Identification of allosteric inhibitors blocking the hepatitis C virus polymerase NS5B in the RNA synthesis initiation step.
Hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B constitutes a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric inhibitors of NS5B, very few are active in clinical applications. We have screened 17,000 compounds in an enzymatic assay involving the purified NS5B in order to increase the therapeutic arsenal. We hoped to shed some light on the precise mechanism of RNA synthesis. We succeeded in isolating a series of 21 original inhibitors of the RNA synthesis by NS5B. Four of these non-nucleoside inhibitors (NNIs) could be mapped to the known binding site called 'B' as judged by the decrease in their inhibition potency when assayed with a 'B' site mutant, M423T NS5B. Incidentally, our in silico model pointed to Y477 as a key residue for inhibitor binding. In vitro, Y477F mutant loses its sensitivity to the newly discovered inhibitors but is unable to extend primers during the elongation phase. Our results demonstrate that elements of the 'B' site are involved in the conformational changes required in the switch between the different RNA synthesis steps and that compounds targeting this site could lock the enzyme in its initiation phase.
Betzi
Stéphane
S
Centre National de la Recherche Scientifique (CNRS), Laboratoire IMR, FRE 3083, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
Eydoux
Cécilia
C
Bussetta
Cécile
C
Blemont
Marilyne
M
Leyssen
Pieter
P
Debarnot
Claire
C
Ben-Rahou
Mohamed
M
Haiech
Jacques
J
Hibert
Marcel
M
Gueritte
Françoise
F
Grierson
David S
DS
Romette
Jean-Louis
JL
Guillemot
Jean-Claude
JC
Neyts
Johan
J
Alvarez
Karine
K
Morelli
Xavier
X
Dutartre
Hélène
H
Canard
Bruno
B
D000495:Allosteric Site; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D016174:Hepacivirus / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D019384:Nucleic Acid Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
D019384:Nucleic Acid Synthesis Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase