title: A time-resolved, internally quenched fluorescence assay to characterize inhibition of hepatitis C virus nonstructural protein 3-4A protease at low enzyme concentrations.
abstract: The hepatitis C virus (HCV) nonstructural protein 3 (NS3) with its cofactor NS4A is a pivotal enzyme for the replication of HCV. Inhibition of NS3-4A protease activity has been validated as an antiviral target in clinical studies of inhibitors of the enzyme. We have developed a sensitive time-resolved fluorescence (TRF) assay capable of detecting very low NS3-4A concentrations. A depsipeptide substrate is used that contains a europium-cryptate moiety and an efficient quenching group, QSY-7. The TRF assay is at least 30-fold more sensitive than a fluorescence energy transfer (FRET) assay and allows evaluation of NS3 protease inhibitors in reactions catalyzed by low enzyme concentrations (30 pM). Use of low enzyme concentrations allows for accurate measurement of inhibition by compounds with subnanomolar inhibition constants. The inhibitory potency of the potent protease inhibitor, BILN-2061, is significantly greater than previously reported. The ability to accurately determine inhibitory potency in reactions with low picomolar concentrations of NS3-4A is crucially important to allow valid comparisons between potent inhibitors. Studies of the interaction of NS3 with its NS4A cofactor at low enzyme concentration also reveal that the protease activity is salt dependent. This salt dependence of the enzyme activity is not present when high enzyme concentrations are used in the FRET assay.
authors.affiliation: Department of Antiviral Research, Merck Research Laboratories, WP 26A-3000, West Point, PA 19486, USA.
authors.forename: Shi-Shan
authors.initials: SS
authors.lastname: Mao
authors.forename: Jillian
authors.initials: J
authors.lastname: DiMuzio
authors.forename: Carolyn
authors.initials: C
authors.lastname: McHale
authors.forename: Christine
authors.initials: C
authors.lastname: Burlein
authors.forename: David
authors.initials: D
authors.lastname: Olsen
authors.forename: Steven S
authors.initials: SS
authors.lastname: Carroll
mesh_terms: D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D016174:Hepacivirus / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*
chemical_list: D011480:Protease Inhibitors; D010447:Peptide Hydrolases