Phosphodiesterase Enzyme Assays

All PDE enzyme activities were measured with a yittrium silicate based scintillation proximity assay that detects radioactive nucleotide monophosphates but not cyclic monophosphates. Assays are conducted in a total volume of 50 μl in 384 well plates (3706, Corning): comprised of 24 μl enzyme, 1 μl compound, and 25 μl of cyclic nucleotide. The general assay buffer consists of 50 mM Tris-HCl (pH 7.5) and 0.1% (w/v) bovine serum albumin (Fatty Acid Free, Sigma-Aldrich). Enzyme concentrations, substrate concentrations, and various PDE-specific buffer additives are described in Supplementary Table 1. Compounds to be tested are diluted in pure dimethyl sulfoxide (DMSO) using 10-point concentration-response curves, and 1 μl is acoustically dispensed into assay plates using the Echo555 (LabCyte). Maximal compound concentration in the reaction mixture is either 10 or 100 μM. Compounds at the appropriate concentration are pre-incubated with either of the PDE enzymes for 30 min before the reaction is started by the addition of substrate ([8-³H]-cAMP, 20.7 Ci/mmol or [8-³H]-cGMP; 6.5 Ci/mmol, Perkin Elmer). Reactions are allowed to proceed for 60 min at room temperature before quenching. Next, reactions are stopped by the addition of 400 μg/per well SPA beads (RPNQ0150, Perkin Elmer) and a potent non-selective PDE inhibitor to quench the reaction. Bead bound radioactivity (product) is quantified 12 h later a Microbeta counter (Perkin Elmer). Data is normalized to % inhibition using standard methods (Campbell et al., 2004), and IC₅₀ values were plotted using PRISM (GraphPad Software) using the 4 parameter logistic equation (Campbell et al., 2004). Data for potency values are expressed as the geometric mean and arithmetic standard deviation.