title: 1a/1b subtype profiling of nonnucleoside polymerase inhibitors of hepatitis C virus.
abstract: The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.
authors.affiliation: HCV Research, Tibotec, 2800 Mechelen, Belgium.
authors.forename: Origène
authors.initials: O
authors.lastname: Nyanguile
authors.forename: Benoit
authors.initials: B
authors.lastname: Devogelaere
authors.forename: Leen
authors.initials: L
authors.lastname: Vijgen
authors.forename: Walter
authors.initials: W
authors.lastname: Van den Broeck
authors.forename: Frederik
authors.initials: F
authors.lastname: Pauwels
authors.forename: Maxwell D
authors.initials: MD
authors.lastname: Cummings
authors.forename: Hendrik L
authors.initials: HL
authors.lastname: De Bondt
authors.forename: Ann M
authors.initials: AM
authors.lastname: Vos
authors.forename: Jan M
authors.initials: JM
authors.lastname: Berke
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
authors.forename: Geneviève
authors.initials: G
authors.lastname: Vandercruyssen
authors.forename: Katrien
authors.initials: K
authors.lastname: Vermeiren
authors.forename: Wendy
authors.initials: W
authors.lastname: Mostmans
authors.forename: Pascale
authors.initials: P
authors.lastname: Dehertogh
authors.forename: Frédéric
authors.initials: F
authors.lastname: Delouvroy
authors.forename: Sandrine
authors.initials: S
authors.lastname: Vendeville
authors.forename: Koen
authors.initials: K
authors.lastname: VanDyck
authors.forename: Koen
authors.initials: K
authors.lastname: Dockx
authors.forename: Erna
authors.initials: E
authors.lastname: Cleiren
authors.forename: Pierre
authors.initials: P
authors.lastname: Raboisson
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Simmen
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Fanning
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000627:therapeutic use*; D001569:Benzodiazepines / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006526:Hepatitis C / Q000188:drug therapy*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000502:physiology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D001569:Benzodiazepines; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase