High-performance liquid chromatography assay with fluorescence detection for the evaluation of inhibitors against human recombinant monoacylglycerol lipase.

A fluorescent assay for the evaluation of inhibitors of monoacylglycerol lipase (MAGL) is described. 1,3-Dihydroxypropan-2-yl 4-pyren-1-ylbutanoate was designed and synthesized as novel fluorogenic substrate. Activity of human recombinant MAGL was determined in the presence of the surfactant Triton X-100 without further sample cleanup by measuring the amount of 4-pyren-1-ylbutanoic acid released by the enzyme with reversed-phase high-performance liquid chromatography (HPLC) and fluorescence detection. The known covalent binding MAGL inhibitors methyl arachidonyl fluorophosphonate (MAFP), 4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]piperidine-1-carboxylate (JZL184), and [4-(5-methoxy-2-oxo-1,3,4-oxadiazol-3-yl)-2-methylphenyl]carbamic acid benzyl ester (CAY10499) were used to validate the test system. Applying an incubation time of 15 min, the IC(50) values obtained for these compounds were 0.16, 3.7, and 1.1 microM, respectively. A prolongation of the incubation to 45 min results in a two- to threefold decrease of the IC(50) values.