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Published online 22 January 2004

Nucleic Acids Research, 2004, Vol. 32, No. 2 422-431
© 2004 Oxford University Press

Specific inhibitors of HCV polymerase identified using an NS5B with lower affinity for template/primer substrate

Ginette McKercher, Pierre L. Beaulieu1, Daniel Lamarre, Steven LaPlante1, Sylvain Lefebvre, Charles Pellerin, Louise Thauvette and George Kukolj*

Department of Biological Sciences and 1 Department of Chemistry, Boehringer Ingelheim Canada Ltd R&D, 2100 Cunard Street, Laval, Québec H7S 2G5, Canada

*To whom correspondence should be addressed. Tel: +1 450 682 4640; Fax: +1 450 682 4642; Email: gkukolj{at}lav.boehringer-ingelheim.com

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 Km) 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.


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