A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α.
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| 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.
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| Authors | Tom van der Wel, Freek J Janssen, Marc P Baggelaar, Hui Deng, Hans den Dulk, Herman S Overkleeft, Mario van der Stelt |
| Journal | Journal of lipid research (J Lipid Res) Vol. 56 Issue 4 Pg. 927-35 (Apr 2015) ISSN: 1539-7262 [Electronic] United States |
| PMID | 25684760 (Publication Type: Journal Article) |
| Copyright | Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc. |
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