Assays for Cyclic Nucleotide‐Specific Phosphodiesterases (PDEs) in the Central Nervous System (PDE1, PDE2, PDE4, and PDE10)

Chengjun Deng1, Daguang Wang1, Bozena Bugaj‐Gaweda1, Michael De Vivo1

1 Memory Pharmaceuticals Corp., Montvale, New Jersey
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.21
DOI:  10.1002/0471142301.ns0721s38
Online Posting Date:  January, 2007
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Abstract

Since the identification of phosphodiesterase activity in brain tissue more than 40 years ago, 11 distinct gene families have been identified, differing with respect to localization, regulation, affinity for cAMP and cGMP, and distinct functions within cells. PDEs 1, 2, 4, and 10 are currently of special interest to CNS pharmacology because of their high expression in specific areas of the brain and the behavioral effects of inhibitors of these enzymes in rodents. Efficient high‐throughput PDE enzyme assays are essential for PDE‐targeted drug discovery, and this unit details two types of assays. The first method is relatively inexpensive and is based on separating radiolabeled cNMPs from degradation products on alumina columns. The second method is fluorescence‐based; it is fast and better accommodates high‐throughput screening, but is more expensive. Although these methods have successfully been used for PDEs 1, 2, 4 and 10, they could be readily adapted to other PDEs.

Keywords: central nervous system; CNS; cyclic nucleotide phosphodiesterase; PDE2; PDE1; PDE4; PDE10; alumina acid; IMAP

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of PDE1 Activity in Vitro Using Radiolabeled Substrate and Alumina Acid Columns
  • Support Protocol 1: Production of PDE Enzyme in Sf 9 Cells
  • Support Protocol 2: Production of PDE Enzyme in Transfected Cells
  • Support Protocol 3: Titration of PDE1 Enzyme to Determine Enzyme Working Concentration
  • Support Protocol 4: Determination of KM of PDE1
  • Alternate Protocol 1: PDE2 Enzyme Inhibition Assay Using Radiolabeled Substrate and Alumina Acid Columns
  • Alternate Protocol 2: PDE4 Enzyme Inhibition Assay Using Radiolabeled Substrate and Alumina Acid Columns
  • Alternate Protocol 3: PDE10 Enzyme Inhibition Assay Using Radiolabeled Substrate and Alumina Acid Columns
  • Basic Protocol 2: Measurement of PDE1 Inhibition in Vitro Using Fluorescence Methods
  • Support Protocol 5: Brain Tissue Dissection and Tissue Lysate Preparation
  • Support Protocol 6: Titration of PDE Activity in Brain Tissue Lysate Using IMAP Method
  • Alternate Protocol 4: Analyze PDE1, 2, 4, and 10 Activities in Dissected Rat Hippocampal and Striatal Tissues Using IMAP Method
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of PDE1 Activity in Vitro Using Radiolabeled Substrate and Alumina Acid Columns

  Materials
  • Sf 9 cells ( protocol 2) or transfected mammalian cells ( protocol 3) expressing PDE1
  • Cell lysis buffer (see recipe)
  • Compound to be tested for PDE inhibition and positive control
  • 100% dimethyl sulfoxide (DMSO)
  • Assay buffer (see recipe)
  • Alumina acid powder
  • 5 mM HCl (Fisher)
  • 1 mCi/ml [2,8‐3H]adenosine 3′, 5′‐cyclic phosphate ammonium salt (30 to 50 Ci/mmol; Amersham) or 1 mCi/ml[8‐3H]guanosine 3′, 5′‐cyclic phosphate ammonium salt (5 to 25 Ci/mmol; Amersham) in ethanol (store at −20°C)
  • 10 mM cAMP or 10 mM cGMP (store in 100‐µl aliquots at −20°C, minimize repeated thaw/freeze cycles)
  • 50 U/ml snake venom 5′‐nucleotidase (Fluka; store in 100‐µl aliquots at −20°C, minimize repeated thaw/freeze cycles)
  • Stop solution (5 mM HCl, boiling)
  • ScintiSafe Plus 50% scintillation fluid (Fisher)
  • Cell homogenizer (Kinematica Polytron 2100 or equivalent)
  • Refrigerated centrifuge (e.g., Beckman)
  • V‐bottom 96‐well polystyrene microtiter plates
  • 96‐well pipettor (Tomtec Quadra96 or equivalent) and reservoir
  • MultiScreen Durapore 96‐well filter plates, pore size 0.65 µm (Millipore)
  • 100‐µl MultiScreen column loader (Millipore)
  • Costar Transtar‐96 dispenser, transplate cartridge, and reservoir
  • 96‐well vacuum manifold system (Biosearch Technologies MicroSync I or equivalent)
  • 96‐well polyethelene terephthalate (PET) collecting plate (e.g., Wallac no. 1450‐401, or other assay plate compatible with racks of Trilux counter and free of cross‐talk)
  • MultiScreen microplate centrifuge alignment frame (Millipore)
  • Centrifuge with microplate rotor (Eppendorf 5804 or equivalent)
  • Microplate liquid scintillation counter (Wallac MicroBeta Trilux or equivalent)
  • Data analysis software (e.g., GraphPad Prism, http://www.graphpad.com)

Support Protocol 1: Production of PDE Enzyme in Sf 9 Cells

  Materials
  • See protocol 1

Support Protocol 2: Production of PDE Enzyme in Transfected Cells

  Materials
  • See protocol 1

Support Protocol 3: Titration of PDE1 Enzyme to Determine Enzyme Working Concentration

  Materials
  • Sf 9 cells ( protocol 2) or transfected mammalian cells ( protocol 3) expressing PDE1
  • Cell lysis buffer (see recipe)
  • Compound to be tested for PDE inhibition
  • 100% DMSO
  • IMAP PDE fluorescence polarization assay kit (Molecular Devices), containing:
    • Fluorescein‐labeled cAMP and cGMP substrates
    • Binding reagent
    • Binding buffer
    • Assay buffer
  • Cell homogenizer (Polytron PT2100 or equivalent)
  • Refrigerated centrifuge
  • V‐bottom 96‐well polystyrene microtiter plates (VWR)
  • 96‐well pipettor (Tomtec Quadra96 or equivalent) and reservoir
  • 96‐well high efficiency microtiter plates (Molecular Devices HE or equivalent)
  • Deep‐well microtiter plates
  • Fluorescence polarization microplate reader (Molecular Devices Analyst HT or Analyst GT, or equivalent)

Support Protocol 4: Determination of KM of PDE1

  • Lysate (supernatant) of dissected hippocampal or striatal tissues (see protocol 10)
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Figures

Videos

Literature Cited

Literature Cited
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