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J. Biol. Chem., Vol. 281, Issue 7, 3793-3799, February 17, 2006

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

Klaus Klumpp¹, Vincent Lévêque, Sophie Le Pogam, Han Ma, Wen-Rong Jiang, Hyunsoon Kang, Caroline Granycome, Margaret Singer, Carl Laxton, Julie Qi Hang, Keshab Sarma, David B. Smith, Dieter Heindl, Chris J. Hobbs, John H. Merrett, Julian Symons, Nick Cammack, Joseph A. Martin, Rene Devos, and Isabel Nájera

From the Roche Palo Alto LLC, Palo Alto, California 94304 and Roche Diagnostics GmbH, 82377 Penzberg, Germany

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 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.

Received for publication, September 16, 2005 , and in revised form, November 28, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Roche Palo Alto LLC, 3431 Hillview Ave., Palo Alto, CA 94304. Tel.: 650-855-6028; Fax: 650-354-7554; E-mail: klaus.klumpp{at}roche.com.

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