HIV Protease Assays

Eric S. Furfine1

1 Glaxo Wellcome Research, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.2
DOI:  10.1002/0471141755.ph0302s00
Online Posting Date:  May, 2001
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Abstract

The fluorescence‐based assay described in this unit is used not only to screen large numbers of compounds in a 96‐well format for the ability to inhibit human immunodeficiency virus‐1 (HIV) protease, but also to determine accurately the affinity of inhibitors for the enzyme. A method for performing an active site titration, using tight‐binding inhibitors, and determining the Km of a substrate is also provided. Because numerous potent inhibitors of HIV protease have been identified, information is provided on the analysis required to deal with these potent compounds and with fluorometric enzyme assay data in general.

     
 
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Table of Contents

  • Basic Protocol 1: Fluorometric Screening to Identify Inhibitors of HIV Protease
  • Support Protocol 1: Assay of HIV Protease to Determine Working Enzyme Concentration
  • Support Protocol 2: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorometric Screening to Identify Inhibitors of HIV Protease

  Materials
  • 2 mM HIV protease inhibitor (test compound) in appropriate solvent (e.g., DMSO)
  • Dimethylsulfoxide (DMSO)
  • Assay buffer with hexapeptide substrate (see recipe)
  • HIV protease working solution (see recipe)
  • 96‐well fluorescence plate reader (PerSeptive BioSystems) with 320 ± 20–nm excitation filter (made by reversing the respective emission filter) and 420 ± 20–nm emission filter
  • 5‐ to 40‐ and 40‐ to 250‐µl 12‐channel pipettors or similar
  • 96‐well polypropylene round‐bottom plates (Costar)
  • Reagent reservoirs suitable for multichannel pipetting of buffers, etc. (Costar)
  • 96‐well flat‐bottom fluorescence assay plate (PerSeptive BioSystems)

Support Protocol 1: Assay of HIV Protease to Determine Working Enzyme Concentration

  • HIV protease dilution buffer (see recipe)
  • HIV protease stock solution (see recipe), when enzyme active site concentration is not known

Support Protocol 2: Data Analysis

  Materials
  • Data from fluorescence assays (see protocol 1 or protocol 2)
  • Microsoft Excel or Sigma Plot (Jandel Scientific) or any database software that allows curve fitting
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Figures

Videos

Literature Cited

Literature Cited
   Condra, J.H., Schleif, W.A., Blahy, O.M., Gabryelski, L.J., Graham, D.J., Quintero, J.C., Rhodes, A., Robbins, H.L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K.E., Deutsch, P.J., and Emini, E. 1995. In vivo emergence of HIV‐1 variants resistant to multiple protease inhibitors. Nature 374:569‐571.
   Dark, P.L. and Huff, J.R. 1994. HIV protease as an inhibitor target of the treatment of AIDS. Adv. Pharmacol. 25:399‐454.
   Ho, D.D., Neumann, A.V., Perelson, A.S., Chen, W., Leonard, J.M., and Markowitz, M. 1995. Rapid turnover of plasma virions and CD4 lymphocytes in HIV‐1 infection. Nature 373:123‐126.
   Huff, J.R. 1991. HIV protease: A novel chemotherapeutic target for AIDS. J. Med. Chem. 34:2305‐2314.
   Jacobsen, H., Brun‐Vezinet, F., Duncan, I., Hanggi, M., Ott, M., Vella, S., Weber, J., and Mous, J. 1994. Genotypic characterizations of HIV‐1 from patients after prolonged treatment with proteinase inhibitor saquinavir. In Third International Workshop on HIV Drug Resistance 3:16.
   Jordan, S.P., Zugau, J., Dark, P.L., and Kuo, L.C. 1992. Activity and dimerization of human immunodeficiency virus protease as a function of solvent composition and enzyme concentration. J. Biol. Chem. 267:20028‐20032.
   Maschera, B., Darby, G., Palú, G., Wright, L.L., Tisdale, M., Myers, R., Blair, E.D., and Furfine, E.S. 1996. Human immunodeficiency virus: Mutations in the protease that confer resistance to saquinavir increase the dissociation rate constant of the protease‐saquinavir complex. J. Biol. Chem. 271:33231‐33235.
   Morrison, J.F. and Walsh, C.T. 1988. The behavior and significance of slow‐binding enzyme inhibitors. Adv. Enzymol. 61:201‐301.
   Norbeck, D.W. and Kempf, D.J. 1991. HIV protease inhibitors. Annu. Rep. Med. Chem. 26:141‐150.
   Richman, D.D. 1995. Protease uninhibited. Nature 374:494.
   Toth, M.V. and Marshall, G.R. 1990. A simple continuous fluorometric assay for HIV protease. Int. J. Pept. Protein Res. 36:544‐550.
   Wei, X., Ghosh, S.K., Taylor, M.E., Johnson, V.A., Emini, E.A., Deutsch, P., Lifson, J.D., Bonhoeffer, S., Nowak, M.A., Hahn, B.H., Saag, M.S., and Shaw, G.M. 1995. Viral dynamics in human immunodeficiency virus type‐1 infection. Nature 373:117‐122.
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