A continuous spectrophotometric assay for the hepatitis C virus serine protease

The hepatitis C virus (HCV) encodes a chymotrypsin-like serine protease res ponsible for the processing of HCV nonstructural proteins and which is a pr omising target for antiviral intervention. Its relatively low catalytic eff iciency has made standard approaches to continuous assay development only m odestly successful. In this report, four continuous spectrophotometric subs trates suitable for both high-throughput screening and detailed kinetic ana lysis are described. One of these substrates, Ac-DTEDVVP(Nva)-O-4-phenylazo phenyl ester, is hydrolyzed by HCV protease with a second-order rate consta nt (k(cat)/K-m) of 80,000 +/- 10,000 M-1 s(-1). Together with its negligibl e rate of nonenzymatic hydrolysis under assay conditions (0.01 h(-1)), anal ysis of as little as 2 nM protease can be completed in under 10 min. (C) 19 99 Academic Press.