SUMMARY The serine esterase monoacylglycerol lipase (MGL) is primarily responsible for deactivating the signaling lipid 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both principal cannabinoid receptors. Although MGL is recognized as a potential therapeutic target, the paucity of structural information on this enzyme has hindered development of MGL-selective inhibitors. Previously, we overexpressed and purified human MGL as the hexa-histidine-tagged recombinant protein (hMGL) and showed that it catalyzed the hydrolysis of both 2-AG and novel fluorogenic reporters. We now characterize by mass spectroscopy the hMGL active site using two chemically distinct inhibitors as direct probes: 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-arachidonylmaleimide (NAM). Suitable conditions were established for hMGL inhibition by AM6701, and the inhibitor-treated enzyme was subjected to trypsin digestion. The tryptic digest of AM6701-inhibited hMGL was analyzed by MALDI-TOF and tandem MS, which showed that AM6701 had carbamylated the serine in a GXSXG motif of the putative MGL catalytic triad. These results provide the first direct confirmation of the essential role of this serine residue for catalysis and establish the mechanism of AM6701 as a high-affinity, covalent hMGL inhibitor. When applied to NAM-treated hMGL, our direct, ligand-assisted approach revealed that partial alkylation of cysteine residues 215 and/or 249 was sufficient to achieve ~ 80% hMGL inhibition. Further alkylation at cysteine 39 did not increase the extent of enzyme inhibition. Although Cys215 and/or Cys249 mutations to alanine(s) did not affect hMGL’s ability to hydrolyze reporter substrate, as compared to nonmutated hMGL the C215A mutant was more sensitive to NAM, whereas the C249A mutation reduced the enzyme’s sensitivity to NAM. These data conclusively demonstrate a sulfhydryl-based mechanism underlying MGL inhibition by this fatty alkyl-maleimide substrate analog in which Cys249 is of paramount importance. Identification of amino acids critical to catalysis by and pharmacological modulation of hMGL provides information useful in the design of selective MGL inhibitors as potential drugs.

Descriptions of materials, metrological methods, computational methods, and supplementary results. Figures of HDX-MS publications and citations versus publication year, histogram of peptide sequence lengths, sequence coverage maps, performance of instrumentsoftware configurations, repeatability plots, %E corrected peptide t HDX versus log 10 (t HDX ) for eight peptides. Tables of instrumentation,software, peptide search methodology, and operating conditions of proteolytic, chromatographic components, and effects of peptide charge on deuterium uptake (PDF)

The serine hydrolase monoacylglycerol lipase (MGL) functions as the main metabolizing enzyme of 2-arachidonoyl glycerol, an endocannabinoid signaling lipid whose elevation through genetic or pharmacological MGL ablation exerts therapeutic effects in various preclinical disease models. To inform structure-based MGL inhibitor design, we report the direct NMR detection of a reversible equilibrium between active and inactive states of human MGL (hMGL) that is slow on the NMR time scale and can be modulated in a controlled manner by pH, temperature, and select point mutations. Kinetic measurements revealed that hMGL substrate turnover is rate-limited across this equilibrium. We identify a network of aromatic interactions and hydrogen bonds that regulates hMGL active-inactive state interconversion. The data highlight specific inter-residue interactions within hMGL modulating the enzymes function and implicate transitions between active (open) and inactive (closed) states of the hMGL lid domain in controlling substrate access to the enzymes active site.