Cyclic Nucleotide Phosphodiesterase Assay Technology

S.J. MacKenzie1, S.F. Hastings1, C. Wells1

1 Scottish Biomedical, Glasgow, Scotland, United Kingdom
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.12
DOI:  10.1002/0471141755.ph0312s49
Online Posting Date:  June, 2010
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Abstract

Because of their critical role in modulating cellular cyclic nucleotide levels, phosphodiesterases (PDEs) are involved in many disease‐related signaling pathways. The PDE family is large and diverse, with members having different tissue distribution, sub‐cellular localizations, and substrate specificities. Because of these characteristics, the PDEs represent a broad group of potential drug targets. Described in the present unit are the assay development and validation procedures needed to establish a high‐throughput screening system for these important enzymes. The assays provide a structured approach for determining the kinetic parameters of related enzyme families to facilitate the characterization of PDE inhibitors. Curr. Protoc. Pharmacol. 49:3.12.1‐3.12.26. © 2010 by John Wiley & Sons, Inc.

Keywords: phosphodiesterase; PDE assay technologies; PDE assay protocols

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Titration of PDE4Cat Enzyme to Determine Optimum Enzyme Working Concentration
  • Basic Protocol 2: Determination of Phosphodiesterase KM
  • Basic Protocol 3: Phosphodiesterase Inhibition Assay Using Radiolabeled Substrate
  • Basic Protocol 4: The IMAP (Immobilized Metal Ion Affintity‐Based Fluorescence Polarization) Method for Titrating Phosphodiesterase Activity
  • Basic Protocol 5: Phosphodiesterase Inhibition Assay Using IMAP Fluorescence
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Titration of PDE4Cat Enzyme to Determine Optimum Enzyme Working Concentration

  Materials
  • Phosphodiesterase enzyme (PDE; available from EMD Biosciences, Cosmo Bio Co., Scottish Biomedical): baculoviral‐expressed PDE enzyme at 0.5 U/µl in 20 mM Tris⋅Cl, pH 7.4
  • 10 mM cAMP or cGMP (Sigma, cat. nos. A9501 and G6129) in 20 mM Tris⋅Cl, pH 7.4
  • [3H]‐labeled substrate (cAMP or cGMP; Perkin Elmer)
  • 0.1 mg/ml snake venom (Sigma, cat. no. V‐7000)
  • 20 mM Tris⋅Cl, pH 7.4 ( appendix 2A)
  • PDE assay buffer (10 mM MgCl 2, 40 mM Tris⋅Cl, pH 7.4; store up to 1 year at 4°C)
  • 50% dimethyl sulfoxide (DMSO)
  • Ice
  • Dowex solution (see recipe)
  • MicroScint‐20 (Perkin Elmer, cat. no. 6005185)
  • Greiner 96 deep well 1‐ml master‐block (Greiner, cat. no. 780201)
  • Multichannel pipettor
  • Standard set of manual Gilson pipets
  • TopSeal (Perkin Elmer)
  • Water baths set at 30°C and 70°C
  • 1‐liter beakers
  • Stir bar
  • Stir plate
  • Microplate shaker
  • Greiner 96‐well Optiplate (Greiner, cat. no. 655075)
  • TopCount scintillation counter
  • Centrifuge
  • GraphPad Prism
NOTE: Use deionized, distilled water in all recipes and protocol steps.

Basic Protocol 2: Determination of Phosphodiesterase KM

  Materials
  • [3H]‐labeled substrate (cAMP or cGMP; Perkin Elmer)
  • PDE assay buffer (10 mM MgCl 2, 40 mM Tris⋅Cl, pH 7.4; store up to 1 year at 4°C)
  • Ice
  • Phosphodiesterase enzyme at optimized concentration ( protocol 1)
  • 0.1 mg/ml snake venom (Sigma)
  • 10 mM cAMP or cGMP, (Sigma, cat. nos. A9501 and G6129)
  • Dowex solution (see recipe)
  • MicroScint‐20 (Perkin Elmer, cat no. 6005185)
  • 96 deep‐well 1‐ml master‐block (Greiner, cat no. 780201)
  • Mutlichannel pipettor
  • Standard set of manual Gilson pipets
  • Water baths set at 30°C and 70°C
  • 1‐liter beakers
  • Stir bar
  • Stir plate
  • TopSeal (Perkin Elmer)
  • 96‐well Optiplate(Greiner, cat no. 655075)
  • Microplate shaker
  • Centrifuge
  • TopCount scintillation counter
  • GraphPad Prism
NOTE: Use deionized, distilled water in all recipes and protocol steps.

Basic Protocol 3: Phosphodiesterase Inhibition Assay Using Radiolabeled Substrate

  Materials
  • Phosphodiesterase enzyme at optimized concentration (see protocol 1)
  • Working stock substrate (cAMP or cGMP) solution as determined from protocol 2 (Table 3.12.1)
  • Test compounds
  • 100%, 50%, and 10% dimethyl sulfoxide (DMSO)
  • Ice
  • 0.1 mg/ml snake venom (Sigma, cat. no. V‐7000)
  • Dowex suspension (see recipe)
  • MicroScint‐20 (Perkin Elmer, cat. no. 6005185)
  • V‐bottom 96‐well plate
  • 96‐deep well 1‐ml master‐block (Greiner 780201)
  • Multichannel pipettor
  • Standard set of manual Gilson pipets
  • TopSeal (Perkin Elmer)
  • Water baths set at 30°C and 70°C
  • 1‐liter beakers
  • Stir bar
  • Stir plate
  • Microplate shaker
  • 96‐well Optiplate (Greiner, cat. no. 655075)
  • Centrifuge
  • TopCount scintillation counter
  • GraphPad Prism
  • Additional reagents and equipment for determining the optimal enzyme working concentration ( protocol 1) and preparing the substrate solution ( protocol 2)
NOTE: Use deionized, distilled water in all recipes and protocol steps.

Basic Protocol 4: The IMAP (Immobilized Metal Ion Affintity‐Based Fluorescence Polarization) Method for Titrating Phosphodiesterase Activity

  Materials
  • IMAP FP Bulk Kit Progressive Binding System with BSA (Molecular Devices, cat. no. R8125) containing:
    • IMAP reaction buffer
    • Binding buffer A
    • Binding buffer B
    • Progressive binding reagent
  • Distilled water
  • Stock fluorescein‐labeled cAMP or cGMP (Molecular Devices, cat. nos. R7506 and R7508)
  • Standard inhibitor solution (see Table 3.12.1)
  • Phosphodiesterase enzyme (PDE; available from EMD Biosciences, Cosmo Bio Co., Scottish Biomedical): baculovirus‐expressed PDE enzyme at 0.5 U/µl in 20 mM Tris⋅Cl, pH 7.4 (1 unit converts 1 pmol of cAMP/cGMP to AMP/GMP per min at 30°C, pH 7.4)
  • 10% DMSO
  • 96‐well, flat bottomed, tissue culture plate (Greiner, cat. no. 655180)
  • Multichannel pipettor
  • Greiner 96‐well, half‐well, black flat‐bottomed plates
  • Packard TopSeal
  • Incubator at 30°C
  • Fluorescence polarization microplate reader
NOTE: Use deionized, distilled water in all recipes and protocol steps.

Basic Protocol 5: Phosphodiesterase Inhibition Assay Using IMAP Fluorescence

  Materials
  • Test compounds
  • 100% and 10% DMSO
  • Stock fluorescein‐labeled cAMP or cGMP (Molecular Devices, cat. nos. R7506 and R7508)
  • Phosphodiesterase enzyme (PDE; available from EMDBiosciences, Cosmo Bio Co., Scottish Biomedical): baculovirus‐expressed PDE enzyme at 0.5 U/µl in 20 mM Tris⋅Cl, pH 7.4 (1 unit converts 1 pmol of cAMP/cGMP to AMP/GMP per min at 30°C, pH 7.4)
  • IMAP PDE assay buffer (10 mM MgCl 2; 40 mM Tris⋅Cl, pH 7.4, 0.33 mg/ml BSA; store up to 1 year at 4°C)
  • Binding solution (prepare as described in step 4 of protocol 4)
  • V‐bottom 96‐well plates
  • Greiner 96‐well, half‐well, black flat‐bottomed plates
  • TopSeal
  • Incubator at 30°C
  • Fluorescence polarization microplate reader
  • GraphPad Prism
  • Additional reagents and equipment for identifying a concentration of enzyme that produces activity at the high end of the linear range ( protocol 4)
NOTE: Use deionized, distilled water in all recipes and protocol steps.
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Figures

Videos

Literature Cited

Literature Cited
   Bella, A.J., Deyoung, L.X., Al‐Numi, M., and Brock, G.B. 2007. Daily administration of phosphodiesterase type 5 inhibitors for urological and nonurological indications. Eur. Urol. 52:990‐1005.
   Conti, M. and Beavo, J. 2007. Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu. Rev. Biochem. 76:481‐511.
   Francis, S.H., Turko, I.V., and Corbin, J.D. 2001. Cyclic nucleotide Phosphodiesterases: Relating structure and function. Prog. Nucleic Acid Res. Mol. Biol. 65:1‐52.
   Gupta, R., Kumar, G., and Kumar, R.S. 2005. An update on cyclic nucleotide phosphodiesterase (PDE) inhibitors: Phosphodiesterases and drug selectivity. Methods Find. Exp. Clin. Pharmacol. 27:101‐118.
   Gibson, L.C., Hastings, S.F., McPhee, I., Clayton, R.A., Darroch, C., Mackenzie, A., MacKenzie, F.L., Nagasawa, M., Stevens, P.A., and MacKenzie, S.J. 2006. The inhibitory profile of Ibudilast against the human phosphodiesterase enzyme family. Eur. J. Pharmacol. 538:39‐42.
   Huang, W., Zhang, Y., and Sportsman, J.R. 2002. A fluorescence polarization assay for cyclic nucleotide phosphodiesterases. J. Biomol. Screen. 7:215‐222.
   Kumar, M. and Bhattacharya V. 2007. Cilostazol: A new drug in the treatment intermittent claudication. Recent Patents Cardiovasc. Drug Discov. 2:181‐185.
   Omori, K. and Kotera, J. 2007. Overview of PDEs and their regulation. Circ. Res. 100:309‐327.
   Thompson, W.J. and Appleman, M.M. 1971. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry 10:311‐316.
   Wu, G., Yuan, Y., and Hodge, C.N. 2003. Determining appropriate substrate conversion for enzymatic assays in high‐throughput screening. J. Biomol. Screen. 8:694‐700.
   Yu, J., Wolda, S.L., Frazier, A.L., Florio, V.A., Martins, T.J., Snyder, P.B., Harris, E.A., McCaw, K.N., Farrell, C.A., Steiner, B., Bentley, J.K., Beavo, J.A., Ferguson, K., and Gelinas, R. 1997. Identification and characterisation of a human calmodulin‐stimulated phosphodiesterase PDE1B1. Cell Signal 9:519‐529.
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