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

George Kukolj,Graham A Mcgibbon,Ginette Mckercher,Martin Marquis,Sylvain Lefèbvre,Louise Thauvette,Jean Gauthier,Sylvie Goulet,Marc-André Poupart,Pierre L Beaulieu

doi:10.1074/jbc.m506407200

George Kukolj, Graham A Mcgibbon + Show 8 more

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https://doi.org/10.1074/jbc.m506407200

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Abstract

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: Pro(495) with substitutions to Ser, Leu, Ala, or Thr; Pro(496) 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 Pro(495) changes provided the greatest shifts in compound potency, followed by moderate changes in potency with the Pro(496) 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.

Highlights

Unmet clinical need and has focused on inhibitors of virally encoded functions
In an effort to further characterize the benzimidazole class of NS5B inhibitors and map the location of inhibitor binding, we describe cross-linking experiments with a photoreactive analogue followed by MS analysis of the enzyme that localize a putative binding site for these inhibitors at the junction of the thumb domain and the NS5B N-terminal ␭1 loop
Based on the accumulated structure-activity relationship with this class of inhibitors, we have shown that the compounds tolerate extensive substitutions off the left-hand side imidazole portion without significantly affecting their inhibition of NS5B (27)

Summary

Introduction

Unmet clinical need and has focused on inhibitors of virally encoded functions. HCV encodes a linear polyprotein of ϳ3010 amino acids that is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins (for review, see Ref. 3). A distinct feature of the HCV polymerase (and closely related RNA-dependent RNA polymerase) active site cavity is the protrusion of a unique ␤-hairpin from the thumb subdomain that apparently plays a role in the initiation of de novo RNA synthesis as demonstrated by both structural and biochemical studies (8 –11). One promiscuous pocket on the thumb domain binds at least three different classes of non-nucleoside inhibitors: the thiophene 2-carboxylic acids, phenylalanine derivatives, and cyclopentyl dihydropyran-2-ones (23, 24) classes of compounds all bind to a pocket with a central Met423 residue Another class of compounds are exemplified by benzothiadiazines that give rise to resistant replicon mutants at position Met414 (25, 26), which is located at the base of the palm and thumb domain in the active site cleft. The compounds have a unique resistance profile and preliminary resistance studies with HCV replicons identified substitutions in one residue, Pro495 in the NS5B thumb domain, as conferring resistance to this class of inhibitor (33)

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