliminate these drawbacks, hairpin oligonucleotides containing two or more fluorophores, but with no quencher moiety, have been designed. The FRET MBs, excimer–monomer switching MBs (EMS MBs), dimer–monomer switching MBs (DMS MBs) are notable among them. In FRET MBs two different fluorophores as a fluorescence acceptor (FA ) and fluorescence donor (FD ) are attached on both of the ends [118]. The FA and FD should have overlapping emission spectra for effective FRET from FD to the FA . The representative FRET MB, shown in Figure 8.19a, consists of two fluorescent dyes: 6-carboxyfluorescein (FAM) is attached at the 5 -end, and coumarin is attached at the 3 -end. FRET MBs containing more than two distinct fluorophores have also been designed and used as fluorescent probes [119]. For instance a hairpin ODN containing fluorophores such as fluorescein (FAM), rhodamine (TMR), and cyanine (Cy5) derivatives has been designed in such a way that FAM is located on one end of ODN, whereas the other two fluorophores (TMR and Cy5) are located on the other end of the ODN. In EMS MB, the same types of two fluorophores are attached at the 5 - and 3 -ends of hairpin ODNs. For example, hairpin oligonucleotides containing two pyrene units on each end are well-known EMS probes. The FRET MB in Figure 8.19b containing pyrene fluorophores was reported by Fujimoto and Inouye [120]. Kim and coworkers have


(a)

(b)

(c)

(d)

317

(e)

Figure 8.19 Representative examples of FRET MB (a) [118], EMS MBs (b and c) [120,121], and DMS MBs (d and e) [123,124].

designed and synthesized dual-labeled quencher-free hairpin ODNs containing pyrene appended deoxyadenosine (Py A) and pyrene appended deoxyuridine (Py U) units in three different combinations, namely, Py U-Py A, Py U-Py U and Py A-Py A in Figure 8.19c [121]. In these dual-labeled MBs, emissive nucleoside units are incorporated in the complementary positions, but within the stem so that the ends are free for further modifications. The practical utility of the free ends in EMS MBs of Figure 8.19c has been demonstrated by attaching a cholesterol moiety at the 5 -end of the ODN. The cellular permeability of the cholesterol-appended ODN was enhanced significantly as compared to that of free ODN [122]. Some fluorophores form nonradiative dimers when they are in close proximity. Such fluorophores have also been used in the design of QF-MBs. The FRET MB in Figure 8.19d containing two MR121 fluorophores attached at the 5 - and 3 ends is self-quenched [123]. Similarly in Figure 8.19e, two dicyanomethylene dihydrofuran (DCDHF) dyes have been attached at the 5 - and 3 -ends using an NHS-ester derivative of the dye [124]. 8.6.4. Aptamer Sensor

Aptamers are short, single-stranded, functional nucleic acids (DNA or RNA) selected from random sequence combinatorial libraries, and they have been used to specifically bind from metal ions and small molecules to peptide, proteins, whole cells, viruses, or parasites [125]. Aptamers, single-stranded nucleic acids isolated from in vitro experiments named as SELEX (systematic evolution of ligands by exponential enrichment), have recently been fully automated [126]. Target binding generally induces structural changes of aptamers, resulting in the formation of well-defined three-dimensional structures responsible for it, and the stem or loop structures are typically active sites for the specific recognition. Having such high affinity, aptamer-based probing systems have been employed for the detection of metal ions, small molecules, nucleic acid, peptide, and proteins.

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Electrochemistry, chemiluminescence, and fluorescence are common detection methods utilized in these probing systems [127]. In the fluorescence probing systems, fluorophores can have minimal perturbations on aptamer binding if their labeling sites are appropriately selected. Fluorophores can associate with nucleic acids through a variety of interactions, such as hydrogen bonding, electrostatic interactions, intercalation, and covalent attachment. Tan and coworkers have applied the conventional molecular beacon system to detect protein-DNA interactions [128]. A well-characterized E. coli protein, single-stranded DNA binding protein (SSB) was the analyte of this molecular beacon system (Figure 8.20a). They observed that the interaction between the molecular beacon and SSB in solution is rapid and tight. When the stem of beacon was dehybridized, fluorescence signal enhancement was observed. They reported that fluorescence from molecular beacons can be recovered by different approaches: hybridization to their complementary cDNAs and denaturation by either heat or high pH. When aptamer-target interactions are involved, such molecular beacons are called aptamer beacons. Using this conventional molecular beacon design, Stanton and coworkers also proposed a thrombin aptamer beacon (Figure 8.20b) [129]. One end of the thrombin binding aptamer strand was extended, and a hairpin structure was constructed. They checked fluorescence increase with changing the number of base pairs in the stem from four to six. The system having five base pairs in the stem could detect less than 10 nM thrombin, with a maximum fluorescence increase of 2.5-fold. (a)

SSB

F

F (b)

Figure 8.20 Schematic diagrams of aptamer beacons for probing (a) single-stranded DNA binding proteins (SSB) [128], and (b) thrombin [129].


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Recently Morse developed tobramycin probe using the loop region of an RNA hairpin structure that could separate the two ends of the strand on targeting [130]. The two ends were labeled with a fluorophore and a quencher, and a detection limit of 30 μM was reported. As mentioned earlier, aptamer beacons can be designed by introducing aptamer sequences with the classic hairpin structure into molecular beacons. Many aptamer beacons were developed based on the FRET or excimer approach but did not require the construction of a full hairpin structure initially. For example, Stojanovic and coworkers made the cocaine aptamer beacon and predicted to form a binding structure of a three-way hairpin (Figure 8.21a) [131]. An unstable state was induced in the opened stem of a three-way hairpin that formed the cocaine-binding pocket, and the resulting stem

(a) F

F Coc

(b)

PDGF

(c) FD

K+

FD

Figure 8.21 Aptamer beacons using DNA secondary structure. (a) Cocaine detection using a fluorophore and a quencher [131], (b) light-switching excimer probes for PDGF [134], and (c) potassium ion analysis by labeling with two fluorophores (fluorescein and TAMRA) [135].

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was end-labeled with a fluorescein (fluorophore) and a dabcyl (quencher) group. This major structural change makes fluorophore and quencher together signaling the presence of cocaine. In the presence of cocaine, the perfect hairpin structure was constructed and the fluorophore was placed close to the quencher. Around 50% quenching of fluorescence was observed with 1 mM of cocaine. In blood serum, 10 μM of cocaine could be detected. Ozaki and coworkers introduced largininamide aptamer into this system [132]. Urata and coworkers also reported a number of adenosine aptamer beacons based on this system [133]. A decrease in fluorescence intensity was observed in the presence of adenosine with a detection limit of around 5 μM and quenching efficiency up to 67%. Tan and coworkers proposed light-switching excimer probes using two pyrene groups at the two ends of the PDGF aptamer (Figure 8.21b) [134]. They also utilized one opened stem of a three-way hairpin structure, which can be constructed as a perfect hairpin structure in the presence of target PDGF. When two pyrenes were positioned in close proximity, the fluorescence wavelength was shifted from ∼400 nm (pyrene monomer) to 485 nm (pyrene excimer). Such a change in emission wavelength allowed quantitative analysis with a detection limit of less than 1 nM PDGF. Importantly, time-resolved fluorescence measurements were used to decrease the biological background signal, because the excimer fluorescence has a longer fluorescence lifetime (∼40 ns) than that of the background signal (∼5 ns). Instead of a quencher, 6-carboxytetramethylrhodamine (TAMRA) as another fluorescence acceptor was linked to the end of a thrombin-binding aptamer (TBA) sequence by Takenaka and coworkers (Figure 8.21c) [135,136]. When fluorescence was excited in the absence of potassium ion, only fluorescein emission was observed. G-quadruplex structure was induced by the potassium ion resulting in close proximity of the two fluorophores, and TAMRA fluorescence was observed. The probe can detect 10 μM of potassium ion if the ratio of the two fluorophore emissions was monitored. They also utilized pyrene excimer instead of FRET phenomenon [137]. Using this sequence, Tan and coworkers developed thrombin recognition probe with detection limits around 0.4 nM [138]. Ono and coworkers reported on a metal ion probe for mercury ion detection based on the phenomenon that mercury ion can be complexed between two thymine bases in DNA [139,140]. Split aptamers have also been applied to aptamer beacons. Kumar and coworkers utilized two split RNA aptamers for detecting Tat protein of HIV [141]. As shown in Figure 8.22a, they developed a molecular beacon system that can be combined by another piece of split aptamer, and the fluorescence was initially quenched. In the presence of the Tat protein of HIV, the two split aptamers were assembled with the target, and thus the fluorophore was positioned far from the quencher. When there are 200 nM of Tat protein in the solution, the fluorescence intensity was increased around 14-fold. They carried out further study using this system and nucleic acid array technology [142]. Stojanovic and coworkers also introduced a split cocaine aptamer into a hybridization system (Figure 8.22b) [143]. The hybridizing event was enhanced by target binding, and the fluorescence


321

(a) Tat Tat

(b)

F

(c)

F

(d)

F

F

F

(e)

Figure 8.22 Schematic diagrams of (a) Splitting type aptamer beacon for HIV Tat protein detection [141], (b) Splitting type aptamer beacon for cocaine or ATP detection [143], and (c, d, e) switching-type aptamer beacons [146,147,149].

intensity was decreased on binding. The fluorescence change was observed in the presence of cocaine with a detection limit of around 10–1000 μM, and quenching efficiency is around 40%. Similarly, they also developed an ATP probe by splitting the ATP aptamer into two parts. Because binding affinity of aptamer may be decreased by such modifications, this type of split aptamer system may not be applicable to all aptamers. Li and coworkers proposed a universal method for detecting aptamer target using aptamer structure-switching from DNA/DNA duplex to DNA/target complex [144,145]. As shown in Figure 8.22c, a DNA including an aptamer fragment was utilized as a template of hybridization system labeled by fluorophore and quencher, resulting in quenched fluorescence [146]. In the presence of target

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molecules, the aptamer sequence switched to the target-binding structure, and the quencher-labeled sequence was dissociated. Aptamer sequences for binding thrombin and ATP were introduced in this study, with detection limits of 10 nM and 10 μM, respectively. Later, they modified the system, which consisted of a fluorophore-labeled aptamer and a quencher-labeled complementary sequence (Figure 8.22d) [147]. Tan and coworkers also designed an intrastrand system linked by a PEG chain between a fluorophore-labeled aptamer and a quencherlabeled complementary sequence [148]. Another modification was achieved by Rankin and coworkers, as shown in Figure 8.22e [149]. This system was composed of an RNA aptamer for theophylline, fluorophore, and quencher labeled short DNA, which is complementary to the theophylline aptamer. Due to the rigidity of duplex, the fluorophore showed high fluorescence intensity. When there were target theophyllines, the DNA fragments were released, resulting in quenched fluorescence. Ho and Li also utilized structure-switching between duplex and aptamer. In the presence of the target analyte, the aptamer-containing duplex was dissociated, and the fluorescence signal was changed [150]. 8.7. CONCLUSION

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CHAPTER 9

LOCKED NUCLEIC ACID OLIGONUCLEOTIDES TOWARD CLINICAL APPLICATIONS RAKESH N. VEEDU School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia

JESPER WENGEL Nucleic Acid Center Department of Physics and Chemistry, University of Southern Denmark, Odense, Denmark

9.1. INTRODUCTION

Nucleic acid–based therapeutic technologies (Figure 9.1) have significantly advanced in the past two decades toward the treatment of many diseases. The first such drug to enter clinic was vitravene®, an antisense oligonucleotide for the treatment of cytomegalovirus retinitis [1]. Later, research on aptamers led to the marketing of macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD) [2]. Nucleic acid–based therapeutic approaches mainly include antisense [3,4], ribozymes [4], small interfering RNA (siRNA) [4–6], microRNA (miRNA) [7–10] targeting and aptamers [11–15]. Oligonucleotides composed of naturally occurring DNA or RNA nucleotides pose some limitations because of their poor RNA binding affinity, low degree of nuclease resistance, and low bioavilability. To overcome these limitations, chemically modified nucleic acids have been introduced, among which locked nucleic acid (LNA) [16–20] proved to be unique and is now used extensively for various applications in chemical biology [21–23]. Medicinal Chemistry of Nucleic Acids, First Edition. Edited by L.H. Zhang, Z. Xi and J. Chattopadhyaya. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

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Figure 9.1 Schematic illustration of various nucleic acid gene silencing techniques. (Fullcolor version of the figure appears in the color plate section.)

9.2. LOCKED NUCLEIC ACID (LNA)

LNA nucleotides are generally considered to be RNA mimicking molecule in which the ribose sugar moiety is locked by an oxymethylene bridge connecting the C2 and C4 carbon atoms, imposing conformational restriction to adopt C3 -endo/N -type furanose conformation (Figure 9.2) [16,19,24]. Structural investigation by NMR spectroscopy has shown that LNA-containing oligonucleotides tend to adopt A-type duplex geometris [25,26]. Commercially available LNA contains natural phosphodiester linkages and therefore resembles natural nucleic acids in terms of aqueous solubility, Watson-Crick mode of binding, and straightforward automated synthesis using standard phosphoramidite chemistry.

O

O

B

O O O P O

O O

O O P O

Base

O

Figure 9.2 Structural representation of LNA monomers.

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TABLE 9.1 Examples of melting temperatures (Tm values) for hybridization of LNA and DNA oligonucleotides to complementary DNA and RNA oligonucleotide sequences. (Data collected from Singh et al . [12]). LNA modifications are represented in bold underlined capital letters; DNA monomers in capital letters; RNA monomers are in capital italic letters DNA/LNA: DNA/RNA duplexes 5 -TTTTTT: AAAAAA 5 -TTTTTT: AAAAAA 5 -TTTTTT: AAAAAA 5 -TTTTTT: AAAAAA 5 -GTGATATGC: CACTATACG 5 -GTGATATGC: CACTATACG 5 -GTGATATGC: CACUAUACG 5 -GTGATATGC: CACUAUACG

Melting temperatures (◦ C) 8 ng/mL) for up to 3 to 4 weeks after dosing, reflecting the slow clearance of Macugen® from the eye into the systemic circulation [59]. Inhalation is also being explored as a possible route of administration. Ali et al . [28] reported the pharmacokinetic study of 35 S-labeled EPI-2010 in rabbits, showing that by using aerosols of ODNs solutions, it was possible to attain significant pulmonary concentrations of ODNs in the lung, but only a small amount of inhaled EPI-2010 was detected in plasma at every time point. With inhalation, the maximal concentration achieved in plasma was 10.3 mg equivalent/total plasma volume at 24 h after drug administration, representing less than 15.0% of the radioactivity associated with the administered dose. The plasma profile for pulmonary administration differs from the typical biphasic profile (rapid distribution phase followed by a prolonged elimination phase) seen with intravenous administration. Similar results were seen with low-dose intratracheal administration in that pulmonary delivery resulted in a slower distribution phase with a much lower systemic availability, as compared with intravenous routes, followed by a prolonged elimination phase [60]. 10.2.1.3. Delivery Vehicles Delivering ODNs in vivo has now become a hot issue. Animal studies of the liposome, nanoparticle, and microsphere showed that delivery vehicles can prolong the half-life of ODNs in plasma, increase the drug concentration in target tissues, and finally, decrease the dosage. Yu et al . [61] examined the pharmacokinetics and tissue distribution of ISIS 2503 formulated in stealth (PEGylated) liposomes (encapsulated) or in phosphate-buffered saline (unencapsulated). Plasma concentrations of encapsulated ISIS 2503 decreased nonexponentially after infusion with a mean half-life of 57.8 h. In contrast, the concentration of unencapsulated ISIS 2503 in plasma decreased rapidly with a mean half-life of 1.07 h. Both encapsulated and unencapsulated ISIS 2503 were widely distributed into tissues. Nuclease-mediated metabolism was extensive for unencapsulated ODNs in plasma and tissues, suggesting that stealth liposomes protect ISIS 2503 from nucleases in blood and tissues.

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THE PHARMACOKINETICS RESEARCH OF NUCLEIC ACID DRUGS

Moreover, biophysical and pharmacokinetic properties of several lipophilic analogs of ODNs have been evaluated and reported [62]. Compared to the unconjugated ODN, the three analogs with lipophilic conjugates, 59-octadecylamine, 59-cholesterol, and 39-cholesterol were more lipophilic than unconjugated ODN but had similar binding affinity for complementary RNA (as measured by thermal melting analysis). Certain lipophilic end modifications can lead to a significant increase in melting temperature (Tm ) [63]. Crooke et al . [62] analyzed tissue distribution and half-life in mice using radioactively labeled ODNs analogs. After bolus intravenous injection, the initial volumes of distribution of the lipophilic conjugates were lower, and the initial clearance from plasma was slower than that of unconjugated ODNs. Conjugation substantially increased the fraction of the dose accumulated by the liver. As discussed, it is not clear whether this change was due to an active transport of the lipophilic conjugates into the liver or if the effects observed were simply due to the increased lipophilicity of the conjugated ODNs. There was no distribution to the central nervous system (CNS). Dizik et al . [64] also studied the pharmacokinetics of cholesterol-conjugated PSODNs. They observed that conjugation of cholesterol to PS-ODNs increased the plasma half-life. Sixty minutes after injection into female mice, the levels of 39cholesterol conjugates were nearly fourfold higher than those of unconjugated ODNs, and the levels of 59-cholesterol and 59, 39-cholesterol-conjugated ODNs were nearly sevenfold higher. Equally, a variety of chemical modification and cationic polymer delivery systems have been developed to enhance the efficiency of siRNA in vivo systems. Kim et al . [65] developed a novel ELISA-based assay to study the pharmacokinetic profiles of siRNAs following intravenous administration, and they found that the higher blood concentrations were achieved using the methylated form of siRNA than using the unmethylated form. Moreover, methylated siRNA complexed to DOTAP-based cationic liposomes showed significantly higher and prolonged blood concentration-time profile, with a 2.2-fold lower clearance rate (0.11 ± 0.02 mL/min) than the uncomplexed form. 10.3. TISSUE DISTRIBUTION

The tissue distribution of nucleic acid–based drugs was closely related to its efficacy and safety. Research indicated that tissue distributions of most nucleic acid drugs are independent of sequence and length. In general organs, the distribution properties of different ODN drugs in its chemical class are remarkably similar across species [46]. The maximum tissue accumulation has been observed in the kidney, liver, spleen, lymph nodes, adipocytes (cell body but not the lipid particle), and bone marrow. ODN drugs do not cross the blood–brain barrier (BBB) and are poorly distributed to the skeletal muscle, heart, lung, and genital glands. The time course of tissue distribution of nucleic acid drugs was very obvious. ODNs were rapidly distributed to various tissues after entering the circulation system before being eliminated gradually. The elimination rate of ODNs in tissues was slower than in blood. Half-lives in tissues were about 1 to 4 weeks due

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to sequence and chemical properties. For example, AR177 (T30177, Zintevir) is a 17-mer ODN that has anti–human immunodeficiency virus activity in vitro. Analysis of the AR177 concentrations in tissues showed that the highest concentrations were achieved in the renal cortex (15.0 mg/g), liver (7.4 mg/g) at 8 h after a single dosage. Forty-eight hours after the last of the seven i.v. doses given every other day, the concentrations in tissues were as follows: 39.9 mg/g in the renal cortex and 33.9 mg/gin the liver [66]. For another PS-ODN ISIS 2302, the accumulation rate of ISIS 2302 in the liver was at its peak when injected intravenously and the accumulation amount could reach 20% after 1 to 2 h. But the elimination rate in the liver was also the most rapid, and the half-life in liver was 62 h compared with 156 h in renal medulla [67]. As the third-generation ODNs, their tissue concentrations are also independent of the sequence and length of PMOs. An estimated total systemic subcutaneous bioavailability of approximately 80% is achieved following subcutaneous injections, with the Cmax occurring within minutes after administration [7]. Unmodified siRNA could be degraded easily by nucleases due to the property in vivo. siRNA after administration was distributed mainly to the liver, spleen, kidney, and lung, but poorly to other tissues. Therefore, siRNAs were often associated with various modifications and delivery methods (for example, coupled with target molecule, antibody, ligand) to prolong half-time in plasma and achieve the expected distribution mode. Studies after multiple systematic administration indicated that nucleic acid drugs were mainly accumulated in the kidney and liver, making them the main toxicity target for these drugs. In fact, patho- and functional alterations were found in these organs after persistent or subchronic administration of a high dose in animals. There were also noticeable accumulations in the spleen, skeleton, bone marrow, and lymph nodes, but these accumulations were significantly lower than in the liver and kidney. Similar results were also observed in mice and nonprimates [68]. 10.3.1. Factors of Influence for Tissue Distribution

Although there are common regularities in tissue distributions among nucleic acid drugs, the difference in structural modifications, routes of administration, and so on assigns each individual drug with its own distribution characteristics. 10.3.1.1. Structural Modification In recent years, a variety of structural modifications have been used to meet diverse purposes. These modifications could alter the time course of nucleic acid drugs in tissues to some extent. But this alteration had definitive regularities. For example, ISIS 16952, a second-generation ODN (PO 2 -O-MOE-full), was distributed to a somewhat less extent in the liver, where the highest concentrations were seen for the counterpart PS-ODN ISIS 2302. The fully PS-modified 2 -O-MOE ODN (ISIS 11159) was distributed to a greater extent to the kidney than either ISIS 2302 or ISIS 14725 (PS 2 O-MOE-partial). ISIS 2302 was substantially degraded in tissues by 24 h with

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only 25 to 30% of the total measurable ODNs in tissues identified as parent 20mer. The partially modified ISIS 14725 was partially protected from exonuclease metabolism and, thus, more slowly degraded in most tissues with fewer measurable metabolites apparent at 24 h (50% intact). The fully 2 -O-MOE-modified ODNs (either PS or PO backbones) appeared to be completely resistant to nuclease degradation even in tissues [55]. PMOs were similar to other kinds of nucleic acid drugs in tissue distributions. There was no particularity about tissue distribution of siRNA compared with other kinds of ODNs. Braasch et al . [69] reported the biodistribution of a phosphodiester (PO) siRNA duplex and examined the effect of phosphorothioate (PS) linkages. Two 125 I-labeled duplex RNAs were synthesized. One contained two phosphodiester (PO/PO) RNA strands, the design commonly used for siRNA. The other siRNA contained one phosphodiester strand and one phosphorothioate strand (PO/PS) to certify the hypothesis that PS linkages would improve the pharmacokinetic properties of the duplex. The result indicated 125 I-labeled siRNA preferentially accumulated in the kidney and liver at all time points. Lesser concentrations were observed in the lungs, spleen, and heart. Very low concentrations were detected in the brain, presumably reflecting a poor ability to penetrate the BBB. Meanwhile, the concentration of (PO/PS) siRNA was higher than that of (PO/PO) siRNA in plasma. The result demonstrated (PO/PS) siRNA was more stable than (PO/PO) RNA in plasma. 10.3.1.2. Route of Administration Routes of administration could noticeably alter distribution characteristics. In recent years, studies on local administration have increased gradually. Nucleic acid drugs can be delivered to a localized therapeutic location by local administration because few or no drugs were systemically absorbed and thus increased the concentration in target organ and validly reduced the toxicity of other organs. Following injection into the vitreous humor, the ODNs are localized in various ocular tissues, and the distributions of other tissues are rare. Among cynomolgus monkeys that received a single intravitreal injection of a high dose ISIS 2922, the uptake and degradation appeared saturated in the retina. The drug concentrations in the vitreous humor increased with the dose, showing linear correlations [32]. Macugen also showed the same property that drug concentrations in plasma and vitreous were linearly related to the dose administered [70]. For another 19-mer AS-ODN, following intravitreal injection in rats, the uptake, distribution, and persistence of the ODN in the retina were demonstrated by fluorescent confocal microscopy. The penetration of the ODN was observed in the ganglion cell layer, the photoreceptor, and retinal pigment epithelial layers. It was demonstrated that the latter is the primary target tissue, and the persistence time was the longest [71]. To improve the ocular retention of the ODNs, chemical modifications on these compounds were employed. The detailed absorption and distribution studies of the first inhaled PS-ODN EPI-2010 revealed a similar pattern of drugs throughout the large and small airways of the rabbit and mouse lung with limited systemic bioavailability [28,30].

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In rabbits, the aerosolized EPI-2010 was delivered into the lung by passive respiration. The Cmax of the drug in the lung at 6 h was 67.0 μg equivalent/organ then decrease. The elimination half-life for EPI-2010 in the rabbit lung was approximately 30 h. About 3% of the delivered drug remained in the lung 72 h after administration, and only 6.9% of EPI-2010 derived radioactivity was found in the heart, liver, and kidney (combined). The postlabeling studies indicated that intact EPI-2010 was detected only in the lung [28]. In the mouse lung, accumulation of inhaled first-generation ODN was most evident in alveolar macrophages, and the elimination half-life was 20–30 h [30]. The second-generation 2 -MOE ODNs also showed the similar distribution characteristics in the mouse lung, but much lower systemic exposure with no measurable levels in the kidney or liver. However, unlike intravitreal injection, pulmonary exposure of 2 -MOE ODNs in mice was dose dependent but was not proportional with dose. For in vivo siRNA delivery, there are two main strategies, systemic or localized, which result in very different profiles of biodistribution. When siRNA is delivered systemically, the typical clearance time without any conjugates or modifications is less than 10 min in the body. This short residence time necessitates the choice of delivery methods and the resulting biodistribution. Systemic delivery is an injection into the systemic circulation system, which provides widespread distribution of siRNA throughout the animal. Localized delivery, which injects siRNA directly, reaches the target sites, thus providing more limited biodistribution. The advantage of local administration is that siRNA will target delivery into the cells or tissues straighter, which expresses the interesting genes. Successful applications of localized siRNA delivery include intranasal [72], intratheca [73], intratesticular [74], intraliver [75], intramuscular [76], intraretinal, and intratumoral [77]. 10.3.1.3. Cell Type Organ uptake is generally heterogenous and cell type specific. For example, the highest concentration of ISIS 22023 was observed in endothelial cells, followed by the Kupffer cells. The concentration of ISIS 22023 observed in hepatocytes was the lowest. The uptake by Kupffer cells appeared to be more delayed than the uptake by hepatocytes or endothelial cells. When the concentrations in hepatocytes diminished, the concentrations of drugs in Kupffer cells appeared to increase. These data suggested that the redistributions of ODNs were diverse between different cell types in the liver. From immunohistochemistry studies with other ODNs, it is clear that Kupffer cells are a repository for nucleic acid drugs long after dosing [18]. Graham et al . studied the distributions of different ODNs in cells, and the results suggested that within 24 h, ODNs could accumulate in renal proximal convoluted tubular cells, skin fibroblasts, dendritic cells, and within the hepatocytes, Kupffer and endothelial cells of the liver. In hepatoctyes, lower concentrations of PS oligonucleotide were observed relative to other cell types [78]. 10.3.1.4. Animal Species Studies showed that tissue distribution was not closely related to animal species. The highest concentrations of ISIS 104838,

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a 20-mer second-generation ODN targeted against human TNF-α mRNA, were measured in the kidney, liver, lymph nodes, and spleen, independent of the species studied. Very low levels were observed in skeletal muscle, and little to no evidence of ISIS 104838 was seen in the brain. The results were similar throughout different animal species [5]. Thus, in combination with the similarity in organ distribution across species, the pharmacokinetic profile in humans infers organ distribution similar to that in the preclinical animal models. However, at a similar mg/kg dose, mice generally exhibit the lowest comparable ODNs concentrations in organs of distribution on a mass/gram tissue basis, although dogs and monkeys exhibit similar organ concentrations given similar doses based on body weights that are 5- to 10-fold higher than rodent tissue. The observation that plasma pharmacokinetics are concordant for monkeys, dogs, and humans when dosed on a weight equivalent basis coupled with the consistency of tissue distribution across several nonclinical models provides further assurance that the underlying tissue distribution in humans is similar to that of preclinical models [5]. The tissue distributions of nucleic acid drugs had some regularity in common, but most of them did not have any distribution target. Recently, more studies on targeted distributions have been conducted for more distributions in targets and more obvious pharmacodynamic action, including structural modifications, conjugates, and carriers. The relationship between tissue distribution and toxicity of nucleic acid drugs remains unknown. There are so many mysteries that it will be several years before the ODN drugs can be understood and used in terms of their therapeutic utility.

10.4. METABOLISM

Nucleic acid–based drugs are metabolized by nucleases and are thus different from most small molecular drugs that are largely metabolized in cytochrome P450–dependent pathways. The metabolic patterns of ODNs are similar no mater how they are administered. The metabolic way analysis by capillary gel electrophoresis (CGE) and liquid chromatography-mass spectrometry (LC-MS) has demonstrated that there is a progressive shortening of the ODN, and the nature of the metabolites clearly depicts the profiles of metabolic pathways. In general, the bases are successively removed from the 3 end of PS-ODN by 3 exonucleases, where is major degradation of ODNs in plasma and tissues [79–81]. Occasionally a few identified metabolites with one or two bases deletions also occur at the 5 end or both ends [82]. The extent of degradation of the ODNs could vary with time and different tissues. Furthermore, a series of metabolites of sulfur and oxygen exchanging at phosphorothiodiester position (oxidative metabolism) could be also detected. All these detected metabolites indicated that the metabolic pathway of ODNs is similar to that of endogenous nucleotides. PS-ODN is degraded quickly in plasma. The chain-shortened metabolites of prototype drugs can be immediately detected after intravenous administration, as in the liver, kidney, and other tissues after subcutaneous administration [83].

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Metabolites of a PS-ODN CPG 7909 identified by matrix-assisted laser desorption ionization–time of flight–mass spectrum (MALDI-TOF-MS) were similar in all analyzed tissues (main metabolites are N-1, N-2, and N-3) for both rodent species, although the relative amounts of the different metabolites varied with tissues [84]. In all organs, the highest proportion of full-length CPG 7909 was detected at the earliest time point (4 h) and at the latest time point (72 h), A large proportion of the ODNs detected was a series of shortened metabolites. PS-ODN metabolism was affected by the length, stereoisomer, sequence, and other factors. Crooke et al . [85] researched the metabolic process of PS-ODN in rat liver homogenate and found that [1] the oligomers’ length affects the rate of degradation; [2] Sp and Rp isomers of PS-ODN display different sensitivities to nucleases, and phosphorothioate linkages with Rp nucleotides are much more liable to be degradated than Sp nucleotides; and [3] the sequences of PS-ODN also affect the metabolism rate, especially pyrimidine-rich sequences, which are easily degradated. Like PS-ODN, the second-generation MBOs are metabolized by nucleases. However, the resistence of different modificated MBOs against nuclease is different. Geary et al . [55] compared the pharmacokinetic properties of 2 -OMOE modified oligonucleotide analogs with first-generation PS-ODN in rats. The unmodified PS-ODN ISIS 2302 was substantially degraded in tissues over 24 h, with only 25–30% of the total measurable ODN in tissue identified as parent. The fully 2 -O-MOE-modified ODNs (either PS or PO backbones) appeared to be completely resistant to nuclease degradation, even in tissues. The partially modified 2 -O-MOE ODN ISIS 14725 was partially protected from exonuclease metabolism. For PMOs, no detectable PMO metabolites have been observed from whole blood, plasma, tissues, cerebrospinal fluid, or urine. Whereas PS-ODNs are degraded in a time- and tissue-dependent manner, PMOs demonstrate sufficient biological stability irrespective of the route of administration. As for siRNAs, their metabolic patterns are similar to those of most ODNs, which is a sequential degradation process. Studies on the pattern of siRNA degradation by analyzing the ocular metabolites of siRNA duplex, SIRNA-027 [86], demonstrated that the duplex was metabolized predominantly from one end, the 3 end of the antisense strand or the 5 end of the sense strand. It was the end of the siRNA duplex with lower binding energy of the two ends by theoretical caculation that indicated the ability of the siRNA to split into single strands was a major factor in its degradation.

10.5. EXCRETION

Urinary excretion represents the major path way of elimination of the ODN. As experiments indicated, the rate of metabolism and excretion varied with ODN modifications. After traditional PS-ODN medicines were delivered into the body, they were cleared quickly from the blood. The levels of ODNs were quite low in the brain and fat, but still existed in the skin, bone, heart, lungs, and tumor

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tissues. The excretion pathway of PS-ODN was similar in different animals and was also time dependent [28,87]. During the research of aerosolized EPI-2010 for asthma, Ali et al . [28] discovered that the excretion pathway of EPI-2010 in the rabbit was very similar to that in mice, rats, and monkeys. Approximately 67.5% of the delivered dose was excreted in urine over a period of 72 h. Excretion in feces represented a minor route of elimination, with 7% eliminated over 72 h. In both cases, excretion of EPI-2010 was time dependent and reached to the maximum level in 6 h after first administration before starting to decline in 48. The excretion process of PS-ODN is associated with the route of administration. In general, compared with the intravenous route, subcutaneous administration produces fewer metabolites. Raynaud et al . [48] evaluated the pharmacokinetics of 35 S-labeled G3139 in female BALB/c mice after a single i.v. bolus administration or s.c. infusion. After both administration routes, most of the radioactivity was eliminated in the urine and to a lesser extent in the feces. Significantly more radioactivity was excreted in the urine after i.v. bolus, compared with s.c. infusion (33% on day 1 and 55% by day 3 for i.v. vs. 7.2% on day 1 and 12.9% by day 3 for s.c.). Like PS-ODN, the prototypes and metabolites of MBOs are excreted with urine and feces. Geary et al . [5] found that the fraction of intact 2 -O-(MOE) modified AS-ODN ISIS 104838 excreted was low in urine after i.v. infusion in all species. The highest rate of excretion occurred 0–24 h after bolus injection in rodents (as high as 17% of the administered 50 mg/kg dose in mouse). In rats, urinary excretion ultimately accounted for approximately 75% of the total radiolabeled dose by 90 days after a single dose of 5 mg/kg ISIS 104838, and a small amount of radioactivity was also tested in feces (10% of administered dose over 90 days), bringing the total excretion to approximately 8085% after a single dose. However, after 1 h infusion in monkeys, less ISIS 104838 and fewer related metabolites were excreted in urine (less than 10% of the total dose was recovered as parent and oligomers metabolites). The fraction of the dose excreted in monkey urine was somewhat lower after s.c. injection at 20 mg/kg when compared with i.v. infusion at 10 mg/kg (4.5% after s.c. vs. 7.7% after i.v.). This is likely due to lower plasma concentrations after s.c. injection and, subsequently, less free drugs available for filtration. For the third generation of nucleic acid drugs in preclinical and clinical trials, PMOs have been shown to be eliminated mainly in urine and feces. For most PMOs, urinary excretion seems to be the major route of elimination, although in some cases fecal excretion has been estimated to be about 30% of administered dose [52].

10.6. PLASMA PROTEIN BINDING

Binding to serum proteins plays a key role in the pharmacokinetics of ODNs and in their toxicologic properties as well, in view of the negative effects of phosphorothioates on clotting and complement activation. PS-ODNs bind nonspecifically

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to various proteins in plasma in a greater affinity than their phosphodiester counterpart. At a clinically relevant concentration, more than 96% of PS-ODNs in plasma are associated with plasma protein in mice, rats, monkeys, and humans [88,89]. The primary ODN binding proteins are albumin and α2 -macroglobulin. The apparent affinity for albumin ranges from 150 to 400 μM, and the binding affinity to α2 -macroglobulin was found to be greater than albumin with less binding capacity. In contrast, binding to α1 -acid glycoprotein was negligible. Geary et al . [5] assessed the protein binding characteristics of ISIS 104838 using ultrafiltration methods. ISIS 104838 was highly bound to plasma proteins and was likely to prevent renal filtration. However, shortened ODN metabolites of ISIS 104838 loosed their affinity to bind plasma proteins. The reduction in protein binding observed for shorter ODN metabolites of ISIS 104838 suggests a mechanism for preferential excretion of the shorter metabolites compared with parent 20-mer. Thus, as the drug is metabolized in tissues, the shortened metabolic products bind with less affinity and are eliminated from tissues and ultimately from the body by urinary excretion. The same results were also attained by Watanabe group [90], who focused on the protein-binding characteristics of ISIS 2302, a PS-ODN targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA. ISIS 2302 was shown to be highly bound (>97%) across species (mouse, rat, monkey, human), with mice having the least degree of binding. Ten shortened ODN metabolites (8,10, and 12–19 nucleotides in length, truncated from the 3 end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Results also suggested that the protein-binding characteristics in human plasma may be sequence dependent, and that other highbinding drugs did not displace ISIS 2302 at supraclinical plasma concentrations, unable to cause any pharmacokinetic interaction clinically as a result of the displacement of binding to plasma proteins. PS-ODNs also exhibit highly nonspecific binding in tissues. The high propensity of PS-ODN drugs bound nonspecifically to proteins may account for, in part, pharmacokinetics and biodistribution properties of the first-generation ODN drugs in animals and humans. Protein-bound drugs serve as a reservoir of circulating ODNs in blood and prevent rapid renal excretion. Excretion of intact drugs in urine is a minor elimination pathway because protein binding prevents it from glomerular filtration. For example, urinary excretion of ISIS 301012, a 2 -OMOE-modified ODN targeting human apolipoprotein B-100 gene [51], increased with the dose in mice. The dose dependence of ISIS 301012 excretion in mice was most probably due to increasing concentrations of unbound drugs at higher plasma drug concentrations, which was reflected in its lower plasma proteinbinding extent. Consistent with this but to a lesser extent, the dose-dependent urinary excretion of ISIS 301012 was observed in monkeys and humans. The dose-dependent difference is less remarkable in monkeys and humans, probably due to the greater plasma protein-binding extent than in mice. As a model for protein binding to human serum albumin (HSA) in plasma, binding constants to bovine serum albumin (BSA) have been measured [62]. The affinities of the lipophilic conjugates were greater at physiologic salt

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concentrations than the affinity of the 29- PS-ODNs. The binding affinity of the conjugates was not salt dependent.More lipophilic PS-conjugates may bind to more than one type of site on BSA or may bind more tightly to the same site as the unmodified PS-ODNs. Srinivasan et al . [91] evaluated interactions of cholesterol conjugated ODNs with BSA and HSA in vitro. When HSA-warfarin complex was incubated with a variety of ODNs, a 59-cholesterol conjugated 20-mer phosphorothioate displaced warfarin to a greater extent than the unconjugated ODN. These two studies indicated that the association of cholesterol conjugates with serum albumin may also contribute to the improved uptake. 10.7. SUBCELLULAR BIODISTRIBUTION OF NUCLEIC ACID–BASED THERAPEUTIC DRUGS

To function as nucleic acid drugs, the ODNs should be able to permeate the cellular membrane and must bind to the target RNA in the cytoplasm or nucleus or both, depending on the efficient uptake by tissue cells and ease of release into the cytoplasmic from endosomes. Therefore, subcellular biodistribution are an important part of pharmacokinetics for ODNs or siRNAs. In this part, we summarized the subcellular distribution profiles and probable mechanisms involving endocytosis and intracellular trafficking of ODNs in various forms. 10.7.1. Subcellular Biodistribution of Naked Oligonucleotides

Early studies focused on the distribution of naked ODNs in specific tissues and cell types. Butler et al . [92] determined the distribution of intravenously injected ODNs in rodent tissues using three histologic methods: immunohistochemistry, direct fluorescence microscopy, and 14 C-labeled autoradiography. All three methods gave the same pattern of ODN distribution that the highest concentrations were observed in proximal tubule cells in the kidney and Kupffer and endothelial cells in the liver. The pharmacokinetics of ISIS 1082, a 21-base heterosequence PS-ODN, was characterized within the rodent whole liver, cellular, and subcellular compartments [93]. Parenchymal and nonparenchymal cells in the liver were isolated and the nuclear, membrane, and cytosolic constituents were further purified to determine the subcellular distribution. Maximum liver concentrations appeared at 8 h and decreased dramatically. Rat nonparenchymal cells retained approximately 80% of the ODN present within all hepatic cell types. This level was equally distributed between Kupffer and endothelial cells, whereas the remaining 20% was localized within hepatocytes. The subcellular distribution profile of MBO was similar. ISIS 22023 was found abundant in endothelial cells with different peak time depending on cell types [18]. Based on the preceding results, it has been suggested that scavenger receptors mediate the primary mechanism of ODN uptake in vivo. To address the role of scavenger receptors (SR-AI/II) in ODN distribution in vivo, cellular distribution profiles were compared in tissues from scavenger receptor knockout mice (SRA-/-) and their wild-type counterparts after i.v. administration. No differences in

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cellular distribution and ODN concentrations in several organs including livers, kidneys, lungs, or spleens taken from SR-A-/- versus wild-type mice could be detected both by the histological method and CGE analysis. Although the concentration of ODNs in isolated Kupffer cells from livers of SR-A-/- mice was 25% lower than that in Kupffer cells from wild-type mice, the target mRNA levels decreased to a similar extent in livers from SR-A-/- (92.8%) and wild-type (88.3%) mice. Taken as a whole, the data suggested that although the SR-AI/II receptor may make some contribution to ODNs, this mechanism alone cannot account for the bulk of ODN distribution into tissues and cells in vivo, including macrophages [94]. A series of ODNs of different lengths (12-mer, 20-mer, and 25-mer) and sequences were evaluated on their binding, internalization, and subcellular distribution properties in vascular smooth muscle cells [95]. The tested ODNs showed striking differences in their uptake and distribution in smooth muscle cells. The affinity of the 20-mer ODN for the cell surface was particularly high, whereas that of the 12-mer one exhibited the lowest affinity. Further results indicated that the ODNs’ affinity for the cell membrane and the efficiency of internalization depended on the sequence, and more likely, on the buildup of a tertiary structure from that sequence, rather than on the size of the oligomer. Therefore, the intracellular distribution fate of naked PS-ODN was probably sequence dependent. Unlike PS-ODNs and other charged ODNs, little is known about the uptake characteristics of the neutral chemistry PMOs except several characteristics [96]. In contrast to primary cells and some transformed cell lines, which were uptake permissive, established cancer cell lines showed very poor uptake with an occasional diffuse intracellular pattern. It seemed that some constitutes necessary for the PMO uptakes were missed during the cell line establishment. Differential PMO uptakes were also observed in immune cells, with dendritic cells and monocytes showing highest uptake compared to T- and B-cells. In addition, PMOs localizations were observed to be heterogeneous within a population of uptakepermissive cells. In vitro experiments showed that the streptolysin O, cationic liposome, and lipofectamine couldn’t improve the intracellular entries of PMOs. In addition, PMO internalizations in uptake-permissive cells were identified to be specific, saturable, and energy dependent, suggesting a receptor-mediated uptake mechanism. However, the mechanisms need to be further investigated. 10.7.2. Subcellular Biodistribution of Oligonucleotide Conjugates

Cell penetrating peptides (CPPs) are small peptides (usually less than 30 amino acids) with net positive charge (in most cases) that could translocate across plasma or endomembranes and transport materials into cytoplasm and nucleus. It is now clear that uptake of ODNs–polycationic CPP conjugates can be enhanced because the polycationic CPPs can bind to cell surface glycosaminoglycans and mediated endocytotic uptake (possibly macropinocytosis) [97]. Specifically, data suggested that CPPs can more effectively deliver ODNs with uncharged backbones, such

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as peptide nucleic acids (PNAs) and morpholino compounds [98,99]. Confocal microscopy combining with endosomal markers showed the delivery of the ODNs into the nucleus as well as to intracellular vesicles and the release of that from the endosomes into the cytoplasm, indicating that the CPP–PNA conjugates were most likely taken up by macropinocytosis (10053 ). Besides CPPs, cellular uptakes and intracellular localizations of ODNs could be achieved by various DNAsignal peptide conjugates [101]. For example, controlled nuclear localization was achieved by ODN-nuclear localization signal (NLS) conjugates (such as SV40 large T-antigen and HIV-1 tat protein) and cytoplasmic localization was achieved by ODN–nuclear export signal (NES; HIV-1 rev) conjugates. Another promising strategy is to deliver ODNs or siRNAs by using cell targeting ligands (CTLs), which are able to bind to certain cell surface receptors with high affinity. Because various receptors are often preferentially expressed on particular cell types, this approach offers the possibility of improved selectivity by targeted-tissue distribution and efficient internalization. Althoughe a rich variety of cell surface receptors are expressed in the human body, work involving delivery of ODNs has so far primarily focused on lipoprotein receptors (particularly those in the liver) [102], integrins [103,104], receptor tyrosine kinases [105], and G-protein coupled receptor (GPCR) [106]. In addition, folateODN conjugates can also penetrate cells exclusively via folate-receptor-mediated endocytosis [107]. For example, the RGD–ODN conjugate delivery system [99,104] consisted of an anionic ODN and a bivalent RGD peptide (Arg-Gly-Asp) that bound to the αvβ3 integrin with high affinity. The delivery system could improve the cellular uptake of the ODN, and the increased uptake could largely be blocked by coincubation with either the excess free cyclic RGD peptide or the monovalent cyclic RGD peptide that is a specific ligand for the αvβ3 integrin. The uptake of the conjugate was higher in a αvβ3-positive melanoma subline than in its αvβ3negative version, suggesting the involvement of the αvβ3 integrin. In addition, the exposure of the cells to RGD-ODN conjugates did not result in any loss of αvβ3 expression on the cell surface, presumably due to efficient recycling of the receptor. Further intracellular distribution showed that at early time points (2 h) there was no colocalization of the RGD-conjugate with transferrin, but there was extensive colocalization with dextran. At later time points (24 h) there was substantial fluorescence overlap in both cases, although it was most pronounced for dextran markers. At the same time, nuclear accumulation of fluorescence appeared. These observations suggested that the RGD-ODN conjugates initially entered cells via a non-clathrin-mediated endocytic process, but eventually trafficked through various endomembrane compartments, including those that can also be accessed by transferrin. 10.7.3. Subcellular Biodistribution of Oligonucleotides in Nanocarriers

A variety of supramolecular nanocarriers including liposome, cationic polymer complexes, and various polymeric nanoparticles have been widely used to deliver

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ODNs and siRNAs. Several cationic nanocarriers, including polyetherimide (PEI) and polyamidoamine dendrimers exerted a so called proton sponge effect that helps to release contents from endosomes [108]. Other nanocarriers such as polymeric nanoparticles, polymer micelles, and lipoplexes, could interact with anionic lipids of the endosome membrane, leading to the formation of nonbilayer structures and consequent endosome instability, and the material release [109]. It is explicit that the transfection reagents representative of cationic liposomes can elicit membrane-fusion-induced endocytosis [110]. Ultrastructural studies clearly showed ODNs in the absence of liposomal delivery to be within endosomal, lysosomal vesicular bodies and, to a lesser extent, in the nucleus and cytoplasm, indicating the endocytic uptake. By employing lipoplexes, enhanced penetration of ODNs within the cytosol and nucleus with a concomitant reduction in vesicular compartments were observed. Efflux studies confirmed that cationic lipoplexes promoted entry of ODNs into deeper cellular compartments, consistent with endosomal release. Bennett et al . also demonstrated that in the presence of the lipoplexes [111,112], the uptake of ODN was increased and the degradation by lysosomes was avoided, which led to the increased activity of AS-ODN ISIS1570 by nearly 1000-fold. 10.7.4. Subcellular Biodistribution of siRNAs

It is traditionally regarded that once siRNA has been introduced directly into the cytoplasm, the guide strand of the siRNA is assembled with other components to form mature RNA-induced silencing complex (RISC). Though the RNA interference (RNAi) pathway occurs mainly in cytoplasm, the subcellular location of siRNA production and degradation of mRNA remains unclear. Recently, several groups have demonstrated that transfection of the naked, unmodified siRNAs can activate potent immune responses through toll-like receptor (TLR)-dependent or -independent pathways. Though the subcellular biodistribution of siRNAs has not been fully clarified, probable localization might be concluded. For the “naked” (whether chemically modified or not) siRNAs, it was affirmative that the fluorescein-conjugated Luc siRNA without cell-permeating entities were not internalized by mammalian cells, including Drosophila melanogaster S2 cells, primary human choroidal endothelial cells (CECs), or mouse retinal pigmented epithelium (RPE) and CECs [113,114]. The subcellular pharmacokinetics of siRNAs were determined by employing various transfection approaches [115]. Cy3-labeled siRNA was delivered to HeLa cells, which were stained with DAPI. Confocal microscopy results showed that using lipofectamine, siRNA localized to the cytoplasm around the periphery of the nucleus, and this pattern was siRNA sequence independent and not altered in the presence of an mRNA target. In contrast, siRNA transfected with nanoparticle NP-45 also localized to perinuclear regions of the cytoplasm, although it appeared to aggregate to more discrete areas within the perinuclear region. Interestingly, using higher concentration of NP-45, a shift in siRNA localization patterns was observed with seemingly more diffuse cytoplasmic localization. In

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addition, siRNA was observed in both the nucleus and nucleolus, indicating that delivering siRNA to cells using NP-45 at this higher concentration altered siRNA subcellular localization. More recently, naked siRNA has been shown to activate TLR3 on the surface of vascular endothelial cells and to trigger the release of interferon (IFN) and interleukin (IL)-12 that mediate nonspecific antiangiogenic effect in vivo [113]. Soluble TLR3, rather than soluble TLR4 or heat-denatured soluble TLR3, abolished choroidal neovascularization (CNV) suppression by Luc siRNA in wild-type mice, suggesting direct interaction of siRNA with TLR3. Fluoresceinconjugated Luc siRNA bound wild-type but not T lr3−/− mouse eye sections in situ. Using flow cytometry to monitor binding of fluorescein-Luc siRNA to the surface of CD 31+ VEGFR+ mouse choroidal endothelial cells, greater fluorescence was detected on wild-type than on T lr3−/− cells. Preincubation with soluble TLR3 or competition with poly (I:C) reduced fluorescein-Luc-siRNA binding to wild-type mouse CECs. These data extended cell-free system data that 20-nucleotide dsRNAs bound TLR3 [113,116]. In addition, unlike the RNAinduced TLR7/8 response, it had been reported that the activation of cell surface TLR3 by naked siRNA was not inhibited by 2 -O-Me modification [113]. Studies also showed that siRNAs were capable of activating the mammalian immune response, primarily through endosomal activation of the TLR7/8 pathway [117,118]. The nucleic acid-sensing TLRs were typically located intracellularly and engaged nucleic acids released from invading pathogens as they were degraded within the endosomal/lysosomal compartment [119]. Plasmacytoid dendritic cells (pDCs) that constitutively express TLR7 in both mice and humans were directly activated by siRNA to secrete high levels of IFN. In vivo experiment results were consistent with the in vitro ones. IFN-α induction by the TLR9 ligand CpG ODN 1826 and siRNA9.2 was not reduced in bone marrow cells derived from TLR7-deficient mice compared with wild-type control mice. Furthermore, no activation was found in spleen cells of TLR7-deficient mice [117]. Altogether, whether the potential mechanisms of siRNAs-based drugs are via RNAi pathway or via TLR-dependent or -independent pathway, more evidence is needed to determine the siRNA subcellular distribution to contribute to the optimizated design of such nucleic acid drugs.

10.8. CONCLUSIONS

As discussed in this section, a great deal of effort is currently focused on the improvement of subcellular pharmacokinetics and intracellular delivery of nucleic acid drugs. However, the mechanisms of subcellular trafficking are not totally clear and may vary, depending on different modifications and delivery systems. Importantly, many of the positive features of delivery strategies are counterbalanced by some important negative ones. For example, though nanoparticles can be designed to release their contents at prescribed rates and can also be engineered to assist in the release of their contents from endosomes, it is important to note that

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the carrier systems themselves can have significant effects on gene expression and may potentially cause toxicity due to the endothelial barrier [120,121]. In contrast, ODN conjugates are usually far smaller than the pores in normal vascular endothelium. Thus, in principle, they should be able to access virtually all tissues, like conventional drugs do (with the exception of the central nervous system) [97]. Further, it seems that the polymolecular complexes of CPPs are more effective than monomolecular ODN conjugates, which is perhaps because it requires multiple copies of CPPs to attain strong endosome destabilizing effects. Also, in most of the examples cited, it is not clear that endosomal release is also enhanced, whereas the targeting ligand can certainly enhance uptake. Perhaps one aspect of the receptor targeting strategy may entail differential opportunities for release from endosomes as the internalized ODN traffics through different endomembrane compartments. Altogether, optimized modification to nucleic acid drugs or an ideal delivery system not only enhances cellular uptake of ODNs, but also facilitates the drug’s escape from subcellular compartments to cytoplasm and nuclear compartment, which is clearly important to achieving maximum therapeutic activity of ODNs and siRNAs. 10.9. QUANTITATIVE ANALYTICAL TECHNIQUES IN PHARMACOKINETICS RESEARCH

Under preclinical and clinical development, quantitative analysis of therapeutic nucleic acid–based drugs and characterization of their metabolites are important aspects of drug development and evaluation. Quantitative analysis of oligonucleotides (ODNs) or small interfering RNA (siRNA) from various biological matrices (plasma, urine, cerebrospinal fluid, tissue, feces, etc.) and identification of their metabolites in vitro and in vivo not only helps investigators to acquire the key information on their kinetics and metabolic profiles in pharmacokinetics (PK) and pharmacokinetics/pharmacodynamics (PK/PD), but also makes possible the development of rational strategies to improve the stability, durability, and bioavailability of these therapeutic compounds. All the preceding assay data will be important in estimating the human doses and clinical dose regimen designs. Before biological quantitative analysis turns into routine analysis, the applicability and reliability of this method should be determined. The ideal bioanalytical methods for carrying out these studies should be highly selective, sensitive, accurate, and robust as well. Furthermore, it should not be disturbed by biological matrix and have high throughput analysis capabilities. Recently, during preclinical and clinical PK studies, many techniques have been applied in the quantitative analysis and metabolism studies of therapeutic ODNs, which include radioisotope tracer technique, hybridization-based enzyme-linked immunosorbent assay (ELISA), capillary gel electrophoresis (CGE), high-performance liquid chromatography (HPLC), and liquid chromatography separation coupled with mass spectrometric detection (LC-MS), etc. [122]. However, there are few quantitative analyses applied on double-stranded siRNA or confined to LC-MS detection

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and ELISA. Each analytical method has its own strengths and weaknesses in quantitative analysis of different nucleic acid drugs. Except for sensitive quantitative methods, extraction is very critical during the study of PK properties of ODNs.The radioactive method need not be sophisticated sample preparation during the assay because it is only to determine the sample radioactivity; besides, there is no biological matrix interference. During radioactive assay, samples usually follow few separation steps and mix with scintillation fluid before counting the radioactivity. For quantitative methods such as hybridization ELISA, CGE, HPLC, and LC-MS, they need to separate samples from biological matrix and add internal standard as quantitative reference during sample pretreatment. Due to ODNs’ property, the combination of liquid extraction and solid phase extraction has always been used. Among them, methanol, or acetonitrile combine with triethylamine acetate (TEAA) or triethylamine bicarbonate (TEAB) to remove proteins are commonly used. Sample preperation will be discussed in detail in the following chapters on quantitative analytical methods. On the whole, hybridization-based ELISA method is highly sensitive, reproducible, and amenable for high throughput assay, which has been applied to studies of multiple preclinical and clinical PK. However, loss of one or more nucleotides also results in cross-hybridization; this method is unable to distinguish prototype ODN drugs from its truncated metabolites, which will be overestimated in the parent ODNs. Such shortcomings narrow this method to be applied widely. 10.9.1. The Radioisotope Tracer Method

In pharmacokinetic studies of biotechnology drugs, the radioisotope tracer method is often adopted, particularly in earlier studies, because of its high sensitivity and simple and quick operation. The radiotracer method is based on the measurement of radioactivity of isotopes of 3 H, 14 C, 35 S or 125 I-labeled ODNs in biological matrices. Thus, labeled ODNs can be quantitated by measuring samples’ total radioactivity [123]. Liquid scintillation counting method is the one mentioned earlier. The samples for radioactive tracer method need nothing more than simple pretreatment, especially in the samples of plasma and urine, which can be directly pipetted to the scintillation vial and whirled with scintillation liquid for counting the radioactivity. The treatment of tissue and fecal samples are relatively complex compared with plasma or urine, involving series pretreatments, which include homogenate, incubation, and decolorization before the total radioactivity of samples is determined with scintillation fluid [28,124,125]. However, without an additional separation via chromatography, the assay result only reflects the overall radioactivity of parent ODN and metabolites, which leads to overestimation of the parent drug. Information on metabolites is limited by the chemical location and metabolic fate of the isotope labels [49]. So the disadvantage of this method is that it cannot distinguish parent ODN, metabolites, and other unknown forms of radioactivity. Although various means are adopted to compensate for the quantitative ability of parent ODN, for example, using SDS, Tris-EDTA to digest tissue, plasma, and


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others, subsequently labeling and employing polyacrylamide gel electrophoresis (PAGE) or HPLC for the determination of every content, or making histological section for autoradiography. But in most cases, pharmacological doses of exogenous labeled biotech drugs will degrade in vivo or be reused by the organism after administration and result in the total radioactivity that cannot represent the concentration of original drugs. Therefore, it is not appropriate for using total radioactivity represent PK process. Generally, PK profiles of nucleic acid drugs cannot only depend on the result of radiolabeled method, but also need combination with other analysis methods. In addition, a radiolabeled ODN has a different physicochemical property from unmodified ODN, which might result in a different disposition in vivo. As Yu et al . [122] mentioned, due to poor selectivity, limited sensitivity, high costs in producing radiolabeled compounds, and limited application to clinical trials, radiotracer techniques are not frequently used in PK studies, with the exception of mass balance studies. 10.9.2. Hybridization-Based ELISA Method

Generally, the hybridization-based ELISA method offered the best assay sensitivity and high throughput. The method requires a minimum of sample cleanup. It has been widely used for quantitative analysis of ODNs in the evaluation of PK/PD, especially in the assessment of terminal elimination phase in plasma [126,62]. Now, hybridization quantitative techniques comprise one-step homologization-competitive hybridization and two-step noncompetitive heterogenesis hybridization. Hybridization technique has been reported in quantitative analysis of ODNs and their analogs [127,58,128]. The lower limit of quantitation (LLOQ) of these techniques ranged from 7 to 30 ng/mL [122]. Competitive hybridization assay with homogeneous format is simply operative and very sensitive, but the matrix effect may interfere with the detection. Consequently, the hybridization method is improved to enhance detection sensitivity to nanogram limit when the nucleic acid locking capture probe has been designed. Yu et al . [128] developed an ultrasensitive noncompetitive hybridizationligation heterogeneous ELISA assay for the quantitation of antisense phosphorothioate oligonucleotides (PS-ODN) ISIS 2302 in plasma using a 96-well plate format. The result showed no significant interference noted from untreated human plasma. The method is selective to the parent drugs with minimal cross-reactivity with 3 -end deletion oligomers [128,129]. LLOQ of the method was 0.05 nM (0.05 pmol/mL), and linear range of 0.05 to 10 nM (r2 > 0.99) was observed for PS-ODN GTI-2040 in a variety of biological matrices. Furthermore, the assay was demonstrated to be quitely accurate and precise for a number of PS-ODNs [128,129]. The validated method is also suitable for quantitation of secondgeneration ODNs and is used to analyze the ODN level of plasma in a phase I trial of ISIS-104838, a 2 -methoxyethyl (2 -MOE) modified ODN, targeting tumor necrosis factor-α [58].

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In addition, the developed novel ELISA-based assay for specific quantifying double-stranded intact siRNA for in vivo pharmacokinetic analysis was reported [65]. This assay was linear over the range of 0.01-0.1 pmol/ml, with a high sensitivity. The coefficient of variation of the ELISA quantification was 9.4% for intra-assay and 12.1% for interassay. On the whole, hybridization-based ELISA method is highly sensitive, reproducible, and amenable for high throughput assay, which has been applied to multiple preclinical and clinical PK studies. However, due to the loss of one or more nucleotides, it can also react as normal during cross-hybridization, so this method is unable to distinguish between prototype ODN drugs or truncated metabolites, which will be overestimated as the parent ODNs. 10.9.3. Capillary Gel Electrophoresis

CGE is widely employed in preclinical and clinical PK studies of nucleic acid drugs. It seems to be the most preferred separation technique and has the highest resolving power [130–132]. Actually CGE is a kind of electrophoresis that moves gel media from the plate to the capillary. Gel is porous, so sample molecules will move differently through the gel matrix, depending on their size: Small molecules will more easily fit through the pores in the gel, whereas larger ones will have more difficulty (they encounter more resistance). The high gel viscosity can reduce the diffusion of solute and help to form a sharp peak shape, which has higher column efficiency than other capillary electrophoresis techniques. Separation matrix of CGE can be divided into two categories, which are gel and nongel sieving. Due to the coagulation properties of gels for filling, CGE is very difficult to prepare and store. Besides, the high cost limits the application of CGE in this field [133]. The capillary electrophoresis mode of nongel sieving was introduced by Zhu et al . for the first time in 1989 [134]. They used low visicosity linear polymers such as methyl cellulose in place of polyacrylamide to form the medium known as nongel sieving. Nongel sieving is less expensive and replaces the media inside more easily than traditional gel, making it easy to prepare with better sensitivity and efficiency. Shang et al . [135] reported that the method was capable of discriminating the parent PS-ODN with single base deletion analogs. The relative standard deviation (RSD) of both intra- and interassay was below 10% for both, and the total mean recovery was about 91%. Extensive sample preparation procedures are required before CGE analysis, which can diminish the endogenous interference. Extraction of ODNs from tissue or plasma is mostly devoted to liquid/liquid extractions, that is, by phenol/chloroform, and/or solid-phase extraction (SPE), which are mostly found in ion-exchange and reversed-phase chromatography [136,127]. These techniques often offer adequate yield and reproducibility. They are, however, laborious and time consuming, requiring a large amount of samples. The sample preparation process is complicated and reduces sample recovery to under 50%. The internal standard is commonly used to overcome this problem [137]. The utilization of appropriate internal standards could normalize the deviations caused by electrokinetic injection and sample extraction.


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CGE provides good resolution in distinguishment of truncated analogs of single nucleotide deletion from full-length ODN. Therefore, CGE not only can determine the parent drug, but also can analyze the species and amounts of various in vivo chain-shortened metabolites [32]. CGE-ultraviolet (UV) has been the main quantitative analysis method for studies on preclinical and clinical PK behavior of the first or second generation of ODN drugs [18,138]. Laser-induced fluorescence instead of ultraviolet detection can improve the detection sensitivity of CGE. The ODNs in biological matrices as low as 20 ng/mL could be detected by postcolumn derivatization followed by fluorescence detector [139]. However, an obstacle to the application of CGE method is that it cannot detect the intake of ODN in vivo, intracellular distribution and metabolic transformation. Intracellular distribution of fluorescein isothiocyanate (FITC) labeled ODN would be detected through laser-induced fluorescence (LIF) technology [140], but its application did not extensive for lack of exclusive functions. 10.9.4. HPLC and LC-MS Methods

HPLC is a traditional quantitative analysis method for PK properties of ODNs and its metabolites. Frequently applied separation techniques include ion pair reversed phase chromatography (IP-RP-HPLC) and strong anion-exchange chromatography (SAX-HPLC). UV is the most widely used means of detection. In addition, fluorescent detecting tracer techniques are yet to be reported in analytical literature [22]. However, SAX-HPLC has poor resolution and sensitivity and can hardly separate full-length ODN from n−1 deletion analogs. The limitation associated with this method of analysis is its disadvantage in discriminating between intact ODN and metabolites [22]. Another limitation of HPLC is its lower sensitivity. HPLC is lower to 10 to 50 times in quantitative detection sensitivity and worse in reproducibility than the radioisotope method. The problem caused by low sensitivity is the determination errors of the end of elimination phase of plasma drug concentration, which shortens the detected elimination half-life. For instance, the plasma elimination half-life of several PS-ODNs has been reported to be in the range of 20–60 h using radioactive techniques for quantitative analysis [52]. However, the plasma elimination half-life of another PS-ODN GEM 91 was shown to be much shorter, with a range of 38–75 min when the parent ODN was analyzed by HPLC instead of by a radioactive technique [141]. Thereby, the problem that researchers are working to solve is to increase the sensitivity of HPLC. Despite the poor sensitivity and resolution of HPLC method in detection of parent drug and its metabolites, it is still widely used in preclinical and clinical PK studies due to its greater availability, ease of operation and robustness. As in the case of CGE, one common problem in developing HPLC methods for quantitative analysis for ODNs is the extensive sample clean-up. HPLC also causes low recovery, limitatied by the serum amount injected into HPLC and the high cost of column stationary phase and mobile phase. An IP-RP-HPLC method was established for the quantitative analysis of GTI-2040 in human plasma. The recovery of GTI-2040 during one-step SPE procedure was significantly improved, and LLOQ was 0.2 μg/mL [142].

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Mass spectrometry (MS) is a recently developed method of quantitative analysis of ODN, with high sensitivity and selectivity and can discriminate chainshortened metabolites truncated from the 3 or 5 terminal [143]. With obvious advantages in selectivity, sensitivity, molecular weight determination and structural information provision, LC-MS can obtain reliable qualitative and quantitative results and therefore has been widely used in studies of biological drug absorption, distribution, and metabolism, including the determination of the structure of metabolites and quantity. According to different LC-MS interface and ionization way, LC-MS can be divided into electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). IP-HPLC-MS technology is an important LC-MS technology for analysis of ODN’s mass and its metabolites in vivo. It has been proven that ion-pair reagents rather than others play the crucial roles in the chromatography performance and MS detection. Triethyl ammonium acetate (TEAA) early used as ion-pair reagent, Because of the poor dissociation of the ion pair formed between TEA and ODN anion after getting into MS ion source, it could cause MS signal suppression and great decrease of detection sensitivity. Apffel et al . [144,145] found an ion-pair reagent that was composed of 1,1,1,3,3,3-hexafluoro-2-propanol/triethylamine (HFIP/TEA) can overcome the “signal suppression” in MS detection. The system provides comparable chromatographic resolution and good MS signal intensity for the TEAA buffer system. A cassette HPLC-MS quantification method for G3139 and three metabolites was developed and validated with LLOQ of 17.6 nM in human and rat plasma with acceptable precision and accuracy by using the above HFIP/TEA ion-pair system [146]. Currently, LC-MS is also being investigated for the quantitative analysis of double stranded siRNA [147,86]. However, LC-MS method still requires a complicated sample pretreatment process. In biological matrices, inorganic and organic small molecular components and macromolecules, such as protein, lipid, and carbohydrate affect the sample separation, which will make difficult quantitative analysis of ODNs using LCMS. Thus, sample cleanup is the critical step to separating the target ODNs from matrix components and bulk proteins before further separation using ion-pair reversed-phase chromatography prior to MS detection. Unfortunately, a general protein precipitation method for ODNs with ammonium acetate, methanol, or acetonitrile results in marked signal suppression in MS detection along with low recovery [123]. Isolation of analytes can be completed via liquid/liquid extraction using phenol/chloroform/isoamyl alcohol [86], solid-phase extraction (SPE) or protein precipitation coupled with SPE [123]. Various SPE phases are available for ODNs’ biological sample preparation, including C18, anion exchange, mixed-mode or polymeric sorbents. But because of complexity of biological matrix, different sorbents and cleanupp techniques might be optimized [123]. In summary, HPLC-MS/MS approaches appear to possess good sensitivity, selectivity, and ability to quantitate metabolites. However, application of this technology to analysis of therapeutic nucleic acid agents in preclinical and clinical PK studies only appearing in decade literature. The utility and robustness of these methods demand further assessment in future.

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CHAPTER 11

INDUCIBLE RNAi AND DRUG TARGET VALIDATION WEI XIONG∗ , JING ZHAO∗ , YUFAN ZHANG, JINXI WANG, YONG-XIANG ZHENG, QIU-CHEN HE, LI-HE ZHANG, and DE-MIN ZHOU State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Beijing China

11.1. INTRODUCTION

RNA interference (RNAi) is a system within living cells that helps to control which genes are active and how active they are. Two types of small RNA molecules—microRNA (miRNA) and small interfering RNA (siRNA)—are central to RNA interference [1]. Historically, RNA interference was known by other names, including posttranscriptional gene silencing and quelling. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm Caenorhabditis elegans, which they published in 1998 [2]. RNAi has an important role in defending cells against parasitic genes—viruses and transposons—but also in directing development as well as gene expression in general. The RNAi pathway is found in many eukaryotes, including animals, and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of ∼20 nucleotides [3]. One of the two strands of each fragment, known as the guide strand , is then incorporated into the RNA-induced silencing complex (RISC) [4]. The most well-studied outcome is ∗

The first two authors contributed equally to this paper.


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posttranscriptional gene silencing [5] which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by argonaute, the catalytic component of the RISC complex (Figure 11.1). This process is known to spread systemically throughout the organism, despite initially limited molar concentrations of siRNA in C. elegans. The selective and robust effect of RNAi on gene expression makes it a valuable research tool for studying gene function through loss-of-function experiments [6] both in cell culture and in living organisms. Double-stranded RNA is synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers can cause a drastic decrease in the expression of a targeted gene. Studying the effects

Figure 11.1 The mechanistic scheme of RNA interference. The natural RNAi pathway is triggered by long double-stranded RNAs that share sequence homology for a target mRNA. The mediators of RNAi are ∼21 nt small interfering RNA duplexes (siRNA), which are derived from the digestion of the long dsRNA by a RNase-β-like enzyme known as Dicer. The siRNAs are then activated by ATP to release the guide strand for target recognition and incorporated into the multiprotein RNA-induced silencing complex (RISC). On identifying a region of complementarity, RISC mediates mRNA cleavage and, consequently, gene silencing.

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of this decrease can show the physiological role of the gene product. Because RNAi may not totally abolish expression of the gene, this technique is sometimes referred as a “knockdown,” to distinguish it from “knockout” procedures, in which expression of a gene is entirely eliminated. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division [7]. Approaches to the design of genomewide RNAi libraries can require more sophistication than the design of a single siRNA for a defined set of experimental conditions [8]. Mass genomic screening is widely seen as a promising method for genome annotation and has triggered the development of high-throughput screening methods based on microarrays.

11.2. APPROACHES FOR siRNA DELIVERY

Unlike small molecules and antibodies, which most likely act as antagonists targeting proteins, RNAi directly targets mRNA rather than its translation product. It is in the cytoplasm wherein RNAi is triggered. There are two common approaches for delivering siRNA into cytoplasm, lipid-mediated transfection and viral-mediated transduction [9]. Determining which approach to use depends on the cell type being studied and whether transient or stable knockdown suffice to achieve the desired effect. 11.2.1. Transient Transfection

Transient transfection is the most common and simplest approach via which exogenous siRNA or siRNA-coding plasmid DNA is introduced into cells by nonviral methods (Table 11.1). Transfection of animal cells typically involves opening transient pores or “holes” in the cell plasma membrane, to allow the uptake of genetic material. Transfection can be carried out using calcium phosphate or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell plasma membrane and deposit their cargo inside. siRNA transfection is always a transient event because the introduced siRNA lacks the ability to replicate itself, except in nematode, and quickly disappears due to degradation and dilution by cell division. Transfected DNA will also be lost at the later stage when the cells undergo mitosis because the foreign TABLE 11.1 Comparisons of RNAi approaches

Transient transfection Stable transduction Inducible RNAi

Advantage

Disadvantage

Simple and common Long-term and permanent Regulable and reversible

Not reproducible and short-term Hysteresis and lethality Time-consuming

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DNA is usually not inserted into the nuclear genome. Once siRNA disappears in transfected cells, the target gene will quickly restore to its normal state. It is well known that maximal gene knockdown by siRNA transfection is typically achieved within 48–72 h and lasts for just 24–96 h, depending on the transfection efficiency [10]. Therefore, siRNA transfection is a useful tool to analyze the short-term impact of an altered gene, most commonly to test designed siRNA work or not. Currently siRNA transfection is the most popular method used for transient gene knockdown because of its easy-to-produce, ready-to-use, and minimal preparation time. 11.2.2. Stable Transduction

However, many assays require a longer time period of gene silencing because oftentimes phenotype change is not contemporary with genotype change [11]. siRNA-directed silencing by transfection is limited by its transient nature, and RNAi with transfection of synthetic siRNA has not been successful in many cell types because of instability and poor transfection efficiency. The utility of plasmids is also limited in cell lines that are difficult to transfect and that cannot be grown for long periods of time in culture, such as primary cells. Long-term gene silencing typically requires development of stable cell lines. If it is desired that the transfected gene actually remains in the genome of the cell and its daughter cells, a stable transfection must occur (Figure 11.2). To accomplish this, another gene, such as resistance toward a certain toxin, is cotransfected, which gives the cell some selection advantage. Some (very few) of the transfected cells will, by chance, have inserted the foreign genetic material into their genome. Viral-mediated delivery of siRNA for gene silencing is a more attractive option for primary cells or other hard-to-transfect cells, mostly because of high efficiency of virus infection [12,13]. To accurately determine the efficacy of knockdown from an siRNA in a population of cells, it is critical to deliver the siRNA to as many cells as possible. Otherwise, when measuring knockdown by quantitative RT-PCR or Western analysis, the background from mRNA or protein present in nontransfected cells will make the knockdown appear less effective than it actually is. Currently, several types of viral vectors are used for hairpin siRNA delivery and expression, including well-characterized retroviral vectors that integrate siRNA expression cassettes into host genome of dividing cells and thus maintains stable [14,15], long-term small hairpin RNA (shRNA) expression and gene silencing under antibiotic selection pressure. Lentiviral vectors have the added benefit of infecting a broad host range, including nondividing, terminally differentiated cells [16]. Once expanded, these transduced cells permanently contain the shRNA-encoding cassette in the host genome and get constitutive expression of shRNA, providing sustained gene silencing. In addition to retroviral and lentiviral delivery systems, the adenoviral system is a commonly used class of non-integrating viruses that are capable of infecting both dividing and nondividing cells, but are maintained in the cell as free episomes [17].

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Figure 11.2 Lentiviral vector-mediated delivery of shRNA. Lentiviral-based shRNA delivery is initiated by a transient transfection in which a packaging cell line is transfected with three separate plasmids, the transfer plasmid containing the shRNA-coding DNA template flanked by LTRs and the Psi-sequence of HIV, the packaging plasmid, and the envelope-encoding plasmid. The components of three plasmids in the packaging cell are put into the HIV virons, and then the lentivirus particles infect target cells per cell surface receptors, releasing the viral RNA together with reverse transcriptase (RT) into the host cell. RT and the host DNA polymerase convert the viral RNA within the target cells into double-stranded DNA, which is then integrated into the host chromosome through the LTRs. From the integrated host genome, shRNA is stably transcribed and therefore keeps long-term gene silencing, making it a very attractive delivery system.

11.3. INDUCIBLE RNAi

The ability of RNAi in mammalian cells will undoubtedly revolutionize the study of functional genomics, the discovery of drug targets, and even the treatment of human diseases. However, RNAi may lead to potential lethality immediately after transduction of a vector when the targeted gene is indispensable, or the phenotype of the knockdown is lethal, or it results in a growth abnormality. In such cases, regulating siRNA expression in mammalian cells has become essential. Having the ability to control when and how much of a particular siRNA is expressed makes it possible to study temporal- and concentration-dependent effects on a host system and allows cells to grow prior to gene silencing by “toxic” siRNAs. Furthermore, studying a host cell’s response to the presence and absence of siRNA often leads to further understanding of the roles of the target gene. 11.3.1. Tet Systems

The most commonly used inducible systems so far are built on the Tet-on/off system. Repression of shRNA expression driven by a Pol III promoter can

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be achieved by placing an Escherichia coli Tet operator (TetO) sequence just upstream of the transcription start site to interfere with the function of the TATA box through recruitment of the tetracycline repressor (TetR). This TetO-chimeric promoter for siRNA transcription is switched off in the presence of TetR, but will be switched on once the bound TetR is released from the chimeric promoter by small molecule tetracycline (Figure 11.3) [18,19]. Earlier versions employ two plasmid systems expressing shRNA and TetR, respectively, which usually encounter problems, including leaky silencing without induction and ineffective silencing when induced, as well as a complex construction process. 11.3.2. Lac Systems

In addition to Tet operon, the lac (lactose) operon, a universal regulatory element in prokaryota [20], can also be utilized in regulating siRNA expression. The regulatory components of this system comprise the lac repressor, LacI, and its DNA-binding sequence, the lac operators (lacOs). In the noninducing condition, the LacI inhibits transcription by binding as a homotetramer to the lacOs located downstream of the lac promoter. In contrast, in the inducing condition, the binding of LacI to the lacOs is relieved by isopropyl thiogalactose (IPTG) [18,21,22], resulting from a conformational change in the LacI and allowing RNA polymerase to initiate transcription (Figure 11.4a) [23,24]. 11.3.3. Ecdysone Systems

An ecdysone-inducible system for transactivation of pol III promoters has been described, which is based on the activation of an engineered U6 promoter by the recombinant transcription factor. Regulated expression of the transcription factor under the control of the ecdysone-dependent regulatory system ultimately allowed regulated production of shRNAs from the engineered U6 promoter [25].

Figure 11.3 Schematic representations of lentivector for constitutive or conditional siRNA expression. pSD31, a constitutive vector, derived from pHIV-7, with a BamHI site for cloning of variant siRNA expression cassettes; pSD400, a conditional vector carrying a bicistronic expression unit that makes repressor protein TetR overexpression.

396


(a)

(b)

Figure 11.4 (a) Lac systems: in the noninducing condition, the LacI inhibits transcription by binding as a homotetramer to the lacOs located downstream of the lac promoter. In contrast, in the inducing condition, the binding of LacI to the lacOs is relieved, resulting from a conformational change in the LacI and allowing RNA polymerase to initiate transcription. (b) KRAB fusion system: in the absence of doxycycline, tTRKRAB binds to tetO and suppresses the expression of the transgene as well as its own via an autoregulatory loop, and prevents the production of shRNAs. The simultaneous repression of both Pol II and Pol III promoters is achieved through KRAB domain inducing epigenetic silencing over a region of 2–3 kilobases. In the presence of doxycycline, tTRKRAB does not bind tetO, thus allowing transgene expression and downregulation of the target gene by RNA interference.

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Using this system, tight and reversible regulation of some target genes has been obtained, but such multicomponent design is prohibitively complex for broad applications [26]. 11.3.4. KRAB Fusion System

The Krüppel-associated box (KRAB) domain found in many zinc-finger proteins can recruit a multimolecular complex that leads to histone deacetylation and methylation and binding of heterochromatin protein 1 (HP-1), thus creating a local heterochromatin state extending over regions of a radius of 2–3 kilobases when tethered to specific DNA regions [27–29]. When KRAB is fused to the TetR DNA binding domain, the resulting tTRKRAB-mediated repression of promoters introduced to TetO sequences can be reversibly controlled by doxycycline (Figure 11.4b). Taking advantage of these properties, a tTRKRAB-based system to regulate the expression of shRNA was developed, which allowed for the controllable suppression of cellular genes both with a high degree of efficacy and without substantial leakiness [30]. 11.3.5. An All-in-One Inducible System

Recently we have developed an all-in-one single system (Figure 11.5) [11] that addressed major issues existing in current various systems, such as leakiness, dual plasmid-based transfection and selection, and time-consuming. The key feature of this system is recruitment of an IRES bicistronic cassette, wherein an antibiotic resistance gene is downstream from the TetR gene that makes TetR overexpression. The high level of TetR expression ensures the inducible promoter to be tightly bound with minimal basal transcription, allowing for the regulation solely dependent on TetR rather than a fusion protein. At the same time, this system contains only a single TetO, thus minimizes the promoter impairment occurring in previous systems due to the incorporation of multiple TetOs, and maximizes the siRNA expression on inductions. In addition, this system combines all the components required for regulation of siRNA expression into a single lentiviral vector, so that stable cell lines can be generated by a single transduction and selection, with significant reduction in time and cost.

11.4. TARGET VALIDATION

A key challenge and essential step in drug development is to identify the right drug targets, providing critical information prior to the lengthy and expensive HTS compound screening and lead development, and this can significantly save cost and time in drug discovery. It is reported that a large percentage of candidate drugs fail during development, due to failures of no-target biological hypothesis as well as safety concerns. The high failure rate of drugs in development, even among drugs that have undergone extensive validation before entering

398


(a)

(b)

Figure 11.5 Overviews of the regular siRNA system versus the inducible siRNA system. (a) The regular siRNA expression system utilizes the wild-type pol III promoter to constitutively transcribe siRNA. (b) The inducible siRNA system employs the bacterial tetracycline resistance operon that one or several tetracycline operators (TetO) have been inserted into the pol III promoter. When the tetracycline repressor protein (TetR) is present in the cell, it binds the TetO site within the pol III promoter and blocks the initiation of shRNA transcription (switch off). When tetracycline (Tet) is added to the culture media, it binds to and changes the conformation of the TetR, causing the TetR to release from the TetO site, and allows shRNA transcription to occur (switch on).

clinical trials, demonstrates the enormous complexity of biological systems and the difficulties in making reliable predictions about the biological role of major therapeutic targets. Target identification through loss-of-function genetic screens using siRNA represents a powerful approach for identification of new drug targets, by screening for target transcripts that, when silenced, affect the phenotype of interest. 11.4.1. Cancer Target Validation

Silencing of cancer therapeutic targets usually results in reduced cell growth/survival in vitro and/or failure to establish tumors in vivo, thus hindering staged tumor response-based efficacy evaluation in animal studies. The Tet inducible RNAi system we developed is for evaluation of in vivo efficacy of cancer targets using a xenograft model system [11], and it enables creation of stably silenced cancer cells in vitro and establishment of tumor in vivo in the absence of the inducer. On induction, tumor response to gene inactivation in vivo can be measured and thus allows rapid advance through the discovery process.

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This all-in-one inducible siRNA system with TetR high level of expression provides a unique and more efficient tool in comparison to the existing systems for conditional gene knockdown, and it displays a high degree of robustness and versatility for regulation of siRNA expression [11]. We have demonstrated the high degree of robustness and versatility of this system as applied to several mammalian cells and xenograft animals. As a proof of principle, we have demonstrated the powerful utility of this all-in-one inducible system for efficacy evaluation of cancer target mTOR, a serine/threonine kinase that functions downstream of Akt to regulate cell growth and proliferation, in xenograft tumor models and provided a direct in vivo efficacy validation of this gene as an effective therapeutic target for pTEN deficient cancer treatment [11]. Our experiments indicated that siRNA-mediated mTOR silencing on Dox induction caused 100% tumor regression for the early-stage PC3 tumors in xenografic mice, with 45% becoming tumor-free (Figure 11.6a). In the advanced-staged tumor model significant Dox-dependent mTOR gene silencing and tumor growth repression were also observed (Figure 11.6b), strongly supporting that mTOR is a cancer therapeutic target. This observation confirmed the utility of the all-in-one system for cancer therapeutic target evaluation in xenograpft animals and raises most exciting perspectives for the development of conditional transgenic and knockdown models in a wide variety of mammals. This also makes it possible to study staged tumor response in the gene silencing, mimicking experiments with antagonist drugs.

(a)

(b)

Control siRNA

mT or siRNA –

+

mTOR expression fold of CNTL (%)

+

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Dox :

200 180 160 140 120 100 80 60 40 20 0

CNTL 0

CNTL 2

mTOR 0

mTOR 0.02

mTOR mTOR 0.1 0.5

mTOR 1

Dox (mg/ml)

Figure 11.6 Cancer target validation via inducible RNAi systems. (a) Response of tumors in xenograft mice to Dox induction. No tumor repression was observed for mice containing control siRNA in either the presence or absence of Dox, while significant tumor shrinking was obtained for mTor conditional siRNA. (b) Taqman analysis of the mTOR mRNA level in xenograft tumors. The expression levels of mTOR mRNA decreased with more Dox being added.

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(a) Parental cells (b) Transduced cells

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Figure 11.7 Viral target validation. (a) and (b) Cell lethality caused by constitutive RNAi when target is an essential gene. (c) and (d) Conditional knockdown of essential genes avoids cellular toxicity.

Inducible transgenic RNAi avoids embryonic lethality, thus rendering on-target evaluation possible in diverse adult disease animal models. Both on-target toxicity and efficacy can potentially be evaluated. Toxicity studies can be conducted at any developmental stage by oral dosing with inducer for example doxycycline. Efficacy can also be assessed through induced silencing of the target at any stage of disease development, even before the disease onset, thus the effectiveness of the targeting strategy for prevention or treatment can be assessed. Multitarget silencing can also help to explore the optimized combination therapy. These ontarget safety and efficacy studies can potentially guide the future drug dosing schedules for lowest toxicity and highest efficacy. 11.4.2. Virus Target Validation

The general idea behind modern antiviral drug design is to identify viral proteins, or parts of proteins, that can be disabled. These “targets” should generally be as

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unlike any proteins or parts of proteins in humans as possible, to reduce the likelihood of side effects. The targets should also be common across many strains of a virus, or even among different species of virus in the same family, so a single drug will have broad effectiveness. For example, a researcher might target a critical enzyme synthesized by the virus, but not the patient, that is common across strains and see what can be done to interfere with its operation. Much ongoing effort in pharmaceutical companies is directed against the viral enzymes (e.g., proteases and polymerases). However, the lesson from the HIV experience is that drug resistance will be a major issue in antiviral drug development because of an extremely high rate of viral RNA mutation. This is also already apparent from the results of the early clinical trials of anti-HCV protease or polymerase drugs [31]. One strategy to avoid drug resistance is to target cellular, instead of viral, proteins that are critical for viral replication. This paradigmatic shift not only opens up vast opportunities for new targets, but also gives hope that viral-resistant mutants against drugs of such targets would be less ready to develop. However, when the target gene is a host essential gene, long-term and dramatic knockdown will become not feasible. The challenge is to identify target genes with desirable therapeutic windows (e.g., those with differential thresholds for their roles in viral replication and normal cellular functions) so that only viral functions but not physiological functions are disrupted For example, human RNA helicase A (RHA) [32], an enzyme involved in viral RNA replication, plays a vital role in cell growth, and dramatic knockdown causes lethal toxicity (Figure 11.7a and b). This poses a fundamental concern—how to elucidate host essential genes as functional components in virus replication [10]. One way is to identify a desirable regulation window (i.e., differential thresholds of the essential gene in viral replication and normal cellular functions) based on our developed inducible RNAi system. The involvement of RHA in HCV replication was verified by our RNAi inducible system that, on the one hand, maintained long-term gene silencing, but on the other hand, alleviated siRNA toxicity during the essential gene silencing [10]. A 21-day follow-up of the response of HCV replication to the presence and absence of RNAi indicated that RHA is a cellular factor involved in HCV replication process (Figure 11.7c). This study, apart from proving RHA is a viral replication factor, also implies that RHA may be a potential HCV drug target. Further elucidation of the host cell lethality versus HCV replication following different extent of RHA suppression may help identify a desirable therapeutic window (Figure 11.7d), which exerts inhibitory effect on HCV replication rather than the vitality of host cells.

11.5. SUMMARY

Over the past few years, various inducible RNAi systems allowing for controllable gene knockdown have been developed, which have affected a wide range of fields, from developmental biology to therapeutic drug screening. Although there are some drawbacks associated with the different inducible RNAi systems, such

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as leakiness, time consuming and expending, they still offer numerous advantages over regular approaches for efficient and robust knockdown of cellular genes. The new users should take into consideration the advantages and drawbacks when choosing a conditional RNAi system. Inducible RNAi technologies hold great promise by targeting the whole human genome for evaluating their function through off/on gene expression.

ACKNOWLEDGMENTS

This work was supported by National Basic Research Program of China (973 Program, 2010CB12300), National Natural Science Foundation of China (20932001 and 20852001), and “985” Project Foundation (985-2-126-121).

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CHAPTER 12

siRNA: THE SPECIFICITY AND OFF-TARGET EFFECTS QUAN DU, HUANG HUANG, and ZICAI LIANG Institute of Molecular Medicine, Peking University, Beijing, China

12.1. INTRODUCTION

Silencing specificity of siRNA is a critical issue for both gene function investigation and potential therapeutic applications. In addition to the silencing of perfectly matched targets, siRNA molecules also compromise the expression of partially matched or even unmatched genes, causing the so-called off-target effects. This chapter summarizes published studies on this issue and categories these effects into two groups, sequence-dependent and sequence–independent off-target effects. The sequence-dependent off-target effects include repression of mismatched genes, miRNA-like gene silencing, asymmetric RISC assembly, or immune response caused by GU/AU-rich elements, whereas the sequenceindependent off-target effects include immune response caused by delivery carriers, as well as the saturation effect of RNAi mechanism by overloaded siRNA. Investigating these effects in detail will not only help in siRNA design and ameliorate siRNA off-target effects in practice, but it also reveals insights of this process and provides us with a theoretic model for better understanding of RNA interference. RNA interference (RNAi) is one of most exciting discoveries in life science in the last 20 years. Since its mechanism was elucidated in 1998, RNAi has penetrated into almost every aspect of biomedical research in a pace that has never been witnessed before the Internet era. RNA is not only successfully applied in many aspects of life science studies as an exceptionally robust research tool for


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deduction of gene functions, but it has also evolved into a strategically important field of therapeutic development in the past few years. RNAi is a process of double-stranded RNA mediated targeting and cleaving homologous RNA transcripts [1]. This process is executed by an endogenous pathway existing in all eukaryotic cells through which a class of small RNAs, namely microRNAs, was found to regulate a majority of the genome [2]. Although genes encoding microRNA were already discovered by 1994 by Ambros and colleagues [3], the elucidation of the RNAi pathway in the cells was not accomplished until eight years later in the “RNA rush” triggered by the discovery of siRNA [4]. In the groundbreaking work published in 1998 for RNAi by Fire and Mello, it was found that long double-stranded RNA (dsRNA) can induce dramatic reduction of cognate genes that share sequence identities to the dsRNA without apparently affecting the expression of other genes [5]. The same dsRNA, however, could not inhibit the expression of genes in mammalian cells, except for germline cells, in such a clean manner due to the fact that long dsRNA can induce strong antiviral responses and production of cytokines that would result in overall shutdown of the gene expression of the cell and eventually kill the cell. This turned out to be a technical issue and was resolved by Tuschl and colleagues in 2001 [4]. In this landmark paper for RNAi, the authors discovered that small dsRNA fragments, namely small interring RNAs (siRNA), generated by RNase III–mediated cleavage of the long double-stranded RNA were the direct mediator of the RNAi process. This work has opened the RNAi toolbos to a vast community that is desperately looking for an efficient way to characterize a large number of genes from genome projects. Within just a few years, RNAi technology has penetrated almost all aspects of life science, in a pace that has never been witnessed before the Internet era. RNAi has also found itself becoming one of the most prominent fields for developing nucleic acid–based therapeutics, with the first siRNA-based drug rolled into clinical trial just three years after the discovery of siRNA. Although RNAi can be elicited by long dsRNAs (in nematodes and insects), siRNA, miRNA, and miRNA-like short hairpin RNAs (shRNA) in almost all eukaryotic cells, the specificity of RNAi is only thoroughly interrogated for siRNA [6–12]. This is perhaps due to the fact that siRNA is most widely applied in functional research and to the relative simplicity of scrutinizing its specificity using readily available technologies. To understand factors affecting siRNA specificity, the process of RNAi by using siRNA is briefly introduced here. siRNA can either be generated by conventional chemical synthesis [13] or by enzymatic cleavage of long dsRNA by RNase III such as Dicer [14], or by vector-mediated expression within cells [15–19]. In a scenario of in vivo application of synthesized siRNA, for example, the nucleic acid duplex has to escape from the scrutiny of Toll-like receptors (TLRs) before entering the cell [20,21]. After penetrating the cytoplasmic membrane, siRNA would escape from endosome and bind to argonaute proteins (AGO), a family of RNA-binding proteins that was found to be the major mediator of RNAi, to

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form the so-called RNA-induced silencing complex (RISC) [22]. Then one strand of the siRNA (the passenger strand) will be removed, and the other strand (the guide strand) will be retained by the AGO to discriminate genes and repress their expression. The process of siRNA binding and removal of the passenger strand is called “RISC loading.” Different RISCs execute the RNAi process in different ways. It had initially been assumed that RISC formed by AGO1 with miRNA binds to mRNA sites sharing partial identity to the siRNA to hinder the translation of the mRNA [23], and RISC formed by AGO2 with siRNA recognizes mRNA sites that complement to the siRNA guide-strand completely and induce efficient cleavage of the cognate mRNA [24]. With more and more data accumulated, the distinction between the miRNA pathway and the siRNA pathway is less and less distinct. Early reports assumed that siRNA mediated target gene silence through a very precise mechanism based on the base-pairing, and single nucleotide mismatch at the target site were found to abolish the silencing effect of siRNAs [25]. The specificity issue came under a spotlight in 2003 when people revealed by using microarray analysis that expression of multiple genes with different levels of sequence identity was knocked down by a single siRNA [7]. This result was corroborated by many subsequent reports [8–12]. The alert of the nonspecific effect of siRNA was heightened again in 2008 when it was discovered that siRNA that was supposed to treat age-related macular degeneration by inhibiting VEGF was actually accomplishing the mission by inducing a generic antiangiogenic effect [26]. In this chapter, we will summarize the current knowledge about matters affecting the specificity of siRNA and major types of off-target effects (e.g., effects of siRNA on unintended genes and processes) for people to configure strategies to minimize or avoid severe adverse effects caused by off-target effects in siRNA drug development or to avoid potential false-positives in characterization of genes.

12.2. FACTORS AFFECTING siRNA SPECIFICITY

Possibly as reminiscent of early challenges of eukaryotic cells by dsRNA viruses, cells carry out constant surveillance of their environment for dsRNAs. Although long dsRNA is the most efficiently detected and responded to most intensively by mammalian cells, the shorter siRNAs are also triggers for antiviral responses, especially if the siRNA contains certain nucleotide motifs that can facilitate such recognition. Toll-like receptors (TLR) are the cell surface receptor family that carries out this surveillance [27]. For siRNAs that have penetrated into the cells, RISC loading is another step that can incur off-target effects. This is normally a result of misloading of the passenger strand of the siRNA into the RISC [28]. For siRNAs with a guide strand loaded into the RISC correctly, the target recognition mediated by the guide strand was also found to be not as specific as people initially observed in limited data sets. Taken

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together, it is obvious that the off-target effects of siRNA can be classified into sequence-dependent effects and sequence-independent effects.

12.3. SEQUENCE-DEPENDENT OFF-TARGET EFFECTS 12.3.1. miRNA-Like Off-Target Gene Silencing

DNA microarray analysis showed in 2003 that a siRNA targeting MAPK14 gene can result in the down-regulation of a much bigger number of genes in addition to the intended target, and the results seem to be a direct effect of the siRNA [6]. Analysis of those genes revealed the phenomena that many of those genes contain motifs that have partial identity to the 5 part of the siRNA guide strand (Figure 12.1a). It was further found that matching between the 5 end of the siRNA guide strand and target transcript was essential for gene silencing, and continuous base pairing of as few as eight nucleotides could result in effective gene silencing [29,30]. Subsequent analysis further showed that mismatches that occurred at the 5 end of siRNA guide strand greatly compromised its silencing efficacy on these genes, suggesting the importance of this 8-nt region in this type of off-target effects [31]. This 8-nt region was named seed region, and this manner of target recognition greatly resembles how miRNA interacts with its target. Thus, this type of off-target effect is by convention called mi-RNA like off-target effects, and such type of effects is normally characterized not only by seed region matches but also by significant reduction of protein synthesis along with or without minor decrease of mRNA level. It is with this observation that people start to appreciate that siRNAs are actually loaded into both AGO1 and AGO2. When it encounters a perfectly matched site, it will act as an siRNA to cleave the site, and when it encounters an imperfectly matched site, it will recognize the site with its seed region and act as an miRNA to reduce the translation of the mRNA. This will only change the mRNA levels to a much less extent. Therefore, the 5 end of siRNA guide strand plays an essential role in off-target gene silencing. Every single miRNA has the potential to target hundreds of genes, mostly due to its partially pairing with the target site on the 3 UTR of RNA transcripts [32–38]. The obvious similarity between the off-target effect of siRNA and the endogenous function mechanism of miRNA makes it easily to understand that many siRNAs can silence the genes different from the designed targets within cells, in silencing efficacies similar to native miRNAs [6,39]. Crystal structure reported recently for RNAi effector molecules showed that in the ternary complex formed by argonaute protein, siRNA guide strand, and target RNA transcript, base pairing between position 2–8 of the guide strand and the target RNA formed a doublehelix duplex, embedded within the ternary complex [40]. This partially explained why the seed region plays an important role in target gene silencing by miRNA and miRNA-like siRNAs. It should be noted that miRNA-mediated gene silencing is not restricted to seed-region-containing sites [41]. For seed-region-containing sites, sequences

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Figure 12.1 Diagram of the RNA interference pathway.

beyond seed-region apparently play important roles in determining the silencing efficacies as well. This was confirmed by random shuffling of the sequence beyond seed region for some miRNAs, where the resulting sites were found to be seldom affected by the miRNA (Zicai Liang, unpublished data). This is, however, a less investigated aspect in RNAi process, and the direct impact of this is that: (i) many potential miRNA-like off-target cannot for the moment be identified yet using available knowledge and (ii) possible integration of a filter for seed-region match into siRNA design software might eliminate too many siRNAs unduly. This situation might be improved when more detailed understanding of the miRNA mediated target recognition process is achieved.

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Figure 12.2 Diagram of the structure of siRNA, and illustration of the nucleotides taking part in the target recognition in siRNA or miRNA mediated silencing of mRNA. (Fullcolor version of the figure appears in the color plate section.)

Figure 12.3 Diagram of pathways of RNA-triggered immune responses. Different types of RNA can illicit immune responses of the cell through different yet overlapping pathways. (Full-color version of the figure appears in the color plate section.)

A recent study reported sequence features of the flanking nucleotides located around the seed region, for example, the AU ratio and the position of target sequence in the 3 -UTR affected the silencing efficacy of siRNA [42]. Considering the limited matches between siRNA guide strand and gene transcript, the miRNA-like off-target gene silencing likely serves as the major mechanism involved in siRNA off-target gene silencing. The effect in each off-target

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gene is relatively weak according to the general characteristics of the miRNA pathway. To answer the question that if this relative weak miRNA-like repression effects has an impact on the biological system and can induce cellular phenotype, a recent report indicated that these effects did repress target gene expression to a certain extent and inhibit the growth of the cells [43]. 12.3.2. Off-target effects on nearly identical sites

Of all genes found to be repressed unintentionally by an siRNA targeting MAPK14 gene as reported by Jackson et al . [6], several genes were found to contain sequences that were highly similar to the siRNA. The results indicated that 11 continuous base pairings can result in efficient target gene silencing, and identified matches between position 2–5 and 7–17 of siRNA guide strand and target site played an essential role in off-target silencing in RNAi [20]. Using an in vitro assay system, Benjamin Haley and Phillip D. Zamore showed that siRNA could cleave target transcripts with as much as five nucleotide mismatchs; however, more than five mismatches on target site render the siRNA inefficient in gene silencing [44]. This is a contradiction to initial assessments showing siRNA activity can be abolished by single-point mutations. To characterize this type of off-target effect, we performed a systemic study on the silencing effects of a single active siRNA on a whole panel of 57 different reporters, each carrying a single nucleotide mutation in the targeting site of the siRNA [11]. Although single-base mismatch is the simplified form among different mismatch types, previous studies indicated that mismatch position and identity has their effects in target silencing and tolerance. This strategy creates a single-base mismatch between siRNA guide strand and target transcript. By avoiding any changes in the siRNA itself, this method was limiting the variables to only the target recognition step. We demonstrated that more than 50% of all single-point mutations in target sites were well tolerated. It was worth noticing that for each siRNA examined, the dinucleotide at each end of the siRNA guide strand can be mutated without affecting the silencing activity of the siRNA. The most striking finding was that mutations that created seed-region mismatches did not affect the silencing activity of the siRNA as much as initially expected, and in contrast, mismatches in the 9 nt to 16 nt region of the siRNA have much higher impact on the silencing efficacy of the siRNA. This is consistent with the judgment that such off-target is executed through the siRNA pathway, which was confirmed by Northern analysis of the transcript. This assessment was also corroborated by independent research done by mutating siRNAs rather than the targeting sites [45]. Further analysis using 706 reporters carrying double-mutated sites for an active siRNA revealed that the terminal regions and the seed region can even tolerate most double-mismatches without sacrificing too much silencing activity, and even for the 8–16 nt region, which is most sensitive to mismatches, many double-mutated sites were silenced to a level that is comfortably comparable to effects of miRNA [12]. This type off-target effect was referred to as siRNA-type of off-target effect. The characteristic of this effect is that siRNA

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affect much fewer off-targets but each off-target can be knocked down much more significantly than miRNA would. Our single- and double-mutation approach, however, did demonstrate that the spectrum of genes affected by siRNA through this type of off-target effect is much broader than people initially thought. For siRNA-mediated silencing of off-targets, we also discovered that mismatches were discriminated differently in RNAi process in comparison to DNA duplex formation or RNA folding, where thermal stability appeared to dominate the game. In the RNAi process, however, thermal stability change seemed to have yield the position to distortion of the structure, and most likely to potential of the AGOs to accommodate such distortions at different positions [46]. For example, A:C mismatches were well tolerated in position 17 nt of the siRNA, but significantly discriminated at position 12 nt. Such a change is not due to the overall discrimination hack at position 12 nt because A:A mismatches were tolerated the same level here as the wild type A:U base pairs, although it was much more discriminated at all other positions, including position 17 nt. Another example is that an A on siRNA antisense strand and a C on target site forms a very tolerant mismatch type A:C, rendering a 70% inhibition on gene expression, and the most sensitive A:G mismatch inhibit gene expression for only 52% on the average [46]. It should be stressed that this differential tolerance of mismatched sites at each position of the siRNA appears to follow a given order for each position and types of mismatches, regardless of surrounding sequence context, although the level of discrimination differed. This might reflect the changing landscape of the RNA-binding pocket of the AGO proteins across the length of the siRNA. So far, this study is the most comprehensive investigation on single-nucleotide mismatched targets. Based on this model, the most tolerant or the most discriminative mismatch types in RNAi were identified, and the strategy has started to greatly facilitate siRNA for silencing of SNP disease alleles. 12.3.3. Off-Target Effects Caused by Misloading of Passenger Strand of siRNA

Both strands of the siRNA duplex have potential to be integrated and retained in the RISC complex to exert gene-silencing functions. This property of siRNA was extensively characterized by Zamore group in 2003, where multiple siRNA were assayed for incorporation of the two strands into AGO2. It was reported that the relative thermodynamic stability of the 5 end of both siRNA component strands determined which strand was selected to be assembled into RISC complex and exerted its silencing activity, and RISC complex prefers to unwind the duplex from a strand whose 5 end has a lower thermodynamic stability, which is assembled into RISC and function. The other strand will be removed from RISC and degraded [28]. Furthermore, it was established that the thermal stabilities of the duplex ends appear to play a role in determining which strand will be selected as the guide strand, but in many cases such selection was found to be less than 100%. Subsequent experiments have further revealed that even the thermal stability rule can only predict the strand selection behavior of only about less than

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60% of all siRNAs [47], thus making it harder to know which strand would serve as the guide strand in a given siRNA. Therefore for each siRNA, there is a chance that the “supposedly” passenger strand will be actually integrated into the RISC and carry out silencing on other genes. This problem can be prevented in several ways. The most straightforward way is to take advantage of the fact that RNAI is very sensitive to modifications on the 5 -end of the guide strand. Several modifications on this end can render one strand inert for RNAi while incurring minimal negative effects on the silencing activity of the other strand.

12.4. SEQUENCE-INDEPENDENT OFF-TARGET EFFECTS 12.4.1. Induction of Immune Responses via TLR

Pattern recognition is an important aspect of the innate immune system, which in higher animals senses and clears pathogens or their products or presents pathogen to the adaptive immune system. It appears that mammalian cells have, during the course of evolution, obtained the capacity to recognize not only patternindicating molecules such as lipopolysaccharide, peptidoglycan, beta-1,3-glucans, and virus glycoproteins, but also bacteria DNA and virus RNAs [48–50]. Due to its small size of less than 30 nucleotides, some siRNA was initially found to be able to escape from immune detection to some extent without causing extensive interferon reactions. As more and more data, especially data from in vivo experiments, were accumulated, people started to collect a more comprehensive view of the interplay between siRNA and the immune system of mammalian animals. Detailed recent in vitro and in vivo experiments consistently showed that even the short synthetic siRNA could mediated some level of immune responses and significantly increase the levels of various cytokines [50]. Mammalian cells express a series of TLRs that are responsible for detection of various pathogens [51]. Each TLR family member has evolved to recognize a specific pathogen structure. During the immune activation elicited by siRNA, several such pattern-recognition receptors, including TLR3, TLR7, and TLR8, were found to be involved and are responsible for RNA detection. They shuttle back and forth between endoplasmic reticulum and other organelles, including endosome and lysosome to detect and response for exogenous nucleic acid materials. As an early detector of virus infection, they activate TNFα and IL6 and knockdown general gene expression. Because of the different expression levels of these receptors in various cell lines, siRNA-mediated immune response exhibits cell-type specificity. This might explain why siRNA-mediated immune responses were overlooked at the beginning. These effects call people to use siRNA as research tool with caution because siRNA-mediated immune responses may make interpretation of siRNA-based assay more complicated. These results also indicate that such off-target effects may potentially cause a serious challenge in clinical application of siRNA therapeutic reagents. For example, in vivo data obtained from mice experiments indicated that this side effect has significant toxicity, increasing the serum level of alanine and aspartate

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aminotransferase and decreasing the level of lymphocytes and thrombocytes [52]. Yet another example toward this end is that siRNA targeting nonmammalian genes, nonexpressed genes, and nongenomic sequences all suppressed neovascularization in mice at comparable levels to siRNAs targeting Vegfa or Vegfr1 by activatiing cell-surface toll-like receptor 3 (TLR3) and induction of interferongamma and interleukin-12. TLR3 recognizes the duplex form of the siRNA, whereas TLR7 and TLR8 recognize both duplex and single-stranded forms of the siRNA. Therefore, both sense and antisense siRNA strands can induce immune response. The activation efficacy of the single-stranded siRNA form is comparable with that of duplex form, and in some cases, much stronger immune response was caused by single-stranded siRNA [53]. Besides the structural properties, the length of siRNA is another important factor determining its immune response. It was found that dsRNA need to have a minimal length of 21 nt to trigger TLR3 by forming a 2:1 receptor:RNA complex, and it is not clear whether this has coevolved with AGOs, which also have lower threshold of such a duplex length. Shorter siRNA, such as 12 or 16 nucleotide, does not induce immune response even if it contains particular immune-activation elements. However, if the length of siRNA is extended to 19 nucleotides, it can induce potent immune responses. When siRNAs of such size are delivered into mice by lipofentamine, they mediated not only whole-body IFNα response, but also activated T lymphocytes and myeloid dendritic cells, whereas injection of siRNA or liposome alone did not cause these effects. In another study, it was also shown that delivery of carrier/siRNA complex caused immune response, but not the siRNA or liposome alone [54]. In summary, these data indicated that delivery of carrier-formulated siRNA, on the one hand, can protect siRNA from serum ribonuclease and accelerate endocytosis; on the other hand, it also facilitate endosome incorporation and detection by TLR located in endosomes, causing potent immune response. It is obvious that carrier/siRNA complex needs to be incorporated into endosomes before activating immune systems; unfortunately most clinically available liposome delivery carriers utilize this route in drug delivery and very likely cause strong immune response if they are used for siRNA therapeutics. On the contrary, those endosome-independent delivery protocols, like electroporation, did not induce immune response even though the same siRNA was delivered into the cells [55]. Although similar sequence similarity has been reported for the activation of TLR7 and TLR8, sequence specificity of siRNA was recently revealed for TLR7 and TLR8 activation, for example, UG-rich sequences including UG dinucleotide and 5 -UGU-3 [56]. In addition, GU-rich elements were also shown to mediate a relatively weak immune response. However, in some cases, GU-lack elements could also activate the immune response [57]. Furthermore, AU-rich elements, and sometimes even a single U, were shown to activate the immune response [58]. Besides the specificity of certain sequences, position of the sequence as well

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as its flanking sequences might interfere with this response, reminding us the situation in the siRNA recognizing process. These exceptions have indicated that the complexity of RNA recognition by cell surface receptors is still not fully uncovered. 12.4.2. Saturation of miRNA Pathway by siRNA or shRNA

Available studies indicated that overdosed siRNA might lead to a interference with endogenous RNAi activity. This novel side effect is mainly caused by the competition of limited RNAi function mechanism between exogenous shRNA or siRNA and endogenous miRNA. 12.4.2.1. siRNA Competes with miRNA on RISC Binding It has been reported that simultaneous transfection of multiple siRNA could lower the inhibitory effects of individual siRNA, indicating that the RNAi mechanism may be saturated by the overdosed siRNA in vivo [59]. Direct evidence has been presented that introduction of exogenous siRNA compromised the normal functionality of miRNA in vivo [60]. Comprehensive analysis of gene expression data after transfection of synthesized siRNA and miRNA revealed that in addition to the repression of target genes, other potential target genes of endogenous miRNA were found to be up-regulated. It was suggested that exogenously transferred siRNA or miRNA saturated RNA interference pathway, likely due to the competition of downstream protein components, for example RISC complex, and led to reduction of the normal function of endogenous miRNA and resulted in nonspecific up-regulation of the target genes of the miRNAs gene. 12.4.2.2. shRNA Competes with miRNA on Exportin 5 Transporation Chemically synthesized siRNAs function within cytoplasm, and thus only act to compete with endogenous miRNA, which normally has an advantage in Dicerassisted delivery into the RISC [61]. This is perhaps the reason why in many cases no severe side effects can be visualized at cell levels for most siRNAs in a large dosing range. Vector-expressed shRNA, however, not only competes with miRNA for RISC entry but also compete with miRNA for the miRNA biogenesis pathway. Viral vector-mediated shRNA were frequently used in transfection experiments with hard-to-transfect cells. After entering into nuclei, it will be transcribed into shRNA, transported by Exportin 5 located on nuclei membrane into cytoplasm, and function in RNAi. Cellular assay indicated that overexpression of shRNA within nuclei might saturate the transportation capability of Exportin 5, compromising the normal transportation activity of pre-miRNA and thereby induce cytotoxicity. Experimental data with adult mice also displayed similar effects in that continuous overexpression of shRNA in mouse liver resulted in animal lethality. Among the 49 siRNAs tested, 36 of them resulted in dose-dependent liver

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injure, and 23 of them resulted in animal death. Further investigation showed that down-regulation of liver-specific miRNA was correlative with the toxicity and the lethality, indicating that overexpression of shRNA in vivo inhibited normal miRNA generation. Besides the reported Exportin 5, it is very likely that other RNAi component might be also saturated [62].

12.5. STRATEGIES TO MINIMIZE OFF-TARGET EFFECTS

Understanding of the types and causes of off-target effects of siRNA has enabled people to strategize measures to avoid or minimize harmful off-target effects. In our hands, three strategies are usually used to minimize the potential off-target effects of siRNA. 12.5.1. Minimizing Off-Target Effects by siRNA Design

In the rational siRNA design, sequence alignment algorithms such as BLASTn or Smith–Waterman Dynamic Programming Algorithm are often used to exclude siRNAs with potential off-target broad on closely related genes. Appropriate algorithms and suitable databases are required for such a purpose. However, with existing software, targeting prediction is mostly based on sequence similarity search for reducing potential off-target effect, without taking enough knowledge about functional relationship between similarity and silencing efficacy into account. Based on our systematic analysis of the silencing effects of an active siRNA on all single nucleotide mismatched target sites [11], Alistair Chalk and Erik Sonnhammer designed a new prediction method for siRNA off-target effects, implementing the WU-Blast algorithm. The prominent advantage of this method is to rate and sort the selected potential nonspecific target sites, thus helping the detection of potential off-target effects [63]. Though similar algorithms are accumulating and the design is continuously improved, off-target gene silencing in siRNA design remains a big challenge in particular for therapeutic application of siRNA. To address this problem, we are in the process of incorporating the double nucleotide mismatch data into a new generation of siRNA designing software. 12.5.2. Chemical Modification of siRNA

Chemical modifications can be used to block off-target effects in three ways. First, the 5 end of passenger strand of the siRNA can be modified so that either loading of this strand into RISC become blocked or a minimal loaded passenger strand would not be able to exert any inhibitory activity. Blocking the potential activity of the loaded passenger strand can also be accomplished by incorporation of multiple modification along the whole length of the passenger strand. Statistical results have shown that this kind of modification reduced 80% of the nonspecific inhibition and can also effectively inhibit the target. Second, modification of the

SUMMARY

417

guide strand within the seed region can to a large extent block the miRNA-like off-target effects that are mediated by this region. Modification with 2 -O-methyl ribosyl substitution at seed region in siRNA antisense strand, for example, was reported to reduce most off-target effects [64]. Third, modifications along the length of the siRNA on both strands can allow the siRNA to escape from the surveillance of TLRs much more efficiently and thus can effectively relieve the growth inhibition caused by the off-target effects of the siRNA. There are also reports showing that by different kind of chemical modification, including 2 O-Methyl, 2 -deoxyuridine, locked nucleic acid (LNA), the immune activation of siRNA can be reduced effectively with no effects to the normal inhibition function of siRNA [64–67]. Early studies have shown that the degree of the siRNA off-target effect was related to the concentration of the siRNA, suggesting that it is possible to decrease siRNA concentration to reduce siRNA off-target effects, while keeping effective inhibition of the target gene. However reported studies indicated that this protocol is not applicable for most siRNAs because reduction of siRNA concentration also decreases siRNA inhibition efficacy on the target gene. To solve this problem, siRNA pool was used in which several different siRNAs targeting the same gene were used instead of a single siRNA species. Although the final siRNA is the same as with one siRNA species, the concentration of each siRNA within the pool is greatly reduced, eliminating many concentration-related off-target effects. In a reported pool with 10 siRNAs, the concentration of each siRNA can be reduced to 10 nM. The off-target effects were significantly minimized in comparison with a single siRNA [74]. Meanwhile, many siRNA manufacturers have come up with similar strategies, including the SMARTselection™ siRNAs of the Dharmacon company and the BLOCK-iT™ Complete Dicer RNAi Kit of the Invitrogen company, both of which are siRNA pools to increase the specificity of siRNA while guaranteeing the inhibition effectiveness.

12.6. SUMMARY

Based on the reported studies, RNAi not only is an endogenous mechanism for cell to regulate its gene expression, but also provides us a tool to repress the expression of a specific target gene, thereby greatly accelerating the pace of function genomics study. Successful construction of RNAi libraries against almost all known genes further facilitates high-throughput gene study. siRNAs have great potential to become the next generation of blockbuster drugs that can be used in treatment of diseases for which people do not have a cure yet. Some 14 siRNA drugs are now in various stages of clinical trial. Off-target effect of siRNA has not only brought uncertainty to gene function studies, but has also cast shadows on therapeutic applications of siRNAs, as it was in 2003 and 2008 that two of the landmark papers were published for various off-target effects. With the great efforts in this field, much more is understood about the nonspecific gene silencing of siRNA on sequence-dependent and

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non-sequence-dependent levels. The knowledge has enabled the development of strategies to monitor and minimize the off-target effects. Off-target effects of siRNA can also be used in a positive way. For instance, siRNAs with mismatch tolerance properties can be designed to fight viruses that have mutations in targeting regions. Rational design of siRNAs that have potent immune stimulatory activities has also been reported [68]. REFERENCES 1. McManus MT and Sharp PA (2002) Gene silencing in mammals by small interfering RNAs. Nature Reviews Genetics 3, 737–747. 2. Ambros V (2004) The functions of animal microRNAs. Nature 431, 350–355. 3. Ambros V (2008) The evolution of our thinking about microRNAs. Nature Medicine 14, 1036–1040. 4. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K and Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498. 5. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE and Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811. 6. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, et al . (2003) Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology 21, 635–637. 7. Saxena S, Jonsson ZO and Dutta A (2003) Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. Journal of Biological Chemistry 278, 44312–44319. 8. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, et al . (2004) Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proceedings of the National Academy of Sciences of the USA 101, 1892–1897. 9. Snove O Jr and Holen T (2004) Many commonly used siRNAs risk off-target activity. Biochemical and Biophysical Research Communications 319, 256–263. 10. Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, et al . (2006) 3 UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nature Methods 3, 199–204. 11. Du Q, Thonberg H, Wang J, Wahlestedt C and Liang Z (2005) A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Research 33, 1671–1677. 12. Dahlgren C, Zhang HY, Du Q, Grahn M, Norstedt G, Wahlestedt C and Liang Z (2008) Analysis of siRNA specificity on targets with double-nucleotide mismatches. Nucleic Acids Research 36, e53. 13. Xu Y, Zhang HY, Thormeyer D, Larsson O, Du Q, Elmén J, et al . (2003) Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs. Biochemical and Biophysical Research Communications 306, 712–717.

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INDEX

A-62176, 225 abacavir, 2, 35, 37, 40–3, 46 aberrant overexpression, 213 acridine derivatives, 215 acridinium salt, 215, 222–23 acridones, 217 ADP-ribose and TRPM2 channels, 147 adriamycin, 223 alkylating agents, 206 all-in-one single system, 397 ALT-positive cells, 229 AMBER, 259–60 anilino group, 217 anionic copper phthalocyanine, 230 anthraquinones, 216 anti, 208 anticancer drugs, 206 anticancer therapy, 206 antiparallel, 207 aplysia ADP-ribosyl cyclase, 143–4 apoptosis, 213, 226 aptamer, 302–4, 317 ara-A, 57 ara-neplanocin, 26–7 aristeromycin, 2–6, 8–15

ascididemin, 222 AutoDOCK, 260, 263 autophagic cell death, 221 azacyanines, 225 Barton-McCombie, 38, 55, 67 (-)-BCA, 42–3 bcl-2, 213, 221, 229 benzo(h)quinoline derivatives, 220 benzoindoloquinolines, 225 benzylamino group, 218 berberine derivatives, 237 biaryl polyamides, 235 biarylpyrimidine derivatives, 236 bimolecular G-quadruplex, 207 binding mechanisms, 215 binding potency, 216 binding sites, 215–16, 238 bioinformatics studies, 210 birch reduction, 55 bis-benzimidazole, 236 bis-indole, 236 bispyrimidinoacridines, 225 BRACO19, 219 branched DNA assay, 298


423

424

INDEX

butterfly-shape, 236 BVDU, 59–60, 63 Ca2+ messenger molecules, 144–6 cADPR and Ca2+ mobilization, 146 cADPR and TRPM2 channels, 147 camptothecin, 223 cancer target validation, via inducible RNAi systems, 398–400 carbocyanine dye, 234 carbodine, 17–20, 62–3 carbovir, 2, 35, 37–44 carcinoid/neuroendocrine tumors, 227 C-BCNA, 64 CD38, 142–54 ablation, 146 antigenic function, 143 catalytic mechanism, 148–51 catalytic residue, 148 cellular location, 151 critical residues, 151 crystal structure, 148, 150 disulfide linkages, 150, 153 enzymatic activities, 143–6 membrane topology, 151–3 positive-inside rule, 153 relation with HIV, 143 type II signaling, 152–3 type III signaling, 152–3 cDNA microarray, 229 cell adhesion, 213 cell cycle control, 211 cell cycle regulation, 213 cell growth, 212 cellular differentiation, 213 cellular immortalization, 211 CHARMM, 259–60 chemiluminescence enzyme immunoassay, 299 chemiluminescence microplate assay, 299 chromophores, 238 cisplatin, 206, 223 c-kit, 213, 219, 234 claisen rearrangement, 44, 46, 48–9 clinical trial, 227 c-myc, 210, 212–13, 215, 221, 226, 229, 233 CNBP, 213 combination drugs, 219

combination therapies, 223 conformationally locked nucleoside, 65, 71 connecting loop, 206–7 conserved noncoding regulatory sequences, 210 conserved transcription factor-binding sites, 210 coronene, 228 corrole derivatives, 230 C-OXT-A, 77, 79 C-OXT-G, 77, 79 CpG islands, 210 crescent-shaped scaffold, 221 crystallographic studies, 215 CX-3543, 226 cyclic ADP-ribose (cADPR), 144 cyclo[n]pyrroles, 232 cylene pharmaceuticals, 226 cytosine-arabinoside, 232 daunomycin, 215 daunorubicin, 232 DB832, 235 D-carba T, 63 3-deazaaristeromycin, 3, 15 3-deazacarbovir, 47–8 deazanaplanocin, 23 2 -deoxy nucleoside, 59, 68 Dess-Martin reagent, 53 diarylethynyl amides, 236 diarylureas, 236 DIBAL-H, 4–5, 30, 36, 41, 51, 74, 76 dibenzophenanthroline derivatives, 224 Dicer, 390 2,6-diphenylthiazolo[3,2-b][1,2,4]triazoles, 236 discrete molecular dynamics (DMD), 264 distamycin, 216, 234 DNA damage response, 212, 231–2 DNA methyltransferase, 17 DNA repair mechanisms, 211 DNA secondary structures, 206 DNA supercoiling, 211 DNA–mGB (minor groove binders), 262 10–23 DNAzyme (deoxyribozyme), 271–92 general structure, 275–81 mechanism of action, 283–8 mutations in catalytic core, 278–9

INDEX

structure of catalytic site, 281–3 DOCK, 260, 263 doxorubicin, 206 druglike molecules, 238 drug resistance, strategy to avoid, 401 ecdysone-inducible system, 395–97 electrochemical probes, 294 electrostatic interactions, 215 enantioselective, 30, 34, 38, 41 end-stacking binding mode, 216 end-to-end fusion, 212, 219 enhancers, 210 entecavir, 2, 50–56, 82 enzyme-linked immunosorbent assay (ELISA), 300 Escherichia coli Tet operator (TetO) sequence, 395 ethidium derivatives, 220 etoposide, 206

425

G-quartet, 206, 215 groove dimensions, 208 groove region, 206, 215 Grubbs’ catalyst, 5–6, 40, 46, 48 guanidinium-modified phthalocyanines, 230 guanine phosphodiester backbones, 208 guanine-rich DNA, 206 guide strand, 390–91 gymnosis, 339

flavopiridol, 219 fluorenones, 220 fluorescence polarization, 307 fluorescence resonance energy transfer (FRET), 305, 309–11, 316 fluoroneplanocin, 21–2, 25–6, 29 fluorophore, 307 fluoroquinolones, 225 four-stranded structures, 206 furan-based cyclic oligopeptides, 234 fused aromatic system, 216

HBV-polymerase, 55–6 HCMV, 23, 46–8, 60, 68, 75–6, 78 HCV RdRp, 62 hepatitis B virus (HBV), 1–2, 18, 23, 32–3, 50, 52–3, 55–6, 58, 60, 75, 77 heterochromatin protein 1 (HP-1), 397 heterocyclic diamidines, 235 hnRNP K, 213 Hoffman elimination, 54 5 -homoaristeromycin, 12 6 -homoneplanocin, 24–5 Hoogsteen hydrogen-bonding, 207 horseradish peroxidase (HRP), 298 HsSAHH, 11, 15–16 hTERT, 213, 221, 223, 229 human RNA helicase A (RHA), 401 hyBeacons, 307–9 hybrid and conjugation molecules, 237 hybrid capture assay, 298 hybridization protection assay (HPA), 297 hydrogen–bonding interactions, 215

γ-H2AX foci, 231 gene evolution, 210 gene expression, 211, 213, 216 gene regulatory elements, 210 gene silencing, 391 Dox-dependent mTOR, 399 involvement of human RNA helicase A (RHA) in, 401 viral-mediated delivery of siRNA for, 393–94 gene silencing techniques, 336 genomic screening, 392 glycosidic torsion angles, 208 gold nanoparticles (AuNPs), 302–4 G-overhang, 219, 230–1 G-quadruplex structures, 206

imatinib, 232 immunoglobulin switch regions, 210 inducible systems, of RNAi, all-in-one single system, 397 ecdysone-inducible system, 395–97 Krüppel-associated box (KRAB) fusion system, 397 lac system, 395–96 tet-on/off system, 394–95 inducible transgenic RNAi, 400 in silico footprinting (ISF), 262 isopropyl thiogalactose (IPTG), 395 insulators, 210 intercalator, 310 intramolecular G-quadruplex, 207

426

INDEX

iodoneplanocin, 26 ion channel, 206 isaindigotone derivatives, 237 isoalloxazine derivatives, 219 6 -isoneplanocin, 24 isotopic labeling, 293, 296 IVDU, 59–60 knockdown procedure, 392 KRAS, 213 Krüppel-associated box (KRAB) fusion system, 397 lac operators (lacOs), 395–96 lentiviral vector-mediated delivery method, of shRNA, 394 LNA, 335 antimirs, 342 aptamers, 342 gapmer, 338 melting temperatures, 337 mixmer, 338 LNAzymes, 339 locked nucleic acid, 335 long-range charge transfer, 297 loop regions, 215 luminescence resonance energy transfer (LRET), 310 luminal, 298–9 macrocyclic system, 216 macronuclei, 210 magnetic relaxation switch (MRS), 306 magnetic resonance imaging (MRI), 305–6 Mannich reaction, 54 meridine, 222 metabolism, 213 metal complexes, 237 metal ions, 208 methylene blue (MB), 294–6 microRNA (miRNA), 390 Mitsunobu reaction, 7, 27, 34, 49, 62, 71 mixed monolayer protected gold clusters (MMPCs), 302 modes of interaction, 238 molecular beacons (MBs), 311–17 molecular docking, 259, 263–4 molecular dynamics (MD) simulation, 259, 264

molecular modeling, 258 molecular recognition with a driven dynamics optimizeR (MORDOR), 264 Monte Carlo (MC) algorithm, 263 M-phase cell cycle arrest, 232 multitarget silencing, 400 N-methyl mesoporphyrin IX, 229 NAADP, 144 NAADP and lysosomal Ca2+ stores, 146 NAADP and two-pores channels, 146 NAMD, 260 Nanoparticle, 300–2 naphthalene diimide, 216 naphthalene, 228 neplanocin A, 2–8, 12, 15, 18, 21–6, 33 neplanocin C, 65–6 neplanocin F, 32–6 NHE III1 , 210, 213 NMR, 206, 215 noncoding DNAs, 211 nontemplate strand, 214 5 -norabacavir, 42 5 -noraristeromycin, 10–12, 15, 42 5 -norcarbovir, 42 5 -norneplanocin, 24, 42 nuclease hypersensitive sites, 210 nucleic acid-targetd drug design, 258 nucleolin, 214 nucleolin/rDNA G-quadruplex complexes, 226 nysted reagent, 53 octacationic quaternary ammonium zinc phthalocyanine, 230 oligoamides, 234 on–off switching mechanism, 213 oxazole-based peptide relatives, 234 oxazole-containing macrocycles, 232 OXT-A, 76 p16, 219, 221 p21, 221 p27, 221 paclitaxel, 219 parallel, 207 Pauson-Khand adduct, 41 pentacationic manganese(III) porphyrin, 229

INDEX

peptide-based macrocyclic compounds, 234 perylene derivatives, 227 phenanthrolines, 220 phosphates backbone, 215 phosphoramidite chemistry, 316 photoluminescence, 304 π-stacking interactions, 215 PIPER, 227 pKa values, 227 plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH), 10–11, 15–16 pol I, 214 pol I-driven transcription, 214, 226 polymorphism, 207 polynucleated cells, 223 porphyrin, 215, 228 POT1, 212, 219, 231 potential G-quadruplex forming sequences, 207, 210 probe, 292 protein-DNA recognition, 211 pseudomonas fluorescence lipase (PFL), 60–1 pyrene, 316–17, 320 QQ58, 225 quantum dots, 304–6 quarfloxin, 226 quencher-free molecular beacons (QF-MBs), 313–17 quenching, 309–10, 312, 316, 320 quinacridine, 224 quindoline derivatives, 221 rDNA, 214, 229 recombination hot spots, 210 Reformatskii-Claisen rearrangement, 49 regioselective, 6, 47–8, 70 regioselectivity, 40, 49, 53, 61 RET, 229 RHPS4, 222–3 ribavirin, 17, 19–20 ribonolactone, 5, 32 ribosomal DNA, 214 ring-closing metathesis (RCM), 44, 48–9 RMNPA, 23–4

427

RNA-induced silencing complex (RISC), 390 RNA interference (RNAi), 390, see also inducible systems, of RNAi; small interfering RNA (siRNA) effect on gene expression, 391–92 mechanistic scheme of, 391 pathway, 390 RNA polymerase I, 214 RNAse H, 338 rRNA, 214 rRNA biogenesis, 215–16 rRNA synthesis, 214–15, 226 saccharomyces cerevisiae, 229 S-adenosyl-L-homocysteine (SAH), 2–4, 8, 11–16, 18, 25–7 S-adenosyl-L-methionine (SAM), 3 SAR investigation, 218 S-derivatization, 103–8 Se-derivatization, 108–15 selectivity, 216 selenium-substituted expanded porphyrin, 232 SELEX, 342 self-aggregation, 227 senescence, 212, 221 side effects, 206 silencing element, 213 siLNA, 340 Simmons-Smith cyclopropanation, 66, 70, 73 single nucleotide polymorphism (SNP), 292–3 sisiRNA, 341 small hairpin RNA (shRNA) expression, 393–94 small interfering RNA (siRNA), 390–91 approaches for delivering into cytoplasm, 392 regular vs inducible, 398 transfection, 392–93 viral-mediated delivery for gene silencing, 393–94 SMNPA, 23 sp1, 213 stereo amine side chains, 234 steroids, 237 strand directivity, 207

428

INDEX

strand stoichiometry, 207 structure, function and drug discovery, 125 stylonychia lemnae, 210 substituted groups, 238 sugar ribose, 208 surflex dock, 260, 263 symmetric phenyl derivatives, 237 syn, 208 systematic evolution of ligands by exponential enrichment (SELEX), 317 SYUIQ-5, 211, 221 target validation, using siRNA, cancer, 398–400 virus, 400–1 taxol, 223 TEBPα, 210 TEBPβ, 210 Te-derivatization, 116–17 telomerase, 210–11 telomerase inhibition, 219 telomerase-positive cells, 229 telomere, 210–12 damage, 221 dysfunction, 212, 223, 232 elongation, 232 end-binding proteins, 210 function, 211 length, 211–12 maintenance, 211–12, 216 shortening, 219, 223 telomeric DNA sequence, 209 telomeric ends, 210 telomeric fusions, 223 telomeric G-quadruplexes, 212 telomestatin, 230 telophase bridge, 223 temozolomide, 223 termini of chromosomes, 211 tet-on/off system, 394–95 tetracycline repressor (TetR), 395

5,10,15,20-tetra[4-hydroxy-3(trimethylammonium)methylphenyl]porphyrin, 229 tetramethylpyridiniumporphyrazines, 230 tetramolecular G-quadruplex, 207 threading intercalation mode, 217 thrombin binding aptamer (TBA), 305, 320 TMPyP4, 211, 215–16, 228 topoisomerase poisons, 206 transcription factor, 213 transcriptional activation, 210 transcriptional regulation, 211 TRF2, 212, 221, 223, 231–2 triarylpyridines, 236 triazacyclopenta[b]phenanthrene, 221 triazine derivatives, 236 1,4-triazoles, 236 tricyclic aromatic molecules, 217 TSS-proximal regions, 210 tTRKRAB-based system, 397 tumorigenesis, 211, 214 unfused aromatic system, 216 van der Waals interactions, 215 VEGF, 229 vinylmagnesium bromide, 5–6, 8 viral-mediated delivery method, for gene silencing, 393–94 virus target validation, using siRNA, 400–1 water-mediated hydrogen-bonding interactions, 215 Watson-Crick base pairs, 216 wavelength-shifting molecular beacons, 313 x-ray crystallography, 206 xylo-nucleoside, 57, 58 zinc complex, 230

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