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 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.