title: Optimized high-throughput screen for hepatitis C virus translation inhibitors.
abstract: Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. The authors developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5' capped mRNA control. The authors screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1700 initial hits. After secondary counterscreening, the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end, the authors found no selective HCV IRES inhibitors but did discover a new scaffold of general translation inhibitor. The analysis of these molecules, as well we the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA, USA.
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Berry
authors.forename: Betty
authors.initials: B
authors.lastname: Peng
authors.forename: David
authors.initials: D
authors.lastname: Koditek
authors.forename: Douglas
authors.initials: D
authors.lastname: Beeman
authors.forename: Nikos
authors.initials: N
authors.lastname: Pagratis
authors.forename: Jason K
authors.initials: JK
authors.lastname: Perry
authors.forename: Jay
authors.initials: J
authors.lastname: Parrish
authors.forename: Weidong
authors.initials: W
authors.lastname: Zhong
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.forename: I-hung
authors.initials: IH
authors.lastname: Shih
mesh_terms: D000818:Animals; D004353:Drug Evaluation, Preclinical; D017930:Genes, Reporter; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D014176:Protein Biosynthesis / Q000187:drug effects* / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D015203:Reproducibility of Results; D012107:Research Design; D054852:Small Molecule Libraries
chemical_list: D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012367:RNA, Viral; D054852:Small Molecule Libraries; D011691:Puromycin