title: Molecular mechanism of a thumb domain hepatitis C virus nonnucleoside RNA-dependent RNA polymerase inhibitor.
abstract: A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique beta-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8- to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 microM. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.
authors.affiliation: Infectious Diseases, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA. howeaym@wyeth.com
authors.forename: Anita Y M
authors.initials: AY
authors.lastname: Howe
authors.forename: Huiming
authors.initials: H
authors.lastname: Cheng
authors.forename: Ian
authors.initials: I
authors.lastname: Thompson
authors.forename: Srinivas K
authors.initials: SK
authors.lastname: Chunduru
authors.forename: Steve
authors.initials: S
authors.lastname: Herrmann
authors.forename: John
authors.initials: J
authors.lastname: O'Connell
authors.forename: Atul
authors.initials: A
authors.lastname: Agarwal
authors.forename: Rajiv
authors.initials: R
authors.lastname: Chopra
authors.forename: Alfred M
authors.initials: AM
authors.lastname: Del Vecchio
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D024882:Drug Resistance, Viral / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D005814:Genes, Viral; D005818:Genetic Engineering; D014644:Genetic Variation; D006160:Guanosine Triphosphate / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors*; D011995:Recombination, Genetic; D012093:Replicon / Q000235:genetics; D012641:Selection, Genetic; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D014779:Virus Replication
chemical_list: D004791:Enzyme Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D006160:Guanosine Triphosphate; D012324:RNA-Dependent RNA Polymerase