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

• Published in: The Journal of biological chemistry 2006-02, Vol.281 (7), p.3793-3799
• Main Authors: Klumpp, Klaus, Lévêque, Vincent, Le Pogam, Sophie, Ma, Han, Jiang, Wen-Rong, Kang, Hyunsoon, Granycome, Caroline, Singer, Margaret, Laxton, Carl, Hang, Julie Qi, Sarma, Keshab, Smith, David B., Heindl, Dieter, Hobbs, Chris J., Merrett, John H., Symons, Julian, Cammack, Nick, Martin, Joseph A., Devos, Rene, Nájera, Isabel
• Format: Article
• Language: English
• Subjects: Antiviral Agents - pharmacology; Cell Line; Cytidine - analogs & derivatives; Cytidine - pharmacology; Hepacivirus - drug effects; Hepacivirus - physiology; Hepatitis C virus; Humans; RNA, Viral - biosynthesis; RNA-Dependent RNA Polymerase - antagonists & inhibitors; Viral Nonstructural Proteins - antagonists & inhibitors; Virus Replication - drug effects
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M510195200

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 (IC50 = 1.28 μm) with similar potency compared with 2′-C-methylcytidine (IC50 = 1.13 μm). 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 Ki 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.