title: Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays.
abstract: Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma's Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50=1.4 μM), suramin sodium salt (IC50=3.6 μM), NF 023 hydrate (IC50=6.2 μM) and tyrphostin AG 538 (IC50=3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.
authors: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA.
authors: Sourav
authors: S
authors: Mukherjee
authors: Alicia M
authors: AM
authors: Hanson
authors: William R
authors: WR
authors: Shadrick
authors: Jean
authors: J
authors: Ndjomou
authors: Noreena L
authors: NL
authors: Sweeney
authors: John J
authors: JJ
authors: Hernandez
authors: Diana
authors: D
authors: Bartczak
authors: Kelin
authors: K
authors: Li
authors: Kevin J
authors: KJ
authors: Frankowski
authors: Julie A
authors: JA
authors: Heck
authors: Leggy A
authors: LA
authors: Arnold
authors: Frank J
authors: FJ
authors: Schoenen
authors: David N
authors: DN
authors: Frick
mesh_terms: D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005454:Fluorescence Polarization; D016174:Hepacivirus / Q000201:enzymology*; D057166:High-Throughput Screening Assays; D020365:RNA Helicases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054852:Small Molecule Libraries; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D004247:DNA; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D020365:RNA Helicases; D054852:Small Molecule Libraries; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C476430:SSB protein, E coli