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J. Biol. Chem., Vol. 282, Issue 31, 22619-22628, August 3, 2007

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Phenotypic and Structural Analyses of Hepatitis C Virus NS3 Protease Arg¹⁵⁵ Variants

SENSITIVITY TO TELAPREVIR (VX-950) AND INTERFERON ^*

From the Vertex Pharmaceuticals Inc., Cambridge, Massachusetts 02139

Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3·4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg¹⁵⁵ of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg¹⁵⁵ was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg¹⁵⁵ with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5Å. The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys¹⁵⁵ side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells.

Received for publication, November 1, 2006 , and in revised form, May 24, 2007.

The atomic coordinates and structure factors (code 2OIN) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Schemes 1 and 2 and Fig. 1.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Infectious Diseases, Vertex Pharmaceuticals Inc., 130 Waverly St., Cambridge, MA 02139. Tel.: 617-444-6202; Fax: 617-444-6210; E-mail: chao_lin{at}vrtx.com.

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