title: Phenotypic and structural analyses of hepatitis C virus NS3 protease Arg155 variants: sensitivity to telaprevir (VX-950) and interferon alpha.
abstract: 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 Arg(155) of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg(155) 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 Arg(155) 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 2.5A. 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 Lys(155) side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon alpha or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells.
authors.affiliation: Vertex Pharmaceuticals Inc., Cambridge, Massachusetts 02139, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhou
authors.forename: Ute
authors.initials: U
authors.lastname: Müh
authors.forename: Brian L
authors.initials: BL
authors.lastname: Hanzelka
authors.forename: Doug J
authors.initials: DJ
authors.lastname: Bartels
authors.forename: Yunyi
authors.initials: Y
authors.lastname: Wei
authors.forename: B Govinda
authors.initials: BG
authors.lastname: Rao
authors.forename: Debra L
authors.initials: DL
authors.lastname: Brennan
authors.forename: Ann M
authors.initials: AM
authors.lastname: Tigges
authors.forename: Lora
authors.initials: L
authors.lastname: Swenson
authors.forename: Ann D
authors.initials: AD
authors.lastname: Kwong
authors.forename: Chao
authors.initials: C
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry*; D018360:Crystallography, X-Ray; D020128:Inhibitory Concentration 50; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D010641:Phenotype; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D012254:Ribavirin / Q000737:chemistry*; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D001120:Arginine; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D009842:Oligopeptides; D011092:Polyethylene Glycols; D011994:Recombinant Proteins; D012254:Ribavirin; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C486464:telaprevir; C100416:peginterferon alfa-2a