Cyclic nucleotide phosphodiesterases (PDEs) represent eleven different gene families of enzymes that are encoded by at least 21 different genes and accounting for at least 55 isoforms. They have been implicated in controlling specific cellular functions including cardiovascular, pulmonary, and inflammatory diseases, and they play potential role as targets for treatment of cancer. Inhibition of PDEs was shown to induce chronic lymphocytic leukemia (CLL) apoptosis and more evidence have evolved supporting targeting PDEs for treatments of several types of cancer. Thus, the development of novel isoform-specific inhibitors may be useful for the development of therapeutic agents against cancer. We have devised two novel strategies to monitor the activity of cAMP-PDE and cGMP-PDE and developed two homogenous, luminescent high through-put screening (HTS) assays to screen for novel inhibitors of PDE. The first strategy relies on the activation of cAMP-Dependent Protein Kinase (PKA) in the presence of low concentration of cAMP or by moderately higher concentration of cGMP. The effect of compounds on the activity of PDE can be monitored by the cAMP-dependent activation of PKA and measuring the active PKA using luminescent kinase assay. The other strategy relies on the phosphorylation of AMP product formed from cAMP in the presence of PDE into ADP and then ATP and measuring the generated ATP using luminescent kinase assay. Both strategies have been developed into HTS formatted assays that can be carried out in two simple steps in 96-, 384-, or 1536-well formatted plates. The assays are luminescent and thus they encounter no interference from fluorescent compounds and the robustness of the assays are indicated by their remarkably high Z’ value (>0.8). In summary, both assays can be used to screen libraries of compounds targeting PDEs that might be developed into potential drugs for treatment of cancer.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA