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.

Keywords: PLG2; bioluminescence; kinetic assay; monoacylglycerol lipase.

Scheme 1

Bioluminescence reactions of compound 6′-O-arachidonoylluciferin (ArLuc-1). The substrate chemical structure and biochemical reactions leading to 562 nm peak light emission (λ max) are shown.

Scheme 2

Chemical synthesis of ArLuc-1.

Figure 1

Comparison of luminescence produced by ArLuc-1 in presence (●) or absence (▪) of Monoacylglycerol lipase (MAGL). Data represent mean ± standard deviation (SD).

Figure 2

PLG2 luminescence emission in the presence of varying concentrations from 0.5 to 5% of DMSO. Error bars represent mean ± SD.

Figure 3

Luminescence (RLU) of PLG2 as a linear function of LH₂ concentration in the presence of 2.5% DMSO. All points are expressed as mean ± SD.

Figure 4

PLG2 activity in presence of the indicated concentrations of ArLuc-1. Error bars represent mean ± SD.

Figure 5

Kinetic study of MAGL activity with ArLuc-1. Data are reported as mean ± SD. The curve model is that of the Michaelis–Menten.

Figure 6

Concentration-dependent inhibition of JZL184 on MAGL. Data, reported as mean ± SD, are expressed as the percentage of controls (without JZL184).

Figure 7

Top scoring docking pose of compound ArLuc-1 (green stick) into the MAGL binding site. Residues are numbered according to the PDB crystal (5ZUN) convention. The triad catalytic residues are shown in orange sticks, those forming the oxyanion hole in magenta sticks. H-bonds and the π–π stacking interaction are represented with yellow and blue dashed lines, respectively. The black arrow represents the distance between the oxygen atom of the hydroxyl group of Ser122 and the carbon of the carbonyl group of ArLuc-1.

Figure 8

2D representation of interactions between MAGL binding site and ArLuc-1.