Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC 50 ‫؍‬ 1.28 M) with similar potency compared with 2-C-methylcytidine (IC 50 ‫؍‬ 1.13 M). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K i of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2-C-MeATP and other 2-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479. Hepatitis C virus (HCV)2 infection is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is currently the leading cause of liver transplantation (1, 2). Viral genome sequence analysis established six HCV genotype classes (HCV genotypes 1-6), with genotypes 1-3 being the most prevalent in the United States, Europe, and Japan. Current treatment options available to HCV-infected persons are limited, and sustained virological response rates are particularly low for HCV genotype 1-infected patients. Only ϳ50% of individuals infected with HCV genotype 1 with serum viral titers of Ͼ2 ϫ 10 6 copies/ml achieved sustained virological response rates when treated with a combination of pegylated interferon-␣ and ribavirin (3, 4). Response rates are even lower in persons with HIV co-infection or cirrhosis and also decrease with age (1, 5-7). Urgently required improvements in anti-HCV therapy will depend on the development of novel therapeutic approaches, especially in difficult to treat populations.HCV is an enveloped (ϩ)-strand RNA virus that enters host cells via receptor-mediated endocytosis and replicates in the host cell cytoplasm. A membrane-associated replicase complex containing HCV genome-encoded nonstructural proteins and HCV genomic RNA in a tight complex is responsible for the formation of viral RNA for packaging into new virus particles during the HCV replication process. The viral NS5B protein contains the HCV polymerase active site within the replicase complex, an RNA-dependent RNA polymerase. The concept of polymerase inhibition to attain antiviral efficacy has been successfully established in other viral infections (human immunodefi...

Specific inhibitors of hepatitis C virus (HCV) replication that target the NS3/4A protease (e.g., VX-950) or the NS5B polymerase (e.g., R1479/R1626, PSI-6130/R7128, NM107/NM283, and HCV-796) have advanced into clinical development. Treatment of patients with VX-950 or HCV-796 rapidly selected for drug-resistant variants after a 14-day monotherapy treatment period. However, no viral resistance was identified after monotherapy with R1626 (prodrug of R1479) or NM283 (prodrug of NM107) after 14 days of monotherapy. Based upon the rapid selection of resistance to the protease and nonnucleoside inhibitors during clinical trials and the lack of selection of resistance to the nucleoside inhibitors, we used the replicon system to determine whether nucleoside inhibitors demonstrate a higher genetic barrier to resistance than protease and nonnucleoside inhibitors. Treatment of replicon cells with nucleoside inhibitors at 10 and 15 times the 50% effective concentration resulted in clearance of the replicon, while treatment with a nonnucleoside or protease inhibitor selected resistant colonies. In combination, the presence of a nucleoside inhibitor reduced the frequency of colonies resistant to the other classes of inhibitors. These results indicate that the HCV replicon presents a higher barrier to the selection of resistance to nucleoside inhibitors than to nonnucleoside or protease inhibitors. Furthermore, the combination of a nonnucleoside or protease inhibitor with a nucleoside polymerase inhibitor could have a clear clinical benefit through the delay of resistance emergence.Hepatitis C virus (HCV) is a positive-strand RNA virus that is a member of the Hepacivirus genus within the Flaviridae family. There are an estimated 170 million individuals chronically infected with HCV worldwide, which amounts to almost 3% of the global population (1). In the United States, an estimated 20,000 new HCV infections occurred in 2005, adding to the approximately 4 million individuals previously infected with HCV (2, 38). Liver cirrhosis, as a result of HCV infection, is currently the leading reason justifying liver transplantation; however, reinfection occurs immediately posttransplantation and can result in graft loss (39).The current treatment of pegylated alpha interferon in combination with ribavirin results in a sustained viral response in approximately 50% of HCV patients infected with genotype (GT) 1 virus, the most prevalent GT worldwide. Therefore, a specific HCV antiviral therapy is highly desirable. Viral proteases and viral polymerases have been validated as clinically effective targets for a number of different viruses, including human immunodeficiency virus, hepatitis B virus, and herpesviruses (6,7,14,15). Two potential drug targets encoded by HCV are the NS3/4A serine protease and the NS5B RNA-dependent RNA polymerase (5). Several anti-HCV compounds that inhibit the activity of either the NS3/4A protease or the NS5B RNA-dependent RNA polymerase have resulted in decreased viral loads when administered to HCV-infected pat...

The HCV polymerase is an attractive target for the development of new and specific anti-HCV drugs. Herein, the characterization of the inhibitory effect of 2'-C-Methyl-Cytidine shows that it is a potent inhibitor of both genotype 1b and 1a HCV replicon replication, both of laboratory-optimized as well as of NS5B clinical isolates-chimera replicons. The corresponding 5'-triphosphate derivative is a potent inhibitor of native HCV replicase isolated from replicon cells and of the recombinant genotype 1b and 1a HCV polymerase-mediated RNA synthesis. Resistance to 2'-C-Methyl-Cytidine was mapped to amino acid substitution S282T in the NS5B coding region. Cross-resistance was observed to 2'-C-Methyl-Adenosine but not to interferon alpha-2a, to non-nucleoside HCV polymerase inhibitors or to R1479, a new and potent nucleoside inhibitor of NS5B polymerase. In vitro studies mapped resistance to R1479 to amino acid substitutions S96T and S96T/N142T of the NS5B polymerase. These mutations did not confer resistance to 2-C-Methyl-Cytidine, thus confirming the lack of cross-resistance between these two HCV inhibitors. These data will allow the optimization of new polymerase inhibitors and their use in combination therapy.

The higher frequency of known NNI resistance mutations or polymorphisms known to affect their antiviral potency when compared with the lack of detection of resistance mutations to the nucleoside analogues suggests a potential for primary reduced responsiveness as well as faster development of clinically significant resistance.

Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV)polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb-and a palmbinding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.Hepatitis C virus (HCV), a positive-strand RNA virus, is a member of the genus Hepacivirus in the Flaviviridae family and is the leading cause of liver disease worldwide. It is estimated that over 170 million individuals are infected with HCV (43). The current standard of care provides good clinical efficacy for patients infected with genotype 2 and 3 but is less efficacious for patients infected with the most prevalent genotype, genotype 1, thereby emphasizing the urgent need for more effective HCV-specific antiviral therapies (15,27).The HCV RNA-dependent RNA polymerase is an essential enzyme for viral RNA replication and represents an attractive therapeutic target. HCV polymerase has the "right-hand" polymerase fold with finger, thumb, and palm domains (22). As with other RNA-dependent RNA polymerases, the extended "fingertips" contact a thicker thumb domain to create an encircled active site constituting the closed, active conformation of the enzyme (7,16,22,32). With the advent of the HCV replicon system there have been extensive developments supporting the discovery of new HCV polymerase nonnucleoside inhibitors (1-3, 5, 6, 11, 36). Several chemical classes of nonnucleoside inhibitors that inhibit the isolated enzyme and replication in the replicon system have been shown to bind at distinct sites on HCV polymerase. These polymerase inhibitors include benzothiadiazines, binding to the palm domain near the active site (38, 40), thiophene carboxylic acids which bind at the...

) is a selective inhibitor of hepatitis C virus (HCV) replication that targets the NS5B polymerase. R7128, the prodrug of PSI-6130, has shown antiviral efficacy in patients chronically infected with HCV genotype 1a (GT-1a) and GT-1b. We observed that the compound exhibited potent in vitro activity against laboratory-optimized HCV replicons as well as against a panel of replicons containing NS5B HCV polymerases derived from GT-1a and GT-1b clinical isolates. We used the HCV replicon cell system to examine the emergence of variants with reduced sensitivity to PSI-6130. Shortterm treatment of cells harboring the HCV subgenomic replicon with PSI-6130 cleared the replicon without generating resistant variants. Long-term culture of the cells under the compound selection generated the S282T substitution in a complex pattern with other amino acid substitutions in the NS5B polymerase. The presence of the coselected substitutions did not increase the moderate three-to sixfold loss of sensitivity to PSI-6130 mediated by the S282T substitution; however, their presence enhanced the replication capacity compared to the replication levels seen with the S282T substitution alone. We also observed a lack of cross-resistance between PSI-6130 and R1479 and demonstrated that long-term culture selection with PSI-6130 in replicon cells harboring preexisting mutations resistant to R1479 (S96T/N142T) results in the emergence of the S282T substitution and the reversion of S96T to wild-type serine. In conclusion, PSI-6130 presents a high barrier to resistance selection in vitro, selects for variants exhibiting only low-level resistance, and lacks cross-resistance with R1479, supporting the continued development of the prodrug R7128 as a therapeutic agent for the treatment of HCV infection.Hepatitis C virus (HCV) is a positive-strand RNA virus and a member of the Hepacivirus genus in the Flaviviridae family. Acute infection with HCV progresses into chronic infection for approximately 80% of infected patients and is a major cause of liver cirrhosis and hepatocellular carcinoma. The current use of pegylated alpha interferon (IFN-␣) in combination with ribavirin for chronic HCV infection treatment (10,14,16) results in a 37 to 50% sustained viral response in patients infected with HCV genotype 1 (GT-1) (15). This suboptimal sustained viral response, combined with the high prevalence of undesirable side effects, has prompted focused efforts to develop specifically targeted antiviral therapies for HCV infections. The HCV NS5B RNA-dependent RNA polymerase (RdRp) is a critical enzyme for viral RNA replication and presents an attractive therapeutic target.

The requirement for a predominant S282T mutant quasispecies, its low replication capacity, and the low-level resistance it confers probably contribute to the lack of RG7128 resistance observed in HCV-infected patients.

We report the synthesis and biological evaluation of a series of 4′-fluoro-2′-C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC50 values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.