The Novel Nucleoside Analog R1479 (4′-Azidocytidine) Is a Potent Inhibitor of NS5B-dependent RNA Synthesis and Hepatitis C Virus Replication in Cell Culture

Klumpp, Klaus; Lévêque, Vincent; Pogam, Sophie Le; Ma, Han; Jiang, Wen-Rong; Kang, Hyunsoon; Granycome, Caroline; Singer, Margaret; Laxton, Carl; Hang, Julie Q.; Sarma, Keshab; Smith, David B.; Heindl, Dieter; Hobbs, Chris; Merrett, John H.; Symons, Julian; Cammack, Nick; Martin, Joseph A.; Devos, René; Nájera, Isabel

doi:10.1074/jbc.m510195200

Cited by 203 publications

(188 citation statements)

References 25 publications

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“…Drug susceptibility studies and selection experiments with the replicon system have shown that a single mutation, i.e., S282T, confers reduced susceptibility to 2Ј-C-Me-adenosine. The S282T mutation confers cross-resistance to other 2Ј-C-Me nucleotides (18,24). Modeling studies and enzyme kinetic analysis suggested that this mutation can affect binding 1 to 7, respectively).…”

Section: Resultsmentioning

confidence: 99%

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

Deval¹,

Powdrill²,

D’Abramo³

et al. 2007

Antimicrob Agents Chemother

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Nonobligate chain terminators, such as 2-C-methylated nucleotides, block RNA synthesis by the RNAdependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3-hydroxyl group can be excised in the presence of pyrophosphate (PP i ), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.Hepatitis C virus (HCV) infection is a serious public health concern that affects 170 million people worldwide (33, 42). Among those infected, approximately 20 to 30% develop severe liver disease, such as chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (2). The combined use of the nucleoside analogue ribavirin and pegylated alpha interferon is the current treatment standard; however, success in treatment depends largely on the viral genotype, and this drug combination has also been associated with severe side effects (30,43). Thus, the development of novel, more potent, specific drugs is urgent.HCV belongs to the Flaviviridae family. The HCV RNA genome consists of approximately 10 kb, encoding a polyprotein which is processed into several smaller polypeptides, including the capsid protein (C), the envelope proteins (E1 and E2), and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Initial cis cleavage through NS2-NS3 releases the NS3 protein, which in turn continues to process the precursor. Promising compounds with the ability to inhibit the viral protease (NS3) and the polymerase (NS5B) have been identified.NS5B is a 65-kDa RNA-dependent RNA polymerase capable of initiating RNA synthesis de novo in the absence of a primer (16,17,25,27,45). Three classes of inhibitors of HCV NS5B have been developed, namely, nucleoside analogue inhibitors, nonnucleoside analogue inhibitors, and pyrophosphate (PP i ) analogues. Nonnucleoside inhibitors and PP i analogues are still under preclinical evaluation, while a 2Ј-modified nucleoside analogue has advanc...

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Section: Resultsmentioning

confidence: 99%

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

Deval¹,

Powdrill²,

D’Abramo³

et al. 2007

Antimicrob Agents Chemother

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“…HCV Replicon Assays-HCV replicon assays were performed using either the 2209-23 cell line or the transient replicon system in cured Huh-7 cells as described (6,19). Nucleoside analogs were synthesized at Medivir, dissolved in Me 2 SO, and then diluted in Dulbecco's modified Eagle's medium with 5% (v/v) fetal bovine serum before addition to cells.…”

Section: Methodsmentioning

confidence: 99%

“…The RNA template was predicted to form a single stemloop with an unpaired 3-nucleotide sequence at the 3Ј-end. The nucleotide incorporation reactions and RNA product separation on denaturing polyacrylamide gel were conducted as described (6), except that the reactions were carried out with 0.25 M GG primer and nucleotide triphosphates at the indicated concentrations for 30 min. The kinetics of the single nucleotide incorporation of CTP and CTP analogs were determined with the same RNA oligonucleotide template and primer pair for 25 min at the following assay conditions: 7.5 M 19-nucleotide RNA template, 10 M unlabeled GG primer, 0.075 M 5Ј-end-labeled GG primer, 3 M NS5B, and serial dilutions of CTP or each CTP analog.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, R1479 was identified as a potent and selective inhibitor of HCV replication in cell culture (6,7). R1626, a prodrug of 4Ј-azidocytidine, is presently in phase II clinical development.…”

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

“…However, the incorporation efficiency of ara-CTP was 140-fold lower as compared with CTP, in line with an important interaction of the 2Ј-␣-hydroxy group. Earlier, we discovered that 4Ј-substituents on ribonucleoside analogs may pick up additional binding interactions in the HCV polymerase active site, with 4Ј-azido being the most effective to allow productive binding to both HCV polymerase and human nucleoside kinases (6,7,9). The addition of a 4Ј-azido moiety to the ara-CTP molecule resulted in an unexpected 15-30-fold increase in inhibitory potency of 2Ј-␣-deoxy-2Ј-␤-hydroxy-4Ј-azido-CTP (RO-9187-TP) as compared with 2Ј-␣-deoxy-ara-CTP.…”

Section: ј-Substitution Can Increase the Inhibitory Potency Of 2ј-␣-mentioning

confidence: 99%

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2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

Klumpp

Kalayanov

et al. 2008

Journal of Biological Chemistry

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RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2-␣-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of ␣-hydroxy moieties. 2-Deoxy-2-␤-fluoro-4-azidocytidine (RO-0622) and 2-deoxy-2-␤-hydroxy-4-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC 50 ‫؍‬ 171 ؎ 12 nM and 24 ؎ 3 nM for RO-9187 and RO-0622, respectively; CC 50 >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4-azidocytidine) or 2-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC 50 values 8 -150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2-␣-deoxy-4-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection. Hepatitis C virus (HCV)3 infection is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation. Current treatment options available to HCV-infected persons have limitations with regard to efficacy and tolerability. Only about 50% of individuals infected with HCV genotype 1 achieve sustained virological response when treated with a combination of pegylated interferon ␣ and ribavirin (1, 2). In addition, high viral load, age, body weight, co-infection with human immunodeficiency virus, and cirrhosis negatively affect the probability of achieving sustained virological response (3, 4). Therefore, there is an urgent need to develop new and more effective therapies for the treatment of HCV infection. A number of new antiviral candidates are currently being evaluated in clinical studies, the majority targeting either the HCV protease or HCV polymerase enzymes, which are essential for viral replication (5). The HCV RNA-dependent RNA polymerase, NS5B, contains the active site responsible for viral RNA synthesis and functions as part of a membrane-associated replicase complex. Nucleoside and non-nucleoside inhibitors of HCV polymerase h...

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Recent Advances in HepatitisCTherapies

Jonathan

Christoph

Levin

et al. 2010

Burger's Medicinal Chemistry and Drug Discovery

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The Novel Nucleoside Analog R1479 (4′-Azidocytidine) Is a Potent Inhibitor of NS5B-dependent RNA Synthesis and Hepatitis C Virus Replication in Cell Culture

Cited by 203 publications

References 25 publications

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

Recent Advances in HepatitisCTherapies

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