title: A collaborative screening program for the discovery of inhibitors of HCV NS2/3 cis-cleaving protease activity.
abstract: This report describes the development of a cell-based assay for high-throughput screening and detection of small-molecule inhibitors for hepatitis C virus (HCV) NS2/3 protease. The HCV NS2/3 protease is essential for the normal infectious cycle of HCV. Generation of a cell-based assay for this cis-acting viral protease involved reporter constructs in which the NS2/3 protease sequence was inserted between the ,B-lactamase (BLA) reporter and a ubiquitin-based destabilization domain. In stable cell lines, NS2/3 cis cleavage of the NS2/3-BLA fusion protein resulted in differential stability of the cleaved versus uncleaved BLA reporter, providing a robust readout for protease activity. BLA reporter activity was shown to be a function of NS2/3-specific protease activity, by using genetic mutants of the NS2/3 sequence. In addition, the cell-based assay was validated and screened in a 384-well format on a fully automated robotic platform where small-molecule inhibitors of NS2/3 protease activity were identified.
authors.affiliation: Aurora Biosciences Corp. San Diego, CA 92121, USA. info@aurorabio.com
authors.forename: Mike
authors.initials: M
authors.lastname: Whitney
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Stack
authors.forename: Paul L
authors.initials: PL
authors.lastname: Darke
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.forename: Joe
authors.initials: J
authors.lastname: Terzo
authors.forename: Jim
authors.initials: J
authors.lastname: Inglese
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Kuo
authors.forename: Brian A
authors.initials: BA
authors.lastname: Pollock
mesh_terms: D001331:Automation; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D017930:Genes, Reporter; D016174:Hepacivirus / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008957:Models, Genetic; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D011480:Protease Inhibitors; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D003513:Cycloheximide; D003546:Cysteine Endopeptidases; C107824:NS2-3 protease