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

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.

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

“…An optimal HTS assay for changes in NAPE-PLD activity requires high reproducibility for replicate samples and sufficient dynamic range to reliably detect activators and inhibitors. Fluorogenic lipid substrates have been previously used successfully in HTS assays for modulators of lipase activity (38)(39). PED-A1 is a quenched fluorogenic NAPE analog that has previously been used to measure phospholipase A1 (PLA 1) activity in vitro and in tissue samples (40).…”

Small-Molecules as Chemiluminescent Probes to Detect Lipase Activity

“…The choice of this acyl chain derives from our previous studies [ 31 , 32 , 33 , 34 , 35 ], which have suggested medium-short chains as a versatile substrate of lipase and esterase activity evaluation, and it is also supported by the recent literature [ 18 ]. Indeed, in a rational design of a substrate for lipolytic activity evaluation, the alkyl chain length should be carefully evaluated.…”

“…In the present study, continuing our interest in lipase selective probe development [ 31 , 32 , 33 , 34 , 35 ] suitable for in vitro and in vivo assays, we investigated a group of phenoxy-1,2-dioxetane luminophores, 2 – 4 , carrying an octanoyl chain as a triggerable substrate. The enzymatic cleavage of these phenoxy-dioxetane luminophores generates a direct chemiluminescence emission which is strongly dependent on the lipase activity.…”

2-Arachidonoylglycerol Synthesis: Facile and Handy Enzymatic Method That Allows to Avoid Isomerization

“…uronium hexafluorophosphate (COMU) in the presence of diisopropylethylamine (DIPEA), or the activation of the acidic moiety passing through chlorine using oxalylchloride, were assessed. At first, COMU, a molecule of a third-generation uronium-type coupling previously used for the synthesis of other EC and NAEs or their derivatives [39][40][41][42][43], was assessed for arachidonate ester placement in the sn-2 position. Unfortunately, this procedure gave low yields (<40%) associated with long reaction times (>24 h).…”