Abstract
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.
Highlights
The endocannabinoid system (ES) is a modulatory system that plays a critical role in a variety of neuronal functions including motor activity, nociception, and appetite control as well as peripheral functions such as energy metabolism and inflammation [1]
We have previously reported an efficient protocol for a Monoacylglycerol lipase (MAGL) continuous assay, based on a new long-wavelength fluorogenic substrate, 7-hydroxyresorufinyl arachidonate 1g [12]
Numerous assay methodologies have been published in the past decades for measurement of MAGL activity, few of them allow the screening of a large number of compounds showing high sensibility, high throughput, and low assay cost
Summary
The endocannabinoid system (ES) is a modulatory system that plays a critical role in a variety of neuronal functions including motor activity, nociception, and appetite control as well as peripheral functions such as energy metabolism and inflammation [1] This wide range of effects makes it an interesting target for drug development a difficult one to act upon due to life-threatening psychological adverse effects [2] linked to the direct antagonist action against the endocannabinoid receptors. Monoacylglycerol lipase (MAGL), a soluble serine hydrolase that associates with cell membranes, is a very promising target for pharmacological inhibition, since it is the main hydrolase implicated in the degradation of the main signaling molecule in the ES, 2-AG.
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