A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase

Abstract

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

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.…”