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...
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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