Specific inhibitors of HCV polymerase identified using an NS5B with lower affinity for template/primer substrate.
The interaction of the hepatitis C virus (HCV) RNA-dependent RNA polymerase with RNA substrate is incompletely defined. We have characterized the activities of the HCV NS5B polymerase, modified by different deletions and affinity tags, with a routinely used homopolymeric substrate, and established apparent affinities of the various NS5B constructs both for the NTP and the template/primer substrates. We identified a uniquely tagged HCV NS5B RNA polymerase construct with a lower affinity (higher K(m)) than mature HCV NS5B for template/ primer substrate and highlighted the use of such a polymerase for the identification of inhibitors of NS5B activity, particularly inhibitors of productive RNA binding. The characterization of specific benzimidazole-5-carboxamide-based inhibitors, identified in a screening campaign, revealed that this class of compounds was non-competitive with regard to NTP incorporation and had no effect on processive elongation, but inhibited an initiation phase of the HCV polymerase activity. The potency of these compounds versus a panel of different NS5B polymerase constructs was inversely proportional to the enzymes' affinities for template/primer substrate. The benzimidazole-5-carboxamide compounds also inhibited the full-length, untagged NS5B de novo initiation reaction using HCV 3'-UTR substrate RNA and expand the diversifying pool of potential HCV replication inhibitors.
McKercher
Ginette
G
Department of Biological Sciences, Boehringer Ingelheim Canada Ltd R&D, 2100 Cunard Street, Laval, Québec H7S 2G5, Canada.
Beaulieu
Pierre L
PL
Lamarre
Daniel
D
LaPlante
Steven
S
Lefebvre
Sylvain
S
Pellerin
Charles
C
Thauvette
Louise
L
Kukolj
George
G
D000577:Amides / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology; D002417:Cattle; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D020128:Inhibitory Concentration 50; D007700:Kinetics; D017955:Poliovirus / Q000201:enzymology; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects
D000577:Amides; D001562:Benzimidazoles; D004791:Enzyme Inhibitors; C034477:RNA primers; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012313:RNA; D012319:RNA Polymerase II; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase