In vitro selected Con1 subgenomic replicons resistant to 2′-C-Methyl-Cytidine or to R1479 show lack of cross resistance

Pogam, Sophie Le; Jiang, Wen-Rong; Lévêque, Vincent; Rajyaguru, Sonal; Ma, Han; Kang, Hyunsoon; Jiang, Sharon; Singer, Margaret; Ali, Samir; Klumpp, Klaus; Smith, Dave; Symons, Julian; Cammack, Nick; Nájera, Isabel

doi:10.1016/j.virol.2006.03.045

Cited by 126 publications

(135 citation statements)

References 32 publications

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“…These mutations were previously shown to confer resistance to R1479 and NM107, respectively (19). Therefore, the combination of 4Ј-and 2Ј-␤-substitution in the absence of a 2Ј-␣-substituent resulted in two compounds, which show no apparent cross-resistance with 4Ј-azidocytidine and 2Ј-␤-methylcytidine, respectively.…”

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

confidence: 92%

“…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%

“…RO-9187 and RO-0622 Are Potent Inhibitors of HCV Replication-The antiviral potencies of the novel nucleoside analogs RO-9187 and RO-0622 were determined in the HCV genotype 1b (Con1) subgenomic replicon cell line 2209-23 and in the transient replicon system as described previously (6,19). Both compounds were potent inhibitors of HCV replication in these systems (Table 4).…”

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

confidence: 92%

Section: Methodsmentioning

confidence: 99%

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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“…EC 50 Determinations in the HCV Replicon Assay-The concentration of compound required to inhibit the expression of the HCV replicon reporter by 50% compared with untreated controls (50% effective concentration [EC 50 ]) was determined as described previously using 2209 -2223 stably transfected replicon cells (23), except that Huh7-Lunet cells were used for transient transfections (24). The replication capacity of the transient replicons was determined as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

Hang

Yang

Harris

et al. 2009

Journal of Biological Chemistry

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Hepatitis C virus (HCV)4 constitutes a global health problem. Current therapies are unable to effectively eliminate viral infection in a significant number of patients. The RNA-dependent RNA polymerase (RdRp) of HCV NS5B is an attractive target for the development of orally bioavailable small molecule inhibitors (1, 2). The structure of the NS5B apoenzyme and the NS5B-RNA complex reveals the characteristic right hand architecture of polymerase enzymes, comprising three distinct domains (palm, thumb, and finger) encircling the enzyme active site located in the palm domain (3-6). The structural and biochemical characterization of HCV NS5B polymerase can provide a basis for drug design efforts, and the elucidation of the mechanism of inhibition can guide the optimization of inhibitor efficiency against wild-type and resistant mutants.Among the extensively investigated non-nucleosides documented to inhibit the RdRp activity of HCV NS5B, derivatives of various benzofuran and benzothiadiazine have been reported to bind to allosteric binding sites in the palm domain of NS5B (7,8). The palm domain, whose geometry is conserved in virtually all DNA and RNA polymerases, contains catalytic aspartic acids responsible for the nucleotidyl transfer reaction. The benzofuran compound HCV-796 has been shown to have significant antiviral effects in patients chronically infected with HCV (9, 10). In addition, two series of compounds based on the thiophene and benzimidazole scaffolds have been reported to inhibit NS5B by binding to two different binding pockets in the thumb domain of NS5B (11,12). The thumb domain is connected to the palm domain by a ␤-hairpin termed the primer grip motif. The C-terminal region of the thumb protrudes toward the active site (3). The thumb binding inhibitors have been proposed to inhibit the RdRp activity of NS5B, perhaps by interfering with template/primer interaction and conformational dynamics of the protein (13,14).Despite the elucidation of a number of NNIs that bind to the thumb and palm binding sites, the mechanism by which NNIs cause inhibition of RNA synthesis is unclear. Also, our understanding of the kinetics of NNI interaction with NS5B, the role of NNI binding and kinetics for inhibition, and the inhibitor efficacy on NS5B-resistant mutations remains incomplete. The four representative palm-and thumb-binding NNIs selected in this study have been reported to effectively inhibit replication of subgenomic replicons with low toxicity. Noncompetitive inhibition of NS5B polymerase activity with respect to NTPs has been reported (2, 15, 16). Based on co-crystallization studies with NS5B, it has been proposed that allosteric inhibitors may lock the NS5B protein in an inactive formation by binding tightly to the protein (16,17). It is important to understand how the binding affinity relates to inhibition potency and resistance to HCV inhibition. Because the intrinsic potency of slowly binding compounds can be underestimated in the short time □ S The on-line version of this article (available at htt...

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“…The analysis of inhibition of HCV replication by nucleoside analogs and IC 50 determinations were performed as described (12).…”

Section: Compounds-␤-d-2ј-deoxy-2ј-fluoro-2ј-c-methylcytidinementioning

confidence: 99%

Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

Jiang

Robledo

et al. 2007

Journal of Biological Chemistry

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In vitro selected Con1 subgenomic replicons resistant to 2′-C-Methyl-Cytidine or to R1479 show lack of cross resistance

Cited by 126 publications

References 32 publications

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

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

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

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