Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

2.26 Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid) E E Swayze, R H Griffey, and C F Bennett, ISIS Pharmaceuticals, Carlsbad, CA, USA & 2007 Elsevier Ltd. All Rights Reserved. 2.26.1

Introduction

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2.26.2

Deoxyribonucleic Acid

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2.26.2.1

Structure and Nomenclature

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2.26.2.2

Prototypical Function and Pharmacology

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2.26.2.3

Prototypical Therapeutics

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2.26.2.3.1 2.26.2.3.2 2.26.2.3.3 2.26.2.3.4 2.26.2.3.5

Transition metal complexes Intercalation Minor groove binders Nonnatural nucleosides Other structures

2.26.3

Ribosomal Ribonucleic Acids

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2.26.3.1

Structure and Nomenclature

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2.26.3.2

Physiological Function

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2.26.3.3

Prototypical Pharmacology

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2.26.3.4

Prototypical Therapeutics

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2.26.3.4.1 2.26.3.4.2 2.26.3.4.3

Aminoglycosides Macrolides Other ribosomal ribonucleic acid binding antimicrobials

2.26.4

Messenger Ribonucleic AcidS

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2.26.4.1

Structure and Nomenclature

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2.26.4.2

Physiological Function

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2.26.4.3

Prototypical Pharmacology

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2.26.4.4

Prototypical Therapeutics

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2.26.4.4.1 2.26.4.4.2 2.26.4.4.3 2.26.4.4.4

Antisense-based strategies Internal ribosomal entry site Human immunodeficiency virus transactivation response Berberine

2.26.5

New Directions

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2.26.5.1

Riboswitches

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2.26.5.2

Signal Recognition Particle and tmRNA

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2.26.5.3

MicroRNA

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References

2.26.1

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Introduction

Nucleic acids present a broad palate of drug targets, based on their function and structure. Nucleic acids are composed of four nucleotide ‘letters’ arranged by sequence into strands. The nucleotides consist of three chemical components: the heterocyclic base that defines the ‘letter’; the sugar; and the internucleotide phosphate diester linker (Figure 1). There are two fundamental types of nucleic acids: ribonucleic acids (RNAs) and deoxyribonucleic acids (DNAs), whose structures are determined by the removal of a hydroxyl group from the ribose sugar and methylation of uracil to give thymine. Cellular DNA exists in a double-stranded form in the chromosomes of the nucleus, serving as the storage media for a cell’s information and programming instructions. The double-stranded DNA is opened transiently to allow

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Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

Y 5

O O

6

O P O

O

4

N 1

O

2

O O

NH

O

3

O

X

Uridine (RNA): X = OH,Y = H Thymidine (DNA): X = H,Y = CH3

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O O P O O

N X

4′

O

1′

3′

2′

O

X

NH2 N

N

O

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NH2 6

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Cytidine (RNA): X = OH 2 ′-Deoxycytidine (DNA): X = H

N

9

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N1

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2

O O

O P O

N

O

O

O

N X

N

NH NH2

Adenosine (RNA): X = OH 2 ′-Deoxyadenosine (DNA): X = H

Guanosine (RNA): X = OH 2′-Deoxyguanosine (DNA): X = H

Figure 1 Common building-blocks of nucleic acids.

transcription into RNA molecules. RNAs have a large range of sizes (20 to 4100 000 nucleotides) and shapes generated by combinations of single, double, and higher-order strands, allowing them to perform a variety of functions in the cell. For example, messenger RNA (mRNA) acts as the template for the linking of amino acids into peptide chains by the ribosome. The mRNAs are produced from longer pre-mRNA transcripts through a series of complex splicing reactions performed by the spliceosome. The ribosome in turn is composed of three ribosomal RNAs (rRNAs) that serve as the macromolecular machine and chemical catalyst for amino acid coupling. The amino acids are delivered to the ribosome by transfer RNAs (tRNAs), which contain a three-nucleotide anticodon sequence complementary to three nucleotides on the mRNA. Additional small RNAs and RNA domains that control and edit RNA and DNA through chemical modifications have been discovered over the last 10 years (e.g., small nucleolar RNAs and guide RNAs). Among the most exciting are the abundant microRNAs (miRNAs), which appear to function as an intermediate level of transcriptional and posttranscriptional control on gene expression. Mitochondrial RNA and DNA have both structural similarities and differences with bacterial and human nucleic acids. The divergence of bacterial and eukaryotic life, the increase in gene complexity in higher organisms, and the central role that nucleic acids play in all aspects of cellular growth, proliferation, and differentiation make them attractive targets for drug discovery. A number of older drugs now are known to be directed against nucleic acids. However, only a limited number of strategies have been employed to target RNAs and DNA with any level of specificity. Gene therapy has been demonstrated as a method to add new DNA to a cell to produce a new protein product, replace a mutated form of a protein, or to alter the sequence of endogenous DNA. The DNA bases have modest reactivity, and can be altered chemically by alkylating agents and metal complexes such as cisplatin. The phosphodiester backbone can be cleaved chemically by compounds such as bleomycin or enzymatically by endogenous nucleases that recognize RNA : RNA or DNA : RNA duplexes produced by administered oligonucleotide agents. Binding of small molecules or oligonucleotides to a sequence of mRNA or DNA can produce steric blocking that prevents subsequent processing or translation. Finally, small molecules and oligonucleotides can induce rearrangements or stabilization of RNA or DNA structures that block specific biological functions. Examples of these mechanisms are described in subsequent sections.

2.26.2 2.26.2.1

Deoxyribonucleic Acid Structure and Nomenclature

Double-stranded (duplex) DNA is organized into chromosomes in the cell nucleus of higher organisms. These chromosomes contain duplex DNA wrapped around the histone proteins. The DNA can be bound to histones and transcriptionally silent or bound to histones in conjunction with transcriptional factors and polymerases that mediate transcription of the DNA into RNA. The normal DNA double helix adopts a supercoiled form from binding to histones.

Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

B form DNA

Minor groove

A form RNA

Major groove

Minor groove

Figure 2 Models of helical B form DNA (left) and A form RNA (right). B DNA has a wide major groove and a narrow minor groove, while A RNA has a very narrow and deep major groove, and a wide and shallow minor groove.

Duplex DNA is a helical structure composed of two antiparallel strands linked through hydrogen bonds formed between the complementary donor/acceptor groups of deoxyadenosine (dA) and deoxythymidine (dT) residues and deoxyguanosine (dG) and deoxycytidine (dC) residues, respectively. These base pairs form a ladder that gives the DNA its natural right-handed twist (Figure 2). The DNA helix has two grooves, the deep and narrow minor groove and the shallow, wide major groove. As discussed below, these grooves present unique hydrophobic, electrostatic, and hydrogen bonding environments where small molecules are known to bind. The nomenclature for nucleic acids has been specified in detail by the International Union of Pure and Applied Chemistry (IUPAC).1 DNA bases, nucleosides, and nucleotides are referred to by their standard common names, and the numbering system for the bases and sugars are presented in Figure 1. The purine heterocycles (adenine and guanine) are attached to the 20 deoxyribose sugars through N9, and the pyrimidine heterocycles (uracil and cytidine) are bonded through N1. In all natural DNA, the 30 hydroxyl and the 50 hydroxyl of the subsequent nucleotide are linked through a phosphate diester. Typically, DNA and RNA strand sequences are written from the 50 end toward the 30 terminus.

2.26.2.2

Prototypical Function and Pharmacology

As noted above, all RNAs and proteins in a cell are derived from information stored in the DNA sequence. In contrast to prokaryotes, the majority of DNA present in eukaryotic cells is not transcribed. Why this is the case is not well understood. Single base changes in DNA sequence are responsible for more than 100 human diseases.2 Transcription of human DNA is regulated by both protein binding to DNA and methylation of specific sites in the DNA. Drugs that target DNA can alter cellular physiology by inhibiting transcription of RNA or by preventing DNA replication in dividing cells. In all cells, irreversible damage to the DNA (alkylation, strand cleavage, etc.) or inhibition of DNA replication leads to cell cycle arrest. In mammalian cells, cell cycle arrest may trigger apoptosis and cell death. A therapeutic index can be realized by exploiting the difference in rates of cell division between rapidly growing cells and normal human tissue. Hence, most agents targeting DNA are used in treatment of bacterial infection or cancer.

2.26.2.3

Prototypical Therapeutics

The DNA strands stored in a supercoiled form are cleaved to allow transcription or replication to initiate, and subsequently ligated back together by a topoisomerase protein. Many therapeutics target this process. As described above, the rate of RNA transcription is controlled by binding of activator and suppressor proteins with specificity for the DNA surfaces of certain sequences. Therapeutic agents such as intercalators can disrupt the shape of the DNA helix and alter transcription. DNA is copied by the enzyme DNA polymerase, and nucleoside analogs can prevent DNA chain extension. Maintaining the fidelity of the DNA sequence is critical, and the cell has multiple enzymes that inspect and repair the DNA duplex.

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2.26.2.3.1 Transition metal complexes The electron-rich environment at N7 of adjacent purine bases provides a good coordination site for transition metals such as platinum, whose affinity is sufficient to block proper DNA replication and transcription. Cisplatin and analogs are among the most effective anticancer drugs in treatment of solid tumors. New platinum complexes with different binding modes and ruthenium–imidazole complexes are in clinical and preclinical development.3,4 Mithramycin has been demonstrated to form a 2 : 1 complex with Fe(II) that induces a conformational change in DNA which may facilitate strand cleavage.5

2.26.2.3.2 Intercalation The DNA duplex can be altered through intercalation of small molecules between the base pairs or in the minor groove. The base pairs have modest separation and are known to open partially at 37 1C. Small, planar, aromatic groups can be inserted between the base pairs, resulting in a local distortion of the helical geometry. Many classes of drugs are known to work through intercalation, which inhibits the ability of DNA topoisomerase and polymerase enzymes to replicate the DNA. Doxorubicin traps a cleavage intermediate between topoisomerase II and torsionally strained DNA. Analogs of anthracycline drugs with attached saccharides have been shown to intercalate and position carbohydrates at specific sites in the groove.6 DNA intercalating drugs also reduce the activity of DNA methyltransferases, and can induce apoptosis through this inhibition.7 Many intercalating drugs reduce levels of mitochondrial DNA in mammalian cells through altered metabolism or inhibition of replication.8

2.26.2.3.3 Minor groove binders The interactions between proteins and DNA are mediated by hydrogen bonding networks of side chains along the minor groove of the DNA. Distamycin-related molecules bind in the narrow minor groove of duplex DNA, often as dimers that widen and distort the shape of the groove. The ability of aromatic polyamides to bind in the hydrophobic minor groove has been exploited to develop compounds that form sequence-specific H-bond donor and acceptor interactions with the bases.9,10 These compounds can have very high (o100 pM) affinity and selectivity for the target sequence, and have been shown to block the binding of transcription factors to the DNA. In addition, conjugation of regulatory peptides to these molecules produces molecules that upregulate gene expression or recruit additional transcription factors.11

2.26.2.3.4 Nonnatural nucleosides Nucleoside analogs can be incorporated into DNA and block natural maturation processes. For example, enzymatic methylation (and demethylation) of dC residues at C5 is a natural tagging process used to silence transcription of genes that are no longer required for cell function. In some cancers, specific methylation of tumor suppressor genes promotes uncontrolled cell growth. Treatment of B cell leukemias with 5-azadeoxycytidine generates DNA containing dC residues that cannot be methylated, and the tumor suppressor genes are left in the ‘on’ state. Other nucleoside drugs can act as inhibitors of DNA replication.

2.26.2.3.5 Other structures DNA can form higher-order structures, such as the G quartets present as telomeres at the ends of genes. These G quartet DNAs are very stable, and are recognized specifically by cellular proteins. Hence, artificial G quartet structures have been prepared and demonstrated to have anticancer activity. DNA : RNA duplexes formed during transcription are potential targets for drug discovery. These structures are present during transcription, and may be long-lived. For example, the transcription of human immunodeficiency virus (HIV) RNA has stall sites where DNA : RNA structures are formed as sites for recruitment of additional protein factors. DNA : RNA duplexes also are formed during trailing strand DNA synthesis (Okazaki fragment) and subsequently cleaved.

2.26.3 2.26.3.1

Ribosomal Ribonucleic Acids Structure and Nomenclature

Ribosomes are large ribonucleoprotein particles, with a bacterial ribosome from Escherichia coli having a molecular mass around 2700 kDa, a diameter of approximately 200 angstroms, and a sedimentation coefficient of 70S. The rRNA constitutes nearly two-thirds of the mass of the ribosome, and makes up nearly all of the key sites for ribosomal function. Because of this, ribosomes can be viewed as RNA enzymes, or ribozymes, and drugs which exert their effects via binding to ribosomes interact predominantly with rRNA.

Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

Bacterial ribosomes are composed of a small (30S) and a large (50S) subunit, The small subunit (30S) consists of a large, highly structured RNA molecule of over 1500 nucleotides (the 16S rRNA), along with 21 proteins (referred to as S1 through S21). The large subunit consists of two RNAs, the 23S (about 2900 nucleotides) and the 5S (120 nucleotides), along with 34 different proteins (designated L1 through L34). The large and small subunits join to provide for three tRNA binding sites at the interface, the A (aminoacyl) site, the P (peptidyl) site, and the E (exit) site. The ‘active site’ of the ribosome, the peptidyl transferase center, is near the P site on the large subunit. A common structural feature to all ribosomes is a tunnel, from the interface at P site through the large subunit to the cytoplasmic side. Eukaryotic ribosomes are slightly larger (80S) than their bacterial counterparts, with a mass of roughly 4200 kDa. The eukaryotic small subunit (40S) contains a rRNA (18S) which is homologous to the slightly smaller bacterial 16S rRNA. The large subunit (60S) contains 28S and 5S homologs of the bacterial rRNAs, as well as an additional RNA, the 5.8S rRNA. Mitochondria also contain ribosomes that contain similar structural features,12 despite consisting of more protein than rRNA. These mitochondrial rRNAs must be considered as potential sites of side effects of antibacterial rRNA acting drugs, as they have rRNA components similar in structure to bacterial ribosomes.

2.26.3.2

Physiological Function

The ribosomal RNA is the cellular machine that synthesizes proteins by reading the genetic code of a particular mRNA. Recent cryoelectron microscopic and crystallographic studies have provided astounding insights into both the structure and function of ribosomes,13 and illustrated how many antibiotics act to inhibit protein synthesis.14 A recent review provides a detailed analysis of how the ribosome functions in light of these studies.15 The rRNA of the small subunit (16S in bacteria) functions primarily to decode the genetic message. This is accomplished by recognition of correct Watson–Crick base pairing between a particular three nucleotide sequence on an mRNA (the codon) with a three-nucleotide sequence on a tRNA which carries the appropriate amino acid. This recognition occurs at a portion of the small subunit termed the A site, which is the binding site of the aminoglycoside antibiotics. The large subunit functions to append the selected amino acid on the A site tRNA onto the growing peptide chain, which releases the chain from the P site tRNA. This task involves movement of the A site tRNA to the peptidyl transferase center, which is the catalytic site for peptide bond formation. The appropriate amino acid is then transferred onto the growing peptide chain, which exits the ribosome through the tunnel. Many antibiotics bind at or near the peptidyl transferase center, including chloramphenicol and the macrolides such as erythromycin. The macrolides function by blocking the exit of the growing peptide chain through the tunnel. A translocation of the P site tRNA to the E site, and the A site tRNA to the P site allows for the cycle to be repeated. The peptidyl transferase and decoding centers consist predominantly of RNA, and the key processes and reactions of protein synthesis are carried out by the rRNA. Prokaryotic 5S rRNA interacts with ribosomal proteins L5, L18, and L25 and enhances protein synthesis by stabilization of the ribosome structure but its exact role in protein synthesis is still not known, and 5S rRNA remains a largely unexplored target.

2.26.3.3

Prototypical Pharmacology

Because of the central importance of ribosomes to the synthesis of all proteins, rRNA is not a target well suited to host diseases. However, because of subtle differences in ribosome structure between prokaryotes and higher eukaryotes, rRNA is an excellent target for antibacterial chemotherapy. Most antibiotics which target the rRNA and inhibit bacterial protein synthesis prevent bacterial growth (bacteriostatic). These include the tetracyclines, chloramphenicol, and the macrolides. While each class of drug has a slightly different mechanism of action, they all interfere with the function of the ribosomal machinery and inhibit protein synthesis. This leads to a reduction or cessation of bacterial growth. In contrast, the aminoglycosides which act at the A site to induce miscoding are rapidly lethal to bacteria (bactericidal). This is likely due to a progressive build-up of aberrant proteins, which ultimately cause a disruption of multiple cellular processes leading to cell death.

2.26.3.4

Prototypical Therapeutics

2.26.3.4.1 Aminoglycosides There are several related structural classes of aminoglycoside antibiotics, representative structures of which are shown in Figure 3.16 Uniquely differentiated from the majority of aminoglycosides is streptomycin, which contains a

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Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

NH2 O H2N HN

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OH HO NH2

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H2N Gentamicin C1a

Figure 3 Aminoglycosides which bind at the bacterial 16S rRNA site.

guanylated streptamine core, as opposed to the deoxystreptamine core of the other aminoglycosides. Streptomycin binds near the A site at different location from that of the other aminoglycosides, but still interferes with decoding. The 2-deoxystreptamine based aminoglycosides can be classified into the 4,5-substituted analogs such as paromomycin and neomycin, and the 4,6-substituted analogs such as kanamycin, tobramycin, and gentamicin. These compounds all bind to the 16S rRNA A site, and force two conserved adenosine residues to swing into the minor groove formed by the mRNA/tRNA codon/anticodon base triplet formed during decoding. This stabilizes both cognate and noncognate tRNA pairings, and leads to miscoding and inhibition of protein synthesis. Tobramycin long has been administered intravenously for treatment of bacterial infections, and has been approved recently as an inhaled formulation for the treatment of cystic fibrosis patients with Pseudomonas aeruginosa infections. Also in the kanamycin family is amikacin, which is a semisynthetic derivative that contains a 2-hydroxy-4-aminobutyryl chain at the 1-amino group of kanamycin. This substitution confers resistance to a large number of aminoglycoside deactivating enzymes, which modify various portions of the aminoglycoside skeleton and render it pharmacologically inactive. Because of this unique resistance to a broad array of resistance enzymes, amikacin is one of the broadest spectrum aminoglycosides known. It has broad-spectrum activity against a variety of Gram-positive and Gram-negative organisms. However, because of the availability of many other safer, orally acting classes of antibiotics, amikacin and other aminoglycosides such as gentamicin are primarily used against serious Gram-negative infections. In particular, amikacin is used either alone or in combination to treat multidrug resistant nosocomial infections such as P. aeruginosa. All aminoglycosides suffer from potentially severe nephro- and ototoxicities, which limit the use of these excellent antibacterial agents. The nephrotoxicity presents as mild renal impairment in approximately 10–25% of patients receiving an aminoglycoside for more than several days. The nephrotoxicity is generally reversible, and can be monitored by common kidney function tests. In contrast, aminoglycoside-induced ototoxicity is not generally reversible. Both vestibular and auditory dysfunction are evident, and the degree and type of ototoxicity vary with the specific aminoglycoside. The ototoxicity has been correlated with mutations in the 12S mitochondrial A site rRNA, which supports aminoglycoside interaction with this rRNA as a source of aminoglycoside toxicity.17

2.26.3.4.2 Macrolides The macrolide antibiotics are an important class of orally active antibiotics.18 Major members of the class include erythromycin and azithromycin, as well as telithromycin, which was approved in 2004 (Figure 4). The macrolides are most commonly used against Gram-positive organisms, as they are weakly active against most Gram-negative bacilli. The macrolides are generally bacteriostatic agents that bind to the 23S rRNA on the large subunit and block the ‘tunnel’ through which the growing peptide chain exits. Resistance to macrolides arises from a methylase which modifies the ribosomal target and reduces binding, along with enzymes that chemically modify the drug structures and more general uptake/efflux resistance mechanisms. Unlike the aminoglycosides, the macrolides are generally well tolerated and give rise to few serious toxicity issues, and as such are used broadly.

2.26.3.4.3 Other ribosomal ribonucleic acid binding antimicrobials Several other classes of antibacterial agents bind rRNA, including the tetracyclines, chloramphenicol, clindamycin, spectinomycin, and the oxazolidinones (Figure 5).18 Tetracycline and its close relatives such as oxytetracycline,

Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

N

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OH Erythromycin

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Telithromycin

Figure 4 Macrolides that bind to the 50S subunit.

HO

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Linezolid

Figure 5 Other antibacterials that bind to the bacterial ribosome.

minocycline, and doxycycline are bacteriostatic agents which function by binding to the small subunit near the A site, hindering movement of the A site tRNA so that it cannot interact with the peptidyl transferase center. Spectinomycin is also bacteriostatic, and binds to the 16S rRNA of the small subunit in a location distinct from the aminoglycosides or tetracycline. It functions to inhibit translocation of the A site tRNA to the P site, presumably by inhibiting a conformational change required to accomplish this translocation. In contrast, clindamycin and chloramphenicol function by binding near the macrolide-binding sites on the rRNA of the large subunit near the peptidyl transferase center. Clindamycin has been useful for the treatment of anaerobes, as has chloramphenicol. However, the toxicity of chloramphenicol to bone marrow restricts its use to serious infections that cannot be otherwise treated. Linezolid (the prototypical oxazolidinone) represents one of the newer chemical classes of antibacterial agents discovered. It binds to the 23S rRNA of the large subunit, and functions to prevent assembly of the functioning 70S ribosome. Linezolid is broadly active against Gram-positive organisms, and is used for the treatment of vancomycin and/or methicillin-resistant infections. Because of the unique structural class and mechanism of action, there is no cross-resistance with other antibacterials; however, resistance due to a mutation of the 23S rRNA has emerged in clinical practice in a surprisingly short period.19,20

2.26.4 2.26.4.1

Messenger Ribonucleic AcidS Structure and Nomenclature

All nucleic acid sequence information is collected and annotated by the National Center for Biotechnology Information (NCBI), and mRNAs are uniquely identified by their accession number from GenBank.21 However, mRNAs are more generally referred to by their common gene name and species. RNA is synthesized on a DNA template by RNA polymerases. Bacteria utilize a single RNA polymerase to make all RNA. Messenger RNA molecules are made as either monocistronic or polycistronic molecules in bacteria. Translation can occur on bacterial RNA coincident with transcription.

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Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

G A

U C

C G A G U

C

U

A G A C C G G

G C U C

G A

Loop

Bulge U C U G G C C

Stem

Figure 6 Two-dimensional representation and a model of the three-dimensional structure of the HIV-1 TAR stem loop RNA (PDB ID 1ANR). The 50 strand of the stem is shown dark gray, and the 30 strand is shown light gray. The loop is medium gray.

In contrast, eukaryotes utilize three distinct RNA polymerases to transcribe rRNA, mRNA, and tRNA plus other small RNAs: RNA polymerase I, II, and III, respectively. Eukaryotes also compartmentalize transcription and translation, with transcription occurring in the cell nucleus and translation occurring in the cytoplasm. Eukaryotic mRNA is transcribed as a monocistronic precursor molecule in the nucleus which must undergo several processing steps before it is translocated into the cytoplasm where it is translated. The 50 terminus of eukaryotic mRNAs is modified with a unique 50 cap structure consisting of an inverted 50 -50 -triphoshate linkage between the first transcribed nucleotide and 7-methyguanine. Many eukaryotic mRNAs are further modified on their 50 terminus by methylation of the 20 oxygen of the ribose nucleotides. The presence of the methyl group adds stability, and also changes the sugar conformation such that it is predominantly (60%) in the C30 endo conformation.22 The 50 cap on an mRNA contributes to export out of the nucleus to the cytoplasm, acts as a recognition element for translation initiation factors, and confers exonuclease stability to the 50 termini of mRNAs. The primary transcript for most eukaryotic mRNAs is significantly larger than the mature mRNA due to the presence of extraneous nucleotides called intervening sequences or introns. These introns are removed by a complex cleavage and reannealing process called RNA splicing.23,24 The reason why eukaryotic RNA contain these intervening sequences is poorly understood. Most eukaryotic RNAs are also modified at their 30 termini by the addition of 50 to 250 adenosines referred as the poly(A) tail. Finally a select few mRNA transcripts are modified by specific deaminases that either deaminate adenosines or cytosines.25,26 Once each of these steps is complete the mRNA can then be exported out of the nucleus through nuclear pores to the cytoplasm. Thus in contrast to prokaryotes, eukaryotic mRNA must undergo a series of complex processing events before they can be translated. Unlike DNA, mRNAs fold into complex three-dimensional structures. These structures tend to be dominated by Watson–Crick base pairing interactions, which drive the folding of mRNAs into self-complementary stemlike structures. Imperfections in the duplex structure lead to unique shapes which can interact with other portions of the mRNA to form more complex structures in a manner similar to protein folding. Expressed in two-dimensional space, RNA structures can be binned into several classes, including stem loops, internal loops, and bulges, junctions, and pseudoknots.27 Stem loops consist of a self-complementary duplex portion, the stem, which is connected by a loop portion of unpaired nucleobases. Internal loops and bulges are formed as a result of imperfect pairing in a stem. This often allows the normally rigid duplex to bend, and the loop or bulge region can be an attractive binding pocket for a potential drug molecule. A representative structure of a stem loop with a bulge is the HIV transactivation response (TAR) element (Figure 6). Junctions typically join three or more stems together, and can form intricate structures.28 Pseudoknots are complex elements in which two stems from nearby regions of sequence stack on top of each other. The stem regions are connected by necessary loops, and the resulting structure is usually tightly folded and stable. Pseudoknot structures are utilized by several viral genomes to promote frameshifting29 and are also found in eukaryotic RNAs such as telomerase RNA.30,31

2.26.4.2

Physiological Function

Messenger RNA, as its name implies, has been well established as the intermediary in the expression of genetic information contained within DNA as proteins. In that every protein produced in a cell derives from a specific mRNA,

Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

targeting mRNA represents a viable alternative to targeting proteins as potential therapeutic agents. However, it is also clear that the role of mRNA extends beyond being simply an information carrier, and that many interactions with mRNAs are important determinants in the level of expression of the encoded protein. Many mRNAs contain complex structures outside of the coding sequence, e.g., the 50 and 30 untranslated regions (UTR). While the precise biochemical functions of these regions are only just being elucidated, it is becoming evident that many of these elements are involved in regulation of the ultimate expression of the encoded gene. These mechanisms are illustrated by the example of iron regulation in eukaryotes. The mRNAs for ferritin, an iron storage protein, and transferrin receptor, a receptor for the iron carrier transferrin, both contain stem loop structures. A structure in the ferritin 50 UTR termed the iron-responsive element (IRE) binds an IRE-binding protein which serves to prevent translation. In the presence of iron, the IRE-binding protein binds iron, which prevents it from binding the IRE, and translation can take place. This produces ferritin to store the excess iron. In contrast, transferrin receptor mRNA contains IREs in the 30 UTR. In iron-free conditions, IRE-binding protein binds the IREs in the transferrin 30 UTR, and stabilizes the mRNA, preventing degradation, increasing the levels of transferrin receptor. This increases the uptake of iron-rich transferrin from outside the cell. Iron levels are therefore tightly controlled by cells using this posttranscriptional regulatory process.

2.26.4.3

Prototypical Pharmacology

Modulating mRNA levels can in theory lead to dramatic pharmacological effects, as the expression of particular proteins can be reduced by inhibiting translation of or destroying the mRNA which codes for that protein. Conversely, the expression of a particular protein can be increased by stabilizing a particular message such that more is produced from each message. These effects can be observed for a great many mRNA targets by direct measurement of RNA levels by quantitative methods, or by quantitation of the encoded protein. The downstream pharmacological consequences are many, and depend on the particular mRNA target. Targeting mRNA, therefore, represents an orthogonal means to attack any potential protein target, and is especially attractive for ‘undruggable’ protein targets strongly implicated in a disease state. Unfortunately the strategies for selectively modulating an mRNA inside a cell are still only nascent and have only started to be developed over the last 15 years. Messenger RNAs present unique challenges as drug target. Current RNA structure prediction programs are unlikely to predict accurately the structure of an mRNA as it exists in a cell. This is due in part to the length limitations of most computational programs, but more importantly mRNAs have dynamic three-dimensional structures as they are being processed and translated. Additionally, mRNAs are bound by cellular proteins that tend to exhibit low sequence selectivity. Thus it is not currently possible to predict structure of an mRNA in a cell nor to identify routinely which regions can be targeted with drug molecules.

2.26.4.4

Prototypical Therapeutics

2.26.4.4.1 Antisense-based strategies One of the most direct methods for selectively targeting RNA is through antisense-based strategies, wherein oligonucleotides are designed to interact with RNA by Watson–Crick base pairing. Representative antisense drugs are shown in Figure 7. Antisense-based approaches are broadly used to modulate expression of genes in cell culture, in animal models, and in the clinic. There are two basic antisense mechanisms: oligonucleotide-induced cleavage of the targeted RNA and steric blocking of RNA. Examples of the former mechanism include ribonuclease (RNase) H mediated cleavage, small interfering RNAs, and ribozymes.32–36 Examples of steric effects of antisense oligonucleotides include inhibition of translation, alternative RNA processing, and disruption of RNA regulatory structures.37–39

2.26.4.4.1.1 Oligodeoxynucleotides The most widely utilized antisense mechanisms are RNase H based cleavage of RNA and the more recently described RNA interference (RNAi) mechanism. RNase H is a ubiquitously expressed enzyme, present in viruses, prokaryotes, and eukaryotes, which hydrolyzes the RNA strand of a DNA–RNA duplex. In general, oligonucleotides that support the RNase H based mechanism are 15–25 nucleotides in length and contain at least 7–10 consecutive deoxynucleotides. RNase H based oligonucleotides (Figure 7) are the most advanced with the approval of one drug that selectively targets a viral mRNA (fomivirsen40) and multiple drugs in clinical trials.41 Fomivirsen, as well the other early antisense agents to enter clinical trials, are phosphorothioate oligodeoxynucleotides, which are short (typically 20 bases) DNA oligomers that contain a phosphorothioate linkage in place of the phosphodiester linkage. These molecules activate RNase H cleavage of the target mRNA after binding, and reduce

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Figure 7 Representative antisense drugs. (a) Fomivirsen; (b) ISIS 113715; (c) cholesterol conjugated siRNA duplex. Key: lowercase, deoxyribose (DNA); UPPERCASE, ribose (RNA); ITALIC, 20 -O-methoxyethylribose; ITALIC, 20 -O-methylribose; m, 5-methyl substitution on cytosine; s, phosphorothioate linkage. All other linkages are phosphodiester, and the letters A, C, G, T, U refer to adenine, cytosine, guanine, thymine, and uracil, respectively.

levels of the target protein. The phosphorothioate serves to stabilize the molecule to nucleolytic degradation, and aid pharmacokinetics by improving binding to plasma proteins which prevents renal excretion, and leads to tissue uptake.42 The properties of the first-generation phosphorothioate oligodeoxynucleotides have been dramatically improved by incorporating nucleosides modified at the 20 position on the sugar into the 50 and 30 ends of the molecule while keeping the 20 deoxy core region, or gap, and the phosphorothioate linkage. These modifications serve to further improve metabolic stability, and also improve potency by increasing the binding affinity for the target mRNA.43,44 They also activate RNase H cleavage of the target mRNA due to the presence of the 20 deoxy gap. Uniformly 20 modified oligonucleotides are not substrates for RNase H, and generally show reduced potency relative to the gapped versions. The most advanced of these chemistries, 20 -methoxyethyl (MOE), typically employs a design of 5 MOE residues at each end, flanking a 10-base deoxy gap. Several MOE oligonucleotides shown to produce the desired pharmacological endpoints in animal models have advanced to human clinical trials and have shown early evidence of efficacy. These include ISIS-113715, an inhibitor of protein-tyrosine phosphatase 1B (PTP1B) which has shown efficacy in models of diabetes,45 and the human specific version (ISIS-301012) of a mouse-specific apolipoprotein B-100 (apoB-100) antisense drug, which has shown benefit in lowering low-density lipoprotein (LDL) cholesterol.46

2.26.4.4.1.2 Ribonucleic acid interference Oligonucleotides based on RNA interference (RNAi), especially small duplexed RNAs of approximately 20 base pairs known as small interfering RNAs (siRNAs), are gaining widespread use as research reagents for cell culture-based experiments.47,48 There are many similarities for RNase H and siRNA oligonucleotides with the key difference being that, in general, double-stranded RNA oligonucleotides are utilized for siRNA. Once delivered inside the cell, the double stranded oligonucleotide interacts with a protein complex called the RNA-induced silencing complex (RISC), which contains a helicase activity and the cleavage enzyme argonaute 2.49 The helicase unwinds the two RNA strands retaining the antisense strand by an as-yet poorly characterized mechanism which may, in part, be dependent on the thermodynamic stability of the base pairs formed at the 50 end of the RNA.50 The RISC complex then facilitates interaction of the antisense strand with the targeted RNA where it binds by Watson–Crick base pairing. The enzyme argonaute 2, which shares structure and biochemical similarities to RNase H, cleaves the RNA approximately 10 nucleotides from the 50 end of the antisense oligonucleotide.51,52 Although there has been tremendous progress in defining the biochemical mechanisms by which siRNA oligonucleotides promote specific degradation of RNA, progress in identifying strategies for systemic use of these molecules as drugs has been slower. Structure–activity relationship studies53 and subsequent optimization have led to motifs with dramatically altered structures that have increased potency and metabolic stability relative to unmodified siRNA oligonucleotides54; however, rigorous in vivo pharmacokinetic studies have not been reported for any siRNA to date. There is one preliminary report demonstrating activity of systemically administered siRNA in vivo, in which

Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

conjugation of cholesterol to the sense strand enhanced delivery to mouse tissue.55 Liposomal and particulate formulations are also being explored for systemic delivery of siRNA, and have shown in vivo effects.56–58 However, more work is needed to further optimize siRNA constructs and/or formulations for broad systemic delivery, and most current therapeutic strategies are focused on local delivery of siRNA molecules.

2.26.4.4.1.3 50 Cap of messenger ribonucleic acid Several investigators have exploited the unique chemical reactivity of the 50 cap structure, identifying molecules that selectively hydrolyze the triphosphate bond.59,60 Indiscriminate interference with function of 50 caps present on all mRNA would globally inhibit translation and would be predicted to be rather toxic. Such a strategy is utilized by several viruses as a strategy to compete for host cell translation factors.61 In contrast, selective interference with the function of the 50 cap can be used to specifically modulate the production of a protein product. Antisense-based strategies represent the most direct way of doing so. Antisense oligonucleotides can be designed to the 50 terminus of a transcript and sterically interfere with the recognition of the 50 cap by translation factors.38 Another strategy is to conjugate reactive groups to an oligonucleotide that induce cleavage of the 50 cap structure, the oligonucleotide providing the specificity. Thus antisense-based strategies represent the most direct and efficient approach for modulating the function of the 50 cap structure present on mammalian mRNAs. 2.26.4.4.1.4 Other antisense strategies In addition to promoting the degradation of a target mRNA, oligonucleotides can be utilized to modulate RNA maturation selectively. The best-characterized example is use of oligonucleotides to selectively modulate RNA splicing.62 Oligonucleotides can be used to correct aberrant splicing events such as occurs in thalassemia.39 Alternatively oligonucleotides can be used to redirect splicing such that different alternative spliced variants are produced.63,64 As the role of alternative spliced protein products in human disease becomes better characterized, the latter approach will be an increasingly important application for oligonucleotide-based drugs.

2.26.4.4.2 Internal ribosomal entry site Translation of most eukaryotic mRNAs is initiated at the 50 cap structure. However, some viral65 and eukaryotic66,67 mRNAs contain a sequence in the 50 UTR which functions as an internal ribosome entry site (IRES), although the exact function of the IRES remains controversial.68 IRES elements were first discovered in picornavirus and later in other viral mRNAs. More recently IRES-like sequences have been found in some eukaryotic mRNAs such as the mRNAs encoding vascular endothelial growth factor, fibroblast growth factor-2, c-myc, N-myc and the antiapoptotic protein Apaf-1.69,70 One of the best-characterized IRESs is that of extrahepatic hepatitis C virus (HCV). The HCV IRES occurs in the 50 UTR of the positive strand polycistronic mRNA. It is approximately 360 nucleotides in length, extending approximately 30 nucleotides 30 to the AUG translation initiation codon for the core protein. The IRES consists of three major structural domains which extend from a pseudoknot structure.71 The pseudoknot and domain IV are centrally located with the other domains radiating out from the central region. Domain III is required for IRES function and contains a large four-way junction and two stem loop structures (IIId and IIIe). Domain IIId contains an E-loop motif presenting a unique narrowed major groove and a distorted phosphodiester backbone.72,73 This structure is similar to the sarcin/ricin loop in 28 S rRNA. Domain IV includes the translation initiation codon. As the HCV IRES is essential for translation of the HCV genome, it represents a unique structure for drug targeting. There are multiple approaches being pursued to interfere with the HCV IRES. Several investigators have designed synthetic oligonucleotides to bind to the IRES and disrupt its function.74–76 These oligonucleotides were shown to block translation of HCV proteins in a cell culture model. In a related approach, oligonucleotide aptamers were selected to bind to the HCV IRES.77 Aptamers selected to bind to domains II–IV had the highest affinity, and were shown to contain a consensus sequence ACCCA which base pairs to the apical loop of domain IIId. Several efforts to identify small molecules have also been reported. Screening of chemical libraries has resulted in the identification of phenazine78 and guanidine79 small molecules capable of inhibiting HCV IRES dependent translation. In related work, a class of benzimidazoles (Figure 8) was found to bind to an internal loop region of the HCV 50 UTR stem II, and subsequently to inhibit HCV replication in a cellular assay.80

2.26.4.4.3 Human immunodeficiency virus transactivation response One of the best-characterized and explored structured RNA drug targets is the TAR within the HIV RNA genome (Figure 6). TAR is recognized and bound by a virally encoded protein, Tat, which promotes transcription of viral RNA. As a correlation between Tat binding to TAR and HIV mRNA transcription has been shown, the Tat–TAR interaction is

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Figure 8 Small molecules that target RNA.

an interesting drug target for HIV chemotherapy. The TAR structure is a stem loop, containing an asymmetric internal loop, or bulge, of three bases. It therefore contains two unique elements for the recognition of drugs and the Tat protein: the terminal stem loop and the internal bulge. To identify small molecule inhibitors of the Tat–TAR interaction, a corporate library of 150 000 compounds was screened in a high-throughput format. A 1–2% hit rate was found for molecules that showed significant inhibition at 20 mM in the screen. From these hits, approximately 20 compounds were identified that had dose–responsive activity in a cellular assay at nontoxic levels.81 In continued investigations of the mode of action using mass spectrometry, two compounds, a quinoxaline-2,3-dione and a 2,4-diaminoquinazoline (Figure 8), were found to bind the TAR RNA in different locations, and one demonstrated antiviral activity in a cellular assay.82 Although this work has not resulted in movement of an RNA-binding drug to clinical trials, it clearly demonstrates that high-throughput screening approaches can be successfully applied to structured RNA drug targets. Computational screening has been used to identify compounds that should bind to the 50 bulge of TAR RNA. An acetylpromazine (Figure 8) lead was identified that inhibited Tat protein binding to TAR RNA at a 100 nM concentration. The nuclear magnetic resonance (NMR) structure of TAR RNA with the ligand showed that binding of the ligand to the bulge induces a modest conformational change but prevents stacking of the upper and lower stems of the RNA via intercalation at the bulge.83

2.26.4.4.4 Berberine Berberine (Figure 8) is an alkaloid present in a number of clinically important medicinal plants, extracts of which have been used in Chinese medicine for many years, predominantly as antimicrobial treatments. Recently, berberine has been shown to lower cholesterol in animal models of hyperlipidemia, as well as in humans.84 This activity has been attributed to the stabilization of the mRNA for low-density lipoprotein receptor (LDLR), resulting in increased production of protein. The LDLR receptor regulates plasma LDL cholesterol levels, and increased expression of LDLR receptor results in increased clearance of LDL cholesterol. That berberine was having a direct effect on the mRNA was further shown by mutational studies in a reporter system where the stabilization effect was mapped to the 50 proximal region of the LDLR 30 UTR. The precise biochemical interactions that underlie berberine’s effects are unknown. However, the observed clinical and mechanism of action data strongly support that increasing the expression of a protein via stabilization of an mRNA is a viable means to achieve a therapeutic result in patients.

2.26.5 2.26.5.1

New Directions Riboswitches

Riboswitches are metabolite-binding domains present within certain mRNAs.85,86 To date riboswitches have been identified as occuring in bacterial, archaeal, fungal and plant mRNAs. Although not identified to date, it is possible that

Nucleic Acids (Deoxyribonucleic Acid and Ribonucleic Acid)

they will also be found to occur in mammalian mRNAs as well. Riboswitches typically contain two domains, an aptameric domain responsible for binding the metabolite and another domain responsible for genetic regulation. Examples of riboswitch ligands include glycine, coenzyme B12, thiamine, flavin mononucleotides, S-adenosylmethionine, and guanine.87,88 As such, riboswitches represent unique target opportunities for drugs. It should, in principle, be possible to identify agonists and antagonists which modulate riboswitch function in an analogous manner to protein receptors. However, studies validating such an approach have yet to be published.

2.26.5.2

Signal Recognition Particle and tmRNA

Additional large RNA targets that may be good therapeutic targets are the bacterial tmRNA and signal recognition particle (SRP) RNA. Both adopt complex tertiary folds stabilized through interactions with the ribosome or cognate proteins. The SRP RNA binds to a stalled ribosome through an unknown structure present in the mRNA for membrane proteins, and translocates the ribosomal machinery to the cell wall to facilitate insertion of hydrophobic proteins directly into the membrane. Knockout of SRP results in bacterial cell death. The tmRNA is a large (B340 nucleotide) RNA that recognizes bacterial ribosomes that have stalled on a damaged mRNA. The tmRNA provides a pseudo-mRNA that swaps into the 30S subunit to replace the damaged mRNA, then presents a tRNA-like domain to the ribosome to add a short peptide sequence that targets the mRNA for degradation.

2.26.5.3

MicroRNA

MicroRNAs (miRNAs) are a newly discovered class of 20–24 nucleotide RNAs critical to a variety of cellular processes in organisms from yeast to humans.89 They appear to work as inhibitors of translation by binding imperfectly to the 30 UTR of mRNAs as part of a complex with RISC. miRNAs have also been shown to result in reduction of certain target mRNAs, and it appears that they may also play roles in the transcriptional regulation of gene expression via DNA methylation90,91 and heterochromatin formation.92,93 miRNAs are generated from structured regions of transcribed RNAs called pri-miRNAs through the action of the double-stranded RNA nuclease Drosha. The resulting immature hairpin RNAs termed pre-miRNAs are then exported from the nucleus and cleaved in the cytoplasm by another double-stranded RNA nuclease (Dicer) to generate the mature miRNA.49 miRNAs are an attractive new class of therapeutic targets, as they are differentially expressed in tissues, and are associated with tumorigenesis, regulation of adipocyte differentiation, insulin secretion, and activation of immune cells.94–96 Since miRNAs are similar in structure to siRNAs, it is possible to envision the use of siRNA analogs as miRNA mimetics, which would presumably inhibit the expression of the target genes. Perhaps more importantly, inhibition of miRNAs would be expected to increase the expression of its target genes. Inhibition of miRNAs can be achieved with antisense oligonucleotides,97 which provides a straightforward way examine the potential of miRNAs as targets, as well as a means to develop therapeutics. This is exemplified by studies in which inhibition of a liver specific microRNA (miR-122) was achieved in vivo via the use of uniformly modified oligonucleotides, either with98 or without99 cholesterol conjugation. These studies showed upregulation of several target genes, and gave a phenotypic effect of lowered cholesterol and decreased steatohepatitis, suggesting a potential therapeutic indication for microRNA modulation. An additional therapeutic indication for inhibitors of miR-122 is suggested by a connection between miR-122 function and HCV RNA replication, and the observation that inhibition of miR-122 with an antisense oligonucleotide resulted in reduction of HCV RNA levels in an HCV replicon assay.100 Furthermore, the pre-miRNA and pri-miRNA structures contain bulges and mismatched bases at key regions. These structures are potential binding pockets for small molecules which could interfere with their processing and alter miRNA levels.

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Biographies

Eric E Swayze received a BS in chemistry degree from the University of Michigan honors college in 1987, and a PhD in organic chemistry from the University of Michigan in 1994 under the guidance of Prof Leroy B Townsend. In 1994, Dr Swayze joined Isis Pharmaceuticals as a Sr Scientist, where he has focused on developing drugs which act on RNA targets, including both oligonucleotide and small molecule therapeutics. Dr Swayze is currently Vice President of Medicinal Chemistry at Isis Pharmaceuticals of Carlsbad, CA.

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Rich H Griffey is currently a Senior Engineer at SAIC in San Diego. He received a BA in chemistry from Rice University in 1978. His PhD in organic chemistry was obtained in 1983 under the guidance of C Dale Pounter at the University of Utah. After a postdoctoral fellowship with Alfred Redfield, Dr Griffey has held jobs in both academia and industry. His interests include analytical and synthetic chemistry at the chemistry/biology interface, cycling, and astrophotography.

C Frank Bennett is Senior Vice President of Research at Isis Pharmaceuticals. He is responsible for preclinical antisense drug discovery research, manufacturing and pharmaceutics. Dr Bennett is one of the founding members of the company. He has been involved in the development of antisense oligonucleotides as therapeutic agents, including research on the application of oligonucleotides for inflammatory and cancer targets, oligonucleotide delivery, and pharmacokinetics. He also runs the company’s antisense mechanism program which is focused on the development of RNase H, RNAi, micro-RNA, and splicing. Dr Bennett has published more than 120 papers in the field of antisense research and development and has more than 100 issued US patents. Prior to joining Isis, Dr Bennett was Associate Senior Investigator in the Department of Molecular Pharmacology at Smith Kline and French Laboratories (currently GlaxoSmithKline). Dr Bennett received his BS degree in pharmacy from the University of New Mexico, Albuquerque, New Mexico and his PhD in pharmacology from Baylor College of Medicine, Houston, Texas. Dr Bennett performed his postdoctoral research in the Department of Molecular Pharmacology at SmithKline and French Laboratories.

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Comprehensive Medicinal Chemistry II ISBN (set): 0-08-044513-6 ISBN (Volume 2) 0-08-044515-2; pp. 1037–1052