A Continuous Assay of Hepatitis C Virus Protease Based on Resonance Energy Transfer Depsipeptide Substrates

Hepatitis C virus (HCV) is the major causative agent of non-A non-B hepatitis, an important health problem with an estimated 50 million people infected worldwide. Among the possible targets for therapeutic intervention, the serine protease contained within the N-terminal region of nonstructural protein 3 (NS3 protease) is so far the best characterized. In vitro characterization of synthetic substrates based on all the natural cleavage sites (as well as a series of analogs) has consistently revealed poor kinetic parameters, making them unsuitable for sensitive high-throughput screening. To overcome these difficulties, we have recently developed depsipeptide substrates incorporating an ester bond between residues P1 and Pprime1. Due to ready transesterification of the scissile bond to the acyl-enzyme intermediate, these substrates showed very high kcat/Km values, enabling detection of activity with subnanomolar NS3 concentrations. We have used the same principle to synthesize internally quenched depsipeptide fluorogenic substrates based on resonance energy transfer between the donor/acceptor couple 5-[(2'-aminoethyl)amino]naphthalene sulfonic acid/4-[[4'-(dimethylamino)phenyl]azo]benzoic acid, and developed a continuous assay for NS3 activity. Substrate cleavage is linear with enzyme concentration: depending on the conditions chosen, we estimated a detection limit for NS3 between 1 nM and 250 pM. The suitability of the assay for evaluation of inhibitors was established using as competitor a tridecapeptide corresponding to the natural NS4A/4B cleavage site; this gave an IC50 of 30 microM, well in agreement with the previously found Km value (40 microM).