A Continuous Spectrophotometric Assay for the Hepatitis C Virus Serine Protease

The hepatitis C virus (HCV) encodes a chymotrypsin-like serine protease responsible for the processing of HCV nonstructural proteins and which is a promising target for antiviral intervention. Its relatively low catalytic efficiency has made standard approaches to continuous assay development only modestly successful. In this report, four continuous spectrophotometric substrates suitable for both high-throughput screening and detailed kinetic analysis are described. One of these substrates, Ac-DTEDVVP(Nva)-O-4-phenylazophenyl ester, is hydrolyzed by HCV protease with a second-order rate constant (kcat/Km) of 80,000 ± 10,000 M−1s−1. Together with its negligible rate of nonenzymatic hydrolysis under assay conditions (0.01 h−1), analysis of as little as 2 nM protease can be completed in under 10 min.