title: A continuous assay of hepatitis C virus protease based on resonance energy transfer depsipeptide substrates.
abstract: 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 P&prime1. 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).
authors.affiliation: Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Via Pontina Km 30.600, Pomezia, Rome, 00040, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Taliani
authors.forename: E
authors.initials: E
authors.lastname: Bianchi
authors.forename: F
authors.initials: F
authors.lastname: Narjes
authors.forename: M
authors.initials: M
authors.lastname: Fossatelli
authors.forename: A
authors.initials: A
authors.lastname: Urbani
authors.forename: C
authors.initials: C
authors.lastname: Steinkühler
authors.forename: R
authors.initials: R
authors.lastname: De Francesco
authors.forename: A
authors.initials: A
authors.lastname: Pessi
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009282:Naphthalenesulfonates; D010455:Peptides / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000032:analysis*; D017361:Viral Nonstructural Proteins / Q000032:analysis*; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010455:Peptides; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D004124:p-Dimethylaminoazobenzene; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; C063127:4-(4-dimethylaminophenylazo)benzoic acid