title: High-throughput screening (HTS) and hit validation to identify small molecule inhibitors with activity against NS3/4A proteases from multiple hepatitis C virus genotypes.
abstract: Development of drug-resistant mutations has been a major problem with all currently developed Hepatitis C Virus (HCV) NS3/4A inhibitors, including the two FDA approved drugs, significantly reducing the efficacy of these inhibitors. The high incidence of drug-resistance mutations and the limited utility of these inhibitors against only genotype 1 highlight the need for novel, broad-spectrum HCV therapies. Here we used high-throughput screening (HTS) to identify low molecular weight inhibitors against NS3/4A from multiple genotypes. A total of 40,967 compounds from four structurally diverse molecular libraries were screened by HTS using fluorescence-based enzymatic assays, followed by an orthogonal binding analysis using surface plasmon resonance (SPR) to eliminate false positives. A novel small molecule compound was identified with an IC50 value of 2.2 µM against the NS3/4A from genotype 1b. Mode of inhibition analysis subsequently confirmed this compound to be a competitive inhibitor with respect to the substrate, indicating direct binding to the protease active site, rather than to the allosteric binding pocket that was discovered to be the binding site of a few recently discovered small molecule inhibitors. This newly discovered inhibitor also showed promising inhibitory activity against the NS3/4As from three other HCV genotypes, as well as five common drug-resistant mutants of genotype 1b NS3/4A. The inhibitor was selective for NS3 from multiple HCV genotypes over two human serine proteases, and a whole cell lysate assay confirmed inhibitory activity in the cellular environment. This compound provides a lead for further development of potentially broader spectrum inhibitors.
authors.affiliation: Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Hyun
authors.initials: H
authors.lastname: Lee
authors.forename: Tian
authors.initials: T
authors.lastname: Zhu
authors.forename: Kavankumar
authors.initials: K
authors.lastname: Patel
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Zhang
authors.forename: Lena
authors.initials: L
authors.lastname: Truong
authors.forename: Kirk E
authors.initials: KE
authors.lastname: Hevener
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Gatuz
authors.forename: Gitanjali
authors.initials: G
authors.lastname: Subramanya
authors.forename: Hyun-Young
authors.initials: HY
authors.lastname: Jeong
authors.forename: Susan L
authors.initials: SL
authors.lastname: Uprichard
authors.forename: Michael E
authors.initials: ME
authors.lastname: Johnson
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors*
chemical_list: D000998:Antiviral Agents; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins