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J. Biol. Chem., Vol. 280, Issue 47, 39260-39267, November 25, 2005

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Binding Site Characterization and Resistance to a Class of Non-nucleoside Inhibitors of the Hepatitis C Virus NS5B Polymerase^*

From the Departments of Biological Sciences and Chemistry, Boehringer Ingelheim (Canada) Ltd., Research and Development, Laval, Québec H7S 2G5,Canada

The virally encoded NS5B RNA-dependent RNA polymerase has emerged as a prime target in the search for specific HCV antivirals. A series of benzimidazole 5-carboxamide compounds inhibit the cellular RNA replication of a HCV subgenomic replicon and we have advanced our understanding of this class of inhibitors through a combination of complementary approaches that include biochemical cross-linking experiments with a photoreactive analogue followed by mass spectrometry analysis of the enzyme. A novel binding site has been localized for these inhibitors at the junction of the thumb domain and the N-terminal finger loop. Furthermore, the isolation and characterization of resistant replicon mutants that co-localize to this region distinguished this class of compounds from other non-nucleoside NS5B inhibitors that bind to distinct allosteric sites. Resistant mutations that emerged with the benzimidazole 5-carboxamide and related compounds were found at three amino acid positions in the thumb domain: Pro⁴⁹⁵ with substitutions to Ser, Leu, Ala, or Thr; Pro⁴⁹⁶ substitutions to Ser or Ala; and a V499A substitution. Mutations at each of these positions conferred different levels of resistance to this drug class: the Pro⁴⁹⁵ changes provided the greatest shifts in compound potency, followed by moderate changes in potency with the Pro⁴⁹⁶ substitutions, and finally only minor shifts in potency with V499A. Combinations that include the benzimidazole 5-carboxamide polymerase inhibitors and compounds that bind other sites or other HCV targets, including HCV protease inhibitors, are complementary in cell culture models of HCV RNA replication at suppressing the emergence of resistant variants. This novel class of compounds and unique binding site expand the diversity of HCV antivirals currently under development and offer the potential to improve the treatment of chronic HCV infection.

Received for publication, June 13, 2005 , and in revised form, September 9, 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: 2100 rue Cunard Laval, Québec H7S 2G5, Canada. Tel.: 450-682-4640; Fax: 450-682-4642; E-mail: gkukolj{at}lav.boehringer-ingelheim.com.

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