2--c-methylcytidine and 4--azidocytidine

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.

Topics: Antiviral Agents; Cytidine; Deoxycytidine; Drug Resistance, Viral; Hepacivirus; Nucleosides; Oligopeptides; Protease Inhibitors; Replicon; Serine Endopeptidases; Viral Nonstructural Proteins; Virus Replication

A series of 4'-substituted ribonucleoside derivatives has been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The most potent and non-cytotoxic derivative was compound 28 (4'-azidocytidine, R1479) with an IC(50) of 1.28 microM in the HCV replicon system. The triphosphate of compound 28 was prepared and shown to be an inhibitor of RNA synthesis mediated by NS5B (IC(50)=320 nM), the RNA polymerase encoded by HCV. Data on related analogues have been used to generate some preliminary requirements for activity within this series of nucleosides.

Topics: Antiviral Agents; Chemistry, Pharmaceutical; Cytidine; Drug Design; Drug Evaluation, Preclinical; Hepacivirus; Inhibitory Concentration 50; Models, Chemical; Molecular Conformation; Nucleosides; Ribonucleosides; RNA; Uridine; Virus Replication

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.

Topics: Antiviral Agents; Cell Line; Cytidine; Drug Resistance, Viral; Genome, Viral; Hepacivirus; Humans; Interferon-alpha; Molecular Structure; RNA, Viral; Virus Replication