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Journal of Virology, December 2003, p. 13225-13231, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13225-13231.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mechanism of Action and Antiviral Activity of Benzimidazole-Based Allosteric Inhibitors of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Licia Tomei,* Sergio Altamura, Linda Bartholomew, Antonino Biroccio,{dagger} Alessandra Ceccacci, Laura Pacini, Frank Narjes, Nadia Gennari, Monica Bisbocci, Ilario Incitti, Laura Orsatti, Steven Harper, Ian Stansfield, Michael Rowley, Raffaele De Francesco, and Giovanni Migliaccio

Istituto di Ricerche di Biologia Molecolare "P. Angeletti," 00040 Pomezia-Rome, Italy

Received 16 June 2003/ Accepted 2 September 2003

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTP-binding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase.

* Corresponding author. Mailing address: Istituto di Ricerche di Biologia Molecolare "P. Angeletti," via Pontina Km 30,600, 00040 Pomezia-Rome, Italy. Phone: 39 06 91093230. Fax: 39 06 91093225. E-mail: Licia_Tomei{at}Merck.com.

{dagger} Present address: Clinical Biochemistry Laboratory, Children Hospital "Bambin Gesú," IRCCS, Vatican State-Rome, Italy.

Journal of Virology, December 2003, p. 13225-13231, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13225-13231.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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