Novel Approach to the Study of Small Molecule Interactions with HCV RNA Polymerase

Nikki Carter. Pfizer Ltd, Sandwich Kent, United Kingdom.

The inhibition of HCV RNA polymerase is a common treatment paradigm for the treatment of Hepatitis C Virus infections. Historically, functional assays measuring the elongation of RNA by the polymerase enzyme via the incorporation of radio-labelled nucleotides have been applied to identify new chemical matter. Although a successful method, the literature evidence suggests this assay preferentially identifies allosteric inhibitors of the initiation complex of the enzyme. We have developed a novel approach using a SPA binding assay to identify diverse chemical matter with new modes of action.

The assay uses biotinylated HCV RNA polymerase bound to a streptavidin SPA bead. The radioligand is a tritiated compound similar to HCV 796 known to bind in NNI pocket palm II (ref.1). The assay was validated using compounds known to bind to palm I, palm II and thumb sites. The assay was shown to have low non-specific binding and good tolerance to DMSO. Our results indicate that compounds binding to palm II show a full sigmoidal concentration response curve (full displacement) whereas compounds binding to the thumb site showed a partial dose response where a sub maximal response was measured at saturating concentrations of test compound.

The assay has shown the power to identify compounds that bind in different domains, enabling its use as a screening tool and a method for the determination of inhibitor MOA.

Ref. 1 Le Pogam et al. (2008) Journal of Antimicrobial Chemotherapy 61, 1205-1216.