title: A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α.
abstract: The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors.
authors: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
authors: Tom
authors: T
authors: van der Wel
authors: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
authors: Freek J
authors: FJ
authors: Janssen
authors: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
authors: Marc P
authors: MP
authors: Baggelaar
authors: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
authors: Hui
authors: H
authors: Deng
authors: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
authors: Hans
authors: H
authors: den Dulk
authors: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
authors: Herman S
authors: HS
authors: Overkleeft
authors: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
authors: Mario
authors: M
authors: van der Stelt
mesh_terms: D000818:Animals; D004075:Diglycerides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051379:Mice; D013050:Spectrometry, Fluorescence
chemical_list: D004075:Diglycerides; D004791:Enzyme Inhibitors; C075809:1-stearoyl-2-arachidonoylglycerol; D008071:Lipoprotein Lipase
keywords: 2-arachidonoylglycerol; cannabinoids; endocannabinoid; enzymology; lipids; obesity