A high-throughput radiometric assay for hepatitis C virus NS3 protease

A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was dev eloped. The NS3 protein of hepatitis C virus (HCV) contains a serine protea se, whose function is to process the majority of the nonstructural proteins of the viral polyprotein. The viral NS4A protein is a cofactor of NS3 prot ease activity in the cleavage of NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-N S5B junctions, To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A-NS4B junction. Upon inc ubation with the enzyme the substrate was separated from the radiolabeled c leavage product by addition of an ion exchange resin. The assay was perform ed in a microtiter plate format and offered the potential for assaying nume rous samples using a laboratory robot. Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components. We showed that physicochemical conditions aff ect NS3 protease activity in a strain-specific way. Furthermore, the sensit ivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV s erine protease. (C) 1999 Academic Press.