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A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase. 2021

Matteo Miceli, and Silvana Casati, and Pietro Allevi, and Silvia Berra, and Roberta Ottria, and Paola Rota, and Bruce R Branchini, and Pierangela Ciuffreda
Dipartimento di Scienze Biomediche e Cliniche "Luigi Sacco", Università degli Studi di Milano, Via G.B. Grassi 74, 20157 Milano, Italy.

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

UI MeSH Term Description Entries
D008156 Luciferases Enzymes that oxidize certain LUMINESCENT AGENTS to emit light (PHYSICAL LUMINESCENCE). The luciferases from different organisms have evolved differently so have different structures and substrates. Luciferase
D008994 Monoacylglycerol Lipases An enzyme that catalyzes the hydrolysis of glycerol monoesters of long-chain fatty acids EC 3.1.1.23. Glycerol Monoester Hydrolase,Glycerol Monoester Hydrolases,Monoacylglycerol Lipase,Monoglyceride Esterase,Monoglyceride Esterases,Monoglyceride Lipase,Monoglyceride Lipases,Monoglyceride Hydrolase,Esterase, Monoglyceride,Esterases, Monoglyceride,Hydrolase, Glycerol Monoester,Hydrolase, Monoglyceride,Hydrolases, Glycerol Monoester,Lipase, Monoacylglycerol,Lipase, Monoglyceride,Lipases, Monoacylglycerol,Lipases, Monoglyceride,Monoester Hydrolase, Glycerol,Monoester Hydrolases, Glycerol
D010880 Piperidines A family of hexahydropyridines.
D004791 Enzyme Inhibitors Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction. Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001681 Biological Assay A method of measuring the effects of a biologically active substance using an intermediate in vivo or in vitro tissue or cell model under controlled conditions. It includes virulence studies in animal fetuses in utero, mouse convulsion bioassay of insulin, quantitation of tumor-initiator systems in mouse skin, calculation of potentiating effects of a hormonal factor in an isolated strip of contracting stomach muscle, etc. Bioassay,Assay, Biological,Assays, Biological,Biologic Assay,Biologic Assays,Assay, Biologic,Assays, Biologic,Bioassays,Biological Assays
D014151 Anti-Anxiety Agents Agents that alleviate ANXIETY, tension, and ANXIETY DISORDERS, promote sedation, and have a calming effect without affecting clarity of consciousness or neurologic conditions. ADRENERGIC BETA-ANTAGONISTS are commonly used in the symptomatic treatment of anxiety but are not included here. Anti-Anxiety Agent,Anti-Anxiety Drug,Anxiolytic,Anxiolytic Agent,Anxiolytic Agents,Tranquilizing Agents, Minor,Anti-Anxiety Drugs,Anti-Anxiety Effect,Anti-Anxiety Effects,Antianxiety Effect,Antianxiety Effects,Anxiolytic Effect,Anxiolytic Effects,Anxiolytics,Tranquillizing Agents, Minor,Agent, Anti-Anxiety,Agent, Anxiolytic,Agents, Anti-Anxiety,Agents, Anxiolytic,Agents, Minor Tranquilizing,Agents, Minor Tranquillizing,Anti Anxiety Agent,Anti Anxiety Agents,Anti Anxiety Drug,Anti Anxiety Drugs,Anti Anxiety Effect,Anti Anxiety Effects,Drug, Anti-Anxiety,Drugs, Anti-Anxiety,Effect, Anti-Anxiety,Effect, Antianxiety,Effect, Anxiolytic,Effects, Anti-Anxiety,Effects, Antianxiety,Effects, Anxiolytic,Minor Tranquilizing Agents,Minor Tranquillizing Agents
D049449 Luminescence Emission of LIGHT when ELECTRONS return to the electronic ground state from an excited state and lose the energy as PHOTONS. It is sometimes called cool light in contrast to INCANDESCENCE. LUMINESCENT MEASUREMENTS take advantage of this type of light emitted from LUMINESCENT AGENTS. Luminescence, Physical,Chemiluminescence,Chemiluminescence, Physical,Physical Chemiluminescence,Physical Luminescence
D052117 Benzodioxoles Compounds based on benzene fused to oxole. They can be formed from methylated CATECHOLS such as EUGENOL. Methylenedioxybenzenes,1,3-Dioxaindans,1,3-Dioxindans,1,3 Dioxaindans,1,3 Dioxindans
D052160 Benzothiazoles Compounds with a benzene ring fused to a thiazole ring.

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