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 K_M
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 MgCl₂, 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 K_M

Materials

[³H]-labeled substrate (cAMP or cGMP; Perkin Elmer)
PDE assay buffer (10 mM MgCl₂, 40 mM Tris×Cl, pH 7.4; store up to 1 year at 4°C)
Ice
Phosphodiesterase enzyme at optimized concentration (Basic 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 Basic Protocol 1)
Working stock substrate (cAMP or cGMP) solution as determined from Basic 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 (Basic Protocol 1) and preparing the substrate solution (Basic 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 MgCl₂; 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 Basic 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 (Basic Protocol 4)

NOTE: Use deionized, distilled water in all recipes and protocol steps.

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Figures

Figure 3.12.1 PDE catalyzes the conversion of 3¢5¢-cAMP to 5¢-AMP followed by conversion of [³H]-5¢-AMP to [³H]-adenosine by nucleotidase A (snake venom).

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Figure 3.12.2 Radiometric enzyme titration assay plate layout (96-well format) as described in Basic Protocol 1.

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Figure 3.12.3 Enzyme titration and standard IC₅₀ value of rat PDE4Cat. Enzyme titration was performed according to Basic Protocol 1. (A) Data display. (B) Data were analyzed with a sigmoidal equation using GraphPad Prism. A total of 2 µl per well in the assay was determined to be an appropriate working concentration for rat PDE4Cat. The working enzyme concentration must fall within the linear range or reach 80% of the maximum response (EC₈₀)

Figure 3.12.4 K_M substrate assay plate layout (96-well format) as described in Basic Protocol 2.

View Image

Figure 3.12.5 Kinetic analysis of rat PDE4Cat was tested according to Basic Protocol 2. A fixed amount of rat PDE4Cat enzyme was incubated with increasing concentrations of cAMP as determined in Basic Protocol 1. Velocity was presented as pmol/µl. (A) An example of how to calculate and present the data for one time point. The enzyme activity was converted from cpm to pmol/µl and initial velocity (slope) at each time point calculated based on the specific activity of [³H]cAMP. The rate constant K was calculated as follows: K = final assay volume (µl)/sample volume (µl) × [100/incubation time (min) × enzyme used (µl)] and the rate pmol/min/µl = (counts – blanks / totals × k × substrate concentration). (B) Calculated data for each time point. (C) The data in pmol/µl from (B) were used to calculate the gradient for each time point using GraphPad Prism. A Michaelis-Menten curve was fitted to the gradient for each time point versus substrate concentration equation using GraphPad Prism.

Figure 3.12.6 Inhibitor IC₅₀ determination plate plan (96-well format) as described in Basic Protocol 3.

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Figure 3.12.7 Enzyme titration and standard IC₅₀ value for rat PDE4Cat. (A) An example of how to transform the data. The average results for blanks (background) and total binding are an average of two data points and are used for every titration point. The sigmoidal curve should fit between the background and the total binding points to determine an accurate IC₅₀ plot. (B) Inhibition curve of rat PDE4Cat with the standard inhibitor rolipram. A fixed amount of pretitrated rat PDE4Cat was incubated with increasing concentrations of rat PDE4Cat inhibitor with the radiometric assay (Basic Protocol 1). Data were analyzed with a sigmoidal equation using GraphPad Prism.

Figure 3.12.8 Fluorescein-labeled cyclic nucleotide substrate is hydrolyzed at the 3¢-ester bond by PDE. The reaction product, fluorescein-nucleotide monophosphate, binds to the IMAP (immobilized metal ion affinity-based fluorescence polarization) binding reagent through the interaction between the phosphate and trivalent cations immobilized on the surface of nanoparticles. The binding results in a decrease in the molecular mobility of the fluorescein-labeled substrate, which corresponds to an increase in FP (fluorescence polarization). The IMAP binding reagent does not bind to the fluorescein-labeled substrate.

Figure 3.12.9 IMAP enzyme titration assay plate layout (96-well format) as described in Basic Protocol 4.

View Image

Figure 3.12.10 Enzyme titration of rat PDE4Cat. Enzyme titration was performed according to Basic Protocol 4. (A) Data display. (B) Data analyzed with a hyperbolic equation using GraphPad Prism. 0.125 µl per well in the assay is an appropriate working concentration for rat PDE4Cat as highlighted in red on panel A. The working enzyme concentration must fall within the linear range or reach 80% of the maximum response (EC₈₀).

Figure 3.12.11 IMAP inhibitor IC₅₀ determination plate plan (96-well format) as described in Basic Protocol 5.

View Image

Figure 3.12.12 Standard IC₅₀ for rolipram of rat PDE4Cat. (A) Data extrapolation. (B) Inhibition curve of rat PDE4Cat with the standard inhibitor rolipram. A fixed amount of pretitrated rat PDE4Cat was incubated with increasing concentrations of rat PDE4Cat inhibitors with the IMAP assay (Basic Protocol 5). Data were analyzed with a sigmoidal equation using GraphPad Prism.

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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