A fluorescence-based assay for monoacylglycerol lipase compatible with inhibitor screening.

A novel fluorescence-based assay of monoacylglycerol lipase (MAGL) activity that is simple, sensitive, and amenable to the screening of small molecule inhibitors is described. Purified recombinant human MAGL protein and 7-hydroxycoumarinyl-arachidonate (7-HCA), a fluorogenic substrate for MAGL, were employed in the assay. MAGL protein catalyzes the hydrolysis of 7-HCA to generate arachidonic acid and the highly fluorescent 7-hydroxyl coumarin (7-HC). Release of 7-HC was measured using a fluorometer. MAGL protein catalyzed the hydrolysis of 7-HCA with an apparent K(m) of 9.8 microM and V(max) of 1.7 mmol/min/mg of protein. The assay is specific for MAGL as assay buffer alone or heat-denatured MAGL protein had no significant activity against 7-HCA. Furthermore, MAGL activity was inhibited in a dose-dependent manner by the specific inhibitor URB602 as well as N-arachidonyl maleimide with 50% inhibitory concentration values of 3.1 microM and 155 nM, respectively. The assay was further optimized under different conditions, including pH range and bovine serum albumin protein and dimethyl sulfoxide concentrations. The assay was found to be reproducible, having Z' values ranging from 0.7 to 0.9, and is therefore suitable for high-throughput screening.