title: A cell-based beta-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication.
abstract: This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.
authors.affiliation: Department of Automated Biotechnology, Merck and Co., 502 Louise Lane., North Wales, PA 19454, USA. paul_zuck@merck.com
authors.forename: Paul
authors.initials: P
authors.lastname: Zuck
authors.forename: Edward M
authors.initials: EM
authors.lastname: Murray
authors.forename: Erica
authors.initials: E
authors.lastname: Stec
authors.forename: Jay A
authors.initials: JA
authors.lastname: Grobler
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Simon
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Osvaldo A
authors.initials: OA
authors.lastname: Flores
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D019818:Clavulanic Acid / Q000494:pharmacology; D004261:DNA Replication; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics*; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006801:Humans; D020128:Inhibitory Concentration 50; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000235:genetics; D012680:Sensitivity and Specificity; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D019818:Clavulanic Acid; D004791:Enzyme Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D001618:beta-Lactamases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase