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Link to original content: https://doi.org/10.1007/s00216-015-8991-9
Synthesis and characterization of a new fluorogenic substrate for monoacylglycerol lipase and application to inhibition studies | Analytical and Bioanalytical Chemistry Skip to main content

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Synthesis and characterization of a new fluorogenic substrate for monoacylglycerol lipase and application to inhibition studies

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Abstract

Human monoacylglycerol lipase (MAGL), a soluble serine hydrolase that belongs to the α/β hydrolase fold superfamily, regulates 2-arachidonoyl glycerol level in the endocannabinoid system, which is implicated in a number of severe diseases, and therefore, inhibition of MAGL activity is crucial in the treatment of these diseases. We have synthesized a red fluorogenic substrate, 7-hydroxyresorufinyl-arachidonate (7-HRA), for a new MAGL assay. This assay is simple, sensitive, and reliable and useful for identifying compounds that modulate MAGL activity. In addition, the assay emits red fluorescence, which can significantly reduce interference due to compound fluorescence and dust or lint, all of which fluoresce in the blue wavelength. MAGL catalyzes the hydrolysis of 7-HRA to generate arachidonic acid and a highly red fluorescent resorufin, excitation at 571 nm and emission at 588 nm, with a Km of 0.87 μM and Vmax of 26 nmol min−1 mg protein−1. The known MAGL inhibitors URB602, methyl arachidonyl fluorophosphonate, and JZL184 were used to validate the test assay. The assay was highly reproducible with an overall average Z′ value of 0.80. This new red fluorogenic substrate and the resulting enzyme assay could be used in high-throughput screening to identify and develop new potential MAGL inhibitors.

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Acknowledgments

We thank the University of Milan for financial support.

Conflict of interest

The authors declare that they have no conflict of interests.

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Authors and Affiliations

  1. Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”, Università degli Studi di Milano, via G. B. Grassi 74, 20157, Milan, Italy

    Simone Lauria, Silvana Casati & Pierangela Ciuffreda

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  1. Simone Lauria
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  2. Silvana Casati
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  3. Pierangela Ciuffreda
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Corresponding author

Correspondence to Pierangela Ciuffreda.

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Lauria, S., Casati, S. & Ciuffreda, P. Synthesis and characterization of a new fluorogenic substrate for monoacylglycerol lipase and application to inhibition studies. Anal Bioanal Chem 407, 8163–8167 (2015). https://doi.org/10.1007/s00216-015-8991-9

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  • DOI: https://doi.org/10.1007/s00216-015-8991-9

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