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Article

Set-Up and Validation of a High Throughput Screening Method for Human Monoacylglycerol Lipase (MAGL) Based on a New Red Fluorescent Probe

1
Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”, Università degli Studi di Milano, Via G.B. Grassi 74, 20157 Milano, Italy
2
Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Via Fratelli Cervi 93, 20090 Segrate (MI), Italy
3
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy
*
Author to whom correspondence should be addressed.
Academic Editor: Anton Simeonov
Molecules 2019, 24(12), 2241; https://doi.org/10.3390/molecules24122241
Received: 7 May 2019 / Revised: 6 June 2019 / Accepted: 14 June 2019 / Published: 15 June 2019
(This article belongs to the Special Issue High Throughput Screening II)
Monoacylglycerol lipase (MAGL) is a serine hydrolase that has a key regulatory role in controlling the levels of 2-arachidonoylglycerol (2-AG), the main signaling molecule in the endocannabinoid system. Identification of selective modulators of MAGL enables both to provide new tools for investigating pathophysiological roles of 2-AG, and to discover new lead compounds for drug design. The development of sensitive and reliable methods is crucial to evaluate this modulatory activity. In the current study, we report readily synthesized long-wavelength putative fluorogenic substrates with different acylic side chains to find a new probe for MAGL activity. 7-Hydroxyresorufinyl octanoate proved to be the best substrate thanks to the highest rate of hydrolysis and the best Km and Vmax values. In addition, in silico evaluation of substrates interaction with the active site of MAGL confirms octanoate resorufine derivative as the molecule of choice. The well-known MAGL inhibitors URB602 and methyl arachidonylfluorophosphonate (MAFP) were used for the assay validation. The assay was highly reproducible with an overall average Z′ value of 0.86. The fast, sensitive and accurate method described in this study is suitable for low-cost high-throughput screening (HTS) of MAGL modulators and is a powerful new tool for studying MAGL activity. View Full-Text
Keywords: high-throughput screening; endocannabinoid; monoacylglycerol lipase; assay development high-throughput screening; endocannabinoid; monoacylglycerol lipase; assay development
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MDPI and ACS Style

Miceli, M.; Casati, S.; Ottria, R.; Di Leo, S.; Eberini, I.; Palazzolo, L.; Parravicini, C.; Ciuffreda, P. Set-Up and Validation of a High Throughput Screening Method for Human Monoacylglycerol Lipase (MAGL) Based on a New Red Fluorescent Probe. Molecules 2019, 24, 2241. https://doi.org/10.3390/molecules24122241

AMA Style

Miceli M, Casati S, Ottria R, Di Leo S, Eberini I, Palazzolo L, Parravicini C, Ciuffreda P. Set-Up and Validation of a High Throughput Screening Method for Human Monoacylglycerol Lipase (MAGL) Based on a New Red Fluorescent Probe. Molecules. 2019; 24(12):2241. https://doi.org/10.3390/molecules24122241

Chicago/Turabian Style

Miceli, Matteo; Casati, Silvana; Ottria, Roberta; Di Leo, Simone; Eberini, Ivano; Palazzolo, Luca; Parravicini, Chiara; Ciuffreda, Pierangela. 2019. "Set-Up and Validation of a High Throughput Screening Method for Human Monoacylglycerol Lipase (MAGL) Based on a New Red Fluorescent Probe" Molecules 24, no. 12: 2241. https://doi.org/10.3390/molecules24122241

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