Endocannabinoid EnzymeEngineering: Soluble HumanThio-Monoacylglycerol Lipase (sol-S-hMGL)

摘要

In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu¹⁶⁹;Leu¹⁷⁶)-to-Ser(Ser¹⁶⁹;Ser¹⁷⁶) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser¹²² with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser¹²² residue as compared to that of Cys¹²² in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys¹²² residue is notcommensurately enhanced within the three dimensional architectureof hMGL. The interaction of the sol-hMGL variants with the irreversibleinhibitors AM6580 and N-arachidonylmaleimide (NAM)and the reversible inhibitor AM10212 was profiled. LC/MS analysisof tryptic digests from sol-S-hMGL directly demonstrate covalent modificationof this variant by NAM and AM6580, consistent with enzyme thiol alkylationand carbamoylation, respectively. These data provide insight intohMGL catalysis, the key role of the nucleophilic character of Ser¹²², and the mechanisms underlying hMGL inhibition by differentclasses of small molecules.