Medicinal Chemistry of Nucleic Acids E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Serie: Wiley series in drug discovery and development
• Sprache: Englisch

Seitenzahl: 791

Veröffentlichungsjahr: 2011

Beschreibung

Complete, up-to-date coverage of the broad area of nucleic acid chemistry and biology Assembling contributions from a collection of authors with expertise in all areas of nucleic acids, medicinal chemistry, and therapeutic applications, Medicinal Chemistry of Nucleic Acids presents a thorough overview of nucleic acid chemistry--a rapidly evolving and highly challenging discipline directly responsible for the development of antiviral and antitumor drugs. This reliable resource delves into a multitude of subject areas involving the study of nucleic acids--such as the new advances in genome sequencing, and the processes for creating RNA interference (RNAi) based drugs--to assist pharmaceutical researchers in removing roadblocks that hinder their ability to predict drug efficacy. Offering the latest cutting-edge science in this growing field, Medicinal Chemistry of Nucleic Acids includes: * In-depth coverage of the development and application of modified nucleosides and nucleotides in medicinal chemistry * A close look at a large range of current topics on nucleic acid chemistry and biology * Essential information on the use of nucleic acid drugs to treat diseases like cancer * A thorough exploration of siRNA for RNAi and the regulation of microRNA, non-coding RNA (ncRNA), a newly developing and exciting research area Thorough in its approach and promising in its message, Medicinal Chemistry of Nucleic Acids probes the new domains of pharmaceutical research--and exposes readers to a wealth of new drug discovery opportunities emerging in the dynamic field of nucleic acid chemistry.

Leseprobe

Table of Contents

Series Page

Title Page

Copyright

Foreword

Contributors

Introduction

Chapter 1: Recent Advances in Carbocyclic Nucleosides: Synthesis and Biological Activity

1.1 Introduction

1.2 Five-membered carbocyclic nucleosides

1.3 Three-Membered Carbocyclic Nucleosides

1.4 Four-Membered Carbocyclic Nucleosides

1.5 Six-Membered Carbocyclic Nucleosides

1.6 Conclusion

Acknowledgment

References

Chapter 2: Structures and Functions of Nucleic Acids Modified with S, Se, and Te and Complexed with Small Molecules

2.1 Introduction

2.2 Sulfur-Derivatized Nucleic Acids

2.3 Selenium-Derivatized Nucleic Acids (SeNA)

2.4 Tellurium-Derivatized Nucleic Acids (TeNA)

2.5 Structural Studies of Nucleic Acids–Small Molecule Complexes

2.6 Perspective

Acknowledgment

References

Chapter 3: Unraveling the NAD Cyclizing and Calcium Signaling Functions of Human CD38

3.1 Introduction

3.2 Antigenic functions of CD38

3.3 Enzymatic functions of CD38

3.4 Ca2+ mobilizating functions of the catalytic products of CD38

3.5 Ca2+ influx activating functions of the catalytic products of CD38

3.6 Structure and catalytic mechanism of CD38

3.7 Cellular location and membrane topology of CD38

Acknowledgments

References

Chapter 4: DNA and RNA binding small molecules

4.1 DNA-binding drugs

4.2 RNA-binding drugs

References

Chapter 5: G-Quadruplex DNA and Its Ligands in Anticancer Therapy

5.1 G-Quadruplex Structures

5.2 Biological Function of G-Quadruplex

5.3 Ligand-Quadruplex Complex

5.4 Small Molecules Binding to G-Quadruplex Structures

5.5 Summary and Outlook

References

Chapter 6: Molecular Modeling in Nucleic Acid-Targeted Drug Design

6.1 Introduction

6.2 Targeting DNA

6.3 Targeting RNA

6.4 Summary

References

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

7.1 Introduction

7.2 Results and Discussion

7.3 Conclusion

Acknowledgments

References

Chapter 8: Labeling Oligonucleotides toward the Biomedical Probe

8.1 Introduction

8.2 Radioactive Labeling

8.3 Electroactive Labeling

8.4 Chemiluminescent Labeling

8.5 Nanoparticle labeling

8.6 Fluorescent Dye Labeling

8.7 Conclusion

References

Chapter 9: Locked Nucleic Acid Oligonucleotides Toward Clinical Applications

9.1 Introduction

9.2 Locked Nucleic Acid (LNA)

9.3 Concluding remarks and future prospects

Acknowledgments

References

Chapter 10: The Pharmacokinetics Research of Nucleic Acid Drugs

10.1 Absorption

10.2 Plasma Kinetics

10.3 Tissue Distribution

10.4 Metabolism

10.5 Excretion

10.6 Plasma Protein Binding

10.7 Subcellular Biodistribution of Nucleic Acid–Based Therapeutic Drugs

10.8 Conclusions

10.9 Quantitative Analytical Techniques in Pharmacokinetics Research

References

Chapter 11: Inducible RNA and Drug Target Validation

11.1 Introduction

11.2 Approaches for RNA Delivery

11.3 Inducible RNA

11.4 Target Validation

11.5 Summary

Acknowledgments

References

Chapter 12: siRNA: The Specificity and Off-Target Effects

12.1 Introduction

12.2 Factors Affecting RNA Specificity

12.3 Sequence-dependent off-target effects

12.4 Sequence-Independent Off-Target Effects

12.5 Strategies to Minimize Off-Target Effects

12.6 Summary

References

Color Plates

Wiley Series

Index

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Published by John Wiley & Sons, Inc., Hoboken, New Jersey.

Published simultaneously in Canada.

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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. 3. Intestinal absorption. 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

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 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 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

Contributors

Jyoti Chattopadhyaya Bioorganic Chemistry Program, Department of Cell & Molecular Biology, Box 581, Biomedical Center, Uppsala University, SE-75123 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

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 Sciences, Sun Yat-sen University, Guangzhou 510006, China

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, SE-75123 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

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

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.

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 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].

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).

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

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 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 (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 oto- and 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 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 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 quadruplex-forming 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.

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). 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.

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Chapter 1

Recent Advances in Carbocyclic Nucleosides: Synthesis and Biological Activity

Jianing Wang Ravindra K. Rawal 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).