Discovery of new scaffolds for rational design of HCV NS5B polymerase inhibitors

Abstract

Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of anti-HCV drugs. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-based virtual screening and in vitro NS5B inhibition assays. One hundred and sixty thousand compounds from the Otava database were virtually screened against the thiazolone inhibitor binding site on NS5B (thumb pocket-2, TP-2), resulting in a sequential down-sizing of the library by 2.7 orders of magnitude to yield 59 NS5B non-nucleoside inhibitor (NNI) candidates. In vitro evaluation of the NS5B inhibitory activity of the 59 selected compounds resulted in a 14% hit rate, yielding 8 novel structural scaffolds. Of these, compound 1 bearing a 4-hydrazinoquinazoline scaffold was the most active (IC(50) = 16.0 μM). The binding site of all 8 NNIs was mapped to TP-2 of NS5B as inferred by a decrease in their inhibition potency against the M423T NS5B mutant, employed as a screen for TP-2 site binders. At 100 μM concentration, none of the eight compounds exhibited any cytotoxicity, and all except compound 8 exhibited between 40 and 60% inhibition of intracellular NS5B polymerase activity in BHK-NS5B-FRLuc reporter cells. These inhibitor scaffolds will form the basis for future optimization and development of more potent NS5B inhibitors.

Fig. 1

Binding mode of NS5B inhibitors and schematic representation of the binding site occupancy. SP1 to SP5 are the five subpockets mapped in the TP-2 of NS5B. G1 to G4 represent the four groups in which the inhibitors were placed, based on their occupancy of one or more distinct pockets. The compounds are represented by their respective numbers 1 to 8. Coloured ovals indicate subpockets appropriate for structural optimization of the inhibitor. Grey ovals reflect the area occupied by the inhibitors. Intermolecular hydrogen bonds/ionic bonds are shown as green dotted lines.