title: High-performance liquid chromatography assay with fluorescence detection for the evaluation of inhibitors against human recombinant monoacylglycerol lipase.
abstract: 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.
authors.affiliation: Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, D-48149 Münster, Germany.
authors.forename: Angela
authors.initials: A
authors.lastname: Holtfrerich
authors.forename: Teona
authors.initials: T
authors.lastname: Makharadze
authors.forename: Matthias
authors.initials: M
authors.lastname: Lehr
mesh_terms: D001095:Arachidonic Acids / Q000737:chemistry / Q000494:pharmacology; D052117:Benzodioxoles / Q000737:chemistry / Q000494:pharmacology; D020148:Butyric Acid / Q000737:chemistry / Q000378:metabolism; D002219:Carbamates / Q000737:chemistry / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008994:Monoacylglycerol Lipases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017830:Octoxynol / Q000737:chemistry; D063065:Organophosphonates / Q000737:chemistry / Q000494:pharmacology; D010069:Oxadiazoles / Q000737:chemistry / Q000494:pharmacology; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D001095:Arachidonic Acids; D052117:Benzodioxoles; C534918:CAY 10499; D002219:Carbamates; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C534333:JZL 184; D063065:Organophosphonates; D010069:Oxadiazoles; D010880:Piperidines; D011994:Recombinant Proteins; C104810:methyl arachidonylfluorophosphonate; D020148:Butyric Acid; D017830:Octoxynol; D008994:Monoacylglycerol Lipases