title: Development and application of a fast, reproducible assay to measure HCV NS3 protease activity using Escherichia coli lysate.
abstract: The hepatitis C virus (HCV) NS3/4A protease is a key target of efforts to develop direct-acting antiviral inhibitors for treatment of chronic HCV infection. In vitro analyses of the effects of NS3/4A mutations and polymorphisms on protease inhibitor (PI) susceptibility are essential to nonclinical and clinical compound characterization, but can be hampered by time and technical limitations of current in vitro methods using replicon or purified protein systems. We have developed a fast and simple method utilizing full-length NS3/4A protease inducibly expressed in Escherichia coli cells. Minimally processed E. coli whole cell lysate was used for analyzing NS3/4A protease activity and inhibition by antiviral compounds. Assay conditions were optimized to develop a reproducible assay that can be used for efficient analysis of NS3 protease mutants with poor replication capacity in the replicon system. IC50 fold-changes for NS3 mutants relative to their wild-types generated by this NS3 assay are comparable to those observed in the replicon system, with an R(2) of 0.82 for the values obtained by the two methods. In addition, we demonstrate that this assay can be successfully used for population and clonal phenotyping of patient samples and characterization of PIs against the NS3/4A protease from HCV genotypes 1-6.
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA. Electronic address: bhan@gilead.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Han
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors.forename: Hadas
authors.initials: H
authors.lastname: Dvory-Sobol
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Greenstein
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors.forename: Joseph F
authors.initials: JF
authors.lastname: McCarville
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors.forename: Magdeleine
authors.initials: M
authors.lastname: Hung
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Liu
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Miller
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Mo
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology*; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D020128:Inhibitory Concentration 50; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D050505:Mutant Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
keywords: E. coli lysate; Enzyme inhibition assay; HCV; NS3; Protease inhibitor; Resistance