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 he patitis, an important health problem with an estimated 50 million peop le infected worldwide. Among the possible targets for therapeutic inte rvention, the serine protease contained within the N-terminal region o f nonstructural protein 3 (NS3 protease) is so far the best characteri zed. In vitro characterization of synthetic substrates based on all th e natural cleavage sites (as well as a series of analogs) has consiste ntly revealed poor kinetic parameters, making them unsuitable for sens itive high-throughput screening. To overcome these difficulties, we ha ve recently developed depsipeptide substrates incorporating an ester b ond between residues P-1 and P-1'. Due to ready transesterification of the scissile bond to the acyl-enzyme intermediate, these substrates s howed very high k(cat)/K-m values, enabling detection of activity with subnanomolar NS3 concentrations. We have used the same principle to s ynthesize internally quenched depsipeptide fluorogenic substrates base d on resonance energy transfer between the donor/acceptor couple 5-[(2 '-aminoethyl)amino] naphthalene sulfonic acid/4-[[4'-(dimethylamino)ph enyl]azo]benzoic acid, and developed a continuous assay for NS3 activi ty. Substrate cleavage is linear with enzyme concentration: depending on the conditions chosen, we estimated a detection limit for NS3 betwe en 1 nM and 250 pM. The suitability of the assay for evaluation of inh ibitors was established using as competitor a tridecapeptide correspon ding to the natural NS4A/4B cleavage site; this gave an IC50 of 30 mu M, well in agreement with the previously found K-m value (40 mu M). (C ) 1996 Academic Press, Inc.