A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α
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 treat...
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| Format: | Article |
| Language: | English |
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Elsevier
2015-04-01
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| Series: | Journal of Lipid Research |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520355735 |
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| author | Tom van der Wel Freek J. Janssen Marc P. Baggelaar Hui Deng Hans den Dulk Herman S. Overkleeft Mario van der Stelt |
| author_facet | Tom van der Wel Freek J. Janssen Marc P. Baggelaar Hui Deng Hans den Dulk Herman S. Overkleeft Mario van der Stelt |
| author_sort | Tom van der Wel |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T03:58:48Z |
| format | Article |
| id | doaj.art-32527724ab1b4d64aa803f3844dc22cb |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-14T03:58:48Z |
| publishDate | 2015-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-32527724ab1b4d64aa803f3844dc22cb2022-12-21T23:18:00ZengElsevierJournal of Lipid Research0022-22752015-04-01564927935A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase αTom van der Wel0Freek J. Janssen1Marc P. Baggelaar2Hui Deng3Hans den Dulk4Herman S. Overkleeft5Mario van der Stelt6Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The NetherlandsDepartment of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The NetherlandsDepartment of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The NetherlandsDepartment of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The NetherlandsDepartment of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The NetherlandsDepartment of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The NetherlandsTo whom correspondence should be addressed; To whom correspondence should be addressed; Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The NetherlandsThe 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.http://www.sciencedirect.com/science/article/pii/S00222275203557352-arachidonoylglycerolcannabinoidsendocannabinoidenzymologylipidsobesity |
| spellingShingle | Tom van der Wel Freek J. Janssen Marc P. Baggelaar Hui Deng Hans den Dulk Herman S. Overkleeft Mario van der Stelt A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α Journal of Lipid Research 2-arachidonoylglycerol cannabinoids endocannabinoid enzymology lipids obesity |
| title | A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α |
| title_full | A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α |
| title_fullStr | A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α |
| title_full_unstemmed | A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α |
| title_short | A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α |
| title_sort | natural substrate based fluorescence assay for inhibitor screening on diacylglycerol lipase α |
| topic | 2-arachidonoylglycerol cannabinoids endocannabinoid enzymology lipids obesity |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520355735 |
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