1a/1b subtype profiling of nonnucleoside polymerase inhibitors of hepatitis C virus.
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.
Nyanguile
Origène
O
HCV Research, Tibotec, 2800 Mechelen, Belgium.
Devogelaere
Benoit
B
Vijgen
Leen
L
Van den Broeck
Walter
W
Pauwels
Frederik
F
Cummings
Maxwell D
MD
De Bondt
Hendrik L
HL
Vos
Ann M
AM
Berke
Jan M
JM
Lenz
Oliver
O
Vandercruyssen
Geneviève
G
Vermeiren
Katrien
K
Mostmans
Wendy
W
Dehertogh
Pascale
P
Delouvroy
Frédéric
F
Vendeville
Sandrine
S
VanDyck
Koen
K
Dockx
Koen
K
Cleiren
Erna
E
Raboisson
Pierre
P
Simmen
Kenneth A
KA
Fanning
Gregory C
GC
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
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