A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma

The activities of the phospholipase C gamma (PLC[gamma]) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLC[gamma]1 or PLC[gamma]2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLC[gamma] family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLC[gamma]1 and PLC[gamma]2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC.sub.1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLC[gamma] family of enzymes.