title: A fluorescence-based high-throughput screen to identify small compound inhibitors of the genotype 3a hepatitis C virus RNA polymerase.
abstract: The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 "lead-like" compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC(50) of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.
authors.affiliation: 1School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Auda A
authors.initials: AA
authors.lastname: Eltahla
authors.forename: Kurt
authors.initials: K
authors.lastname: Lackovic
authors.forename: Christopher
authors.initials: C
authors.lastname: Marquis
authors.forename: John-Sebastian
authors.initials: JS
authors.lastname: Eden
authors.forename: Peter A
authors.initials: PA
authors.lastname: White
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005456:Fluorescent Dyes; D016174:Hepacivirus / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology; D057166:High-Throughput Screening Assays; D058889:Molecular Typing; D012330:RNA, Double-Stranded / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D012367:RNA, Viral / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase
keywords: NS5B; RNA-dependent RNA polymerase; fluorescence; hepatitis C virus; high throughput; inhibitors