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, 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.

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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. 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


vi

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


viii

FOREWORD

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


wwwwwww


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


xii

CONTRIBUTORS

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

xiii

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


wwwwwww

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


xvi

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

xvii

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.


xviii

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


INTRODUCTION

xix

(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


xx

INTRODUCTION

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


INTRODUCTION

xxi

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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