High-Throughput Screening (HTS) and Hit Validation to Identify Small Molecule Inhibitors with Activity against NS3/4A proteases from Multiple Hepatitis C Virus Genotypes

Hyun Lee, Tian Zhu, Kavankumar Patel, Yan Yan Zhang, Lena Truong, Kirk Hevener, Joseph L. Gatuz, Gitanjali Subramanya, Hyun Young Jeong, Susan L. Uprichard, Michael E. Johnson

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 IC 50 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.

Original languageEnglish (US)
Article numbere75144
JournalPLoS ONE
Volume8
Issue number10
DOIs
StatePublished - Oct 9 2013
Externally publishedYes

Fingerprint

Hepatitis C virus
Viruses
Hepacivirus
Screening
Peptide Hydrolases
proteinases
Genotype
Throughput
screening
Molecules
genotype
Pharmaceutical Preparations
Assays
drugs
Surface plasmon resonance
Serine Proteases
mutation
surface plasmon resonance
Mutation
Surface Plasmon Resonance

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

High-Throughput Screening (HTS) and Hit Validation to Identify Small Molecule Inhibitors with Activity against NS3/4A proteases from Multiple Hepatitis C Virus Genotypes. / Lee, Hyun; Zhu, Tian; Patel, Kavankumar; Zhang, Yan Yan; Truong, Lena; Hevener, Kirk; Gatuz, Joseph L.; Subramanya, Gitanjali; Jeong, Hyun Young; Uprichard, Susan L.; Johnson, Michael E.

In: PLoS ONE, Vol. 8, No. 10, e75144, 09.10.2013.

Research output: Contribution to journalArticle

Lee, Hyun ; Zhu, Tian ; Patel, Kavankumar ; Zhang, Yan Yan ; Truong, Lena ; Hevener, Kirk ; Gatuz, Joseph L. ; Subramanya, Gitanjali ; Jeong, Hyun Young ; Uprichard, Susan L. ; Johnson, Michael E. / High-Throughput Screening (HTS) and Hit Validation to Identify Small Molecule Inhibitors with Activity against NS3/4A proteases from Multiple Hepatitis C Virus Genotypes. In: PLoS ONE. 2013 ; Vol. 8, No. 10.
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