Biochemical Evaluation of HCV NS3 Protease Inhibitors

Brian Schultz1, Huiling Yang1, William E. Delaney1

1 Gilead Sciences, Foster City, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13B.7
DOI:  10.1002/0471141755.ph13b07s54
Online Posting Date:  September, 2011
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Abstract

This unit describes assays for characterizing the potency and mechanism of action of NS3 protease inhibitors. Determination of IC50 values is described using in vitro expressed and purified NS3 protease. This assay can also be used for the rapid exploration of structure‐activity relationships. Another protocol describes using the full‐length NS3/4A complexes expressed in HCV replicon cell lines for a rapid alternative method for assessing protease activity without requiring conventional protein expression and purification. A method is then provided for determination of inhibitor Ki, which more accurately assesses the potency of inhibitors compared to the IC50 assay, particularly for potent inhibitors that reach the sensitivity limit for the basic IC50 assay. The final protocol describes how to determine the reversibility of inhibitor binding to the enzyme, an important parameter that can affect the pharmacodynamic properties of a compound. Curr. Protoc. Pharmacol. 54:13B.7.1‐13B.7.22. © 2011 by John Wiley & Sons, Inc.

Keywords: hepatitis C virus; NS3 protease; inhibitor

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of Inhibitory Activity Against Purified NS3 Protease (Standard IC50 Assay)
  • Basic Protocol 2: Determination of Inhibitory Activity Against Endogenous NS3 Protease (Cell Lysate IC50 Assay)
  • Basic Protocol 3: Determination OF Ki for HCV Protease Inhibitors
  • Basic Protocol 4: Determining the Reversibility of Inhibitor Binding
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Inhibitory Activity Against Purified NS3 Protease (Standard IC50 Assay)

  Materials
  • Antiviral test compounds: suggested control compounds are the peptide inhibitor N‐1725 (BACHEM) or the macrocyclic peptidomimetic BILN‐2061 (Acme Biosciences)
  • DMSO
  • 1 M DTT (see recipe)
  • HEPES assay buffer (see recipe)
  • NS3 protease protein (genotype 1a or 1b purified from E. coli culture; unit 13.6)
  • HCV NS3 FRET substrate RET S1 (AnaSpec, cat. no. 22991‐5)
  • NS4A synthetic peptide (Pep4A: NH2‐KKGSVVIVGRIVLSGKPAIIPKK‐COOH; see recipe) or similar commercially available peptide (GenScript, cat. no. RP11668)
  • 96‐well plates (V‐bottom and white, flat‐bottom plates)
  • Multi‐channel pipettors (10 µl, 200 µl)
  • Fluorescence plate reader (e.g., PerkinElmer Victor3 or equivalent, e.g., Biotek Synergy, Molecular Devices Analyst)
  • 37°C incubator

Basic Protocol 2: Determination of Inhibitory Activity Against Endogenous NS3 Protease (Cell Lysate IC50 Assay)

  Materials
  • HCV replicon cell line (e.g., genotype 1b (con‐1) replicon cell line, Huh‐luc, available from Dr. Ralf Bartenschlager, University of Heidelberg, Germany; or any cell line with a similar or higher level of HCV replicon expression)
  • Complete Dulbecco's modified Eagle medium (DMEM) cell culture medium (see recipe)
  • Phosphate buffered saline (PBS; Invitrogen)
  • Trypsin (10× concentration; Invitrogen)
  • FBS ( appendix 2A)
  • Cell lysis buffer (Promega, cat. no. E1531)
  • NaCl (see recipe)
  • Europium‐labeled NS3 protease substrate (see recipe)
  • Antiviral (test) compounds
  • DMSO
  • 37°C, 5% CO 2 cell culture incubator
  • Cell culture flasks (e.g., 162‐cm2)
  • 10‐ml pipets
  • 50‐ml polypropylene centrifuge tubes
  • Beckman refrigerated tabletop centrifuge
  • Hemacytometer
  • Microscope
  • 96‐well plates (V‐bottom and white cell culture plates with clear bottoms)
  • Multi‐channel pipettors (10‐µl, 200‐µl)
  • Microplate shaker
  • Fluorescence plate reader (Victor3 or other)

Basic Protocol 3: Determination OF Ki for HCV Protease Inhibitors

  Materials
  • NS3 protease protein (genotype 1a or 1b purified from E. coli culture; see unit 13.6; also see Critical Parameters)
  • NS4A synthetic peptide (Pep4A: NH2‐KKGSVVIVGRIVLSGKPAIIPKK‐OH or similar peptide available from GenScript, cat. no. RP11668)
  • Test compounds
  • HCV NS3 FRET substrate (AnaSpec)
  • DMSO
  • Glycerol
  • HEPES buffer (see recipe)
  • DTT (see recipe)
  • 96‐well, V‐bottom plates
  • 96‐well, white plate with flat bottom
  • Multi‐channel pipettors (10‐µl, 200‐µl)
  • Fluorescence plate reader (e.g., Tecan Safire2, Perkin‐Elmer Envision, or Molecular Devices Analyst)

Basic Protocol 4: Determining the Reversibility of Inhibitor Binding

  Materials
  • NS3 protease protein (genotype 1a or 1b, purified from E. coli culture; see unit 13.6; also see Critical Parameters)
  • Test compounds
  • HEPES assay buffer (see recipe)
  • DMSO
  • NS4A synthetic peptide (Pep4A: NH2‐KKGSVVIVGRIVLSGKPAIIPKK‐OH; see recipe)
  • HCV NS3 FRET substrate (AnaSpec)
  • 96‐well V‐bottom plates for drug dilution
  • 96‐well white plates with flat bottoms for fluorescence assays
  • Multi‐channel pipettors (10‐µl, 200‐µl)
  • Fluorescence plate reader
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Figures

Videos

Literature Cited

Literature Cited
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