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Antimicrobial Agents and Chemotherapy, December 2006, p. 4103-4113, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00365-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Mechanism of a Thumb Domain Hepatitis C Virus Nonnucleoside RNA-Dependent RNA Polymerase Inhibitor

Anita Y. M. Howe,^1* Huiming Cheng,¹ Ian Thompson,¹ Srinivas K. Chunduru,^3, Steve Herrmann,² John O'Connell,¹ Atul Agarwal,^2, Rajiv Chopra,² and Alfred M. Del Vecchio^3,

Infectious Diseases, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426,¹ Biological Technology and Structural Biology, Wyeth Research, 85 Bolton Street, Cambridge, Massachusetts 02140,² ViroPharma Incorporated, 405 Eagleview Boulevard, Exton, Pennsylvania 19341³

Received 24 March 2006/ Returned for modification 26 May 2006/ Accepted 21 August 2006

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 ß-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 µM. 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.

Published ahead of print on 28 August 2006.

Present address: TetraLogic Pharmaceuticals, 365 Phoenixville Pike, Malvern, PA 19355.

Present address: Centocor, Inc., 145 King of Prussia Road, Radnor, PA 19087.

Present address: Achillion Pharmaceutical Inc., 300 George Street, New Haven, CT 06511.

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