Discovery Of Small Molecular Inhibitors Targeting Monoacylglycerol Lipase

Monoacylglycerol lipase is a metabolic enzyme which is responsible for hydrolyzing monoacylglycerols to fatty acids and glycerol.2-arachidonoylglycerol（2-AG）is one kind of substrate of MAGL,which is both a special monoacylglycerol and a vital endogenous ligand in the endocannabinoid system（ECS）.2-AG plays an important role in physiological process through activating CB receptor and participating in the lipid metabolism process.A number of studies have manifested that inhibition of MAGL could exerting anti-tumor and anti-inflammatory effects by regulating the fatty acid network and the metabolism of 2-AG in the ECS.Therefore,the discovery of MAGL inhibitors is of great significance.In this study,firstly virtual screening and enzyme inhibition assay were conducted to screen compounds and determine the activity of hits.Then the binding affinity between compound and protein was measured through surface plasma resonance experiment（SPR）,and the molecular docking was utilized to predict the interaction mode.Through the quantitative determination of intracellular 2-AG and RT-q PCR technology,we detected the effects of the hit on intracellular 2-AG level and the intervention on inflammatory response.In addition,we screened the in-house compound library via enzyme inhibition assay,and identified compounds with potent activity and new scaffolds.Then the binding affinity between the most potent compound and protein was measured through the microscale thermophoresis experiment（MST）.Finally,the inhibition mechanism of the compound was confirmed by MAGL dilution assay,preincubation assay and enzyme kinetics experiment.In the work of using virtual screening to search for MAGL inhibitors,a non-covalent inhibitor DC630-8 was obtained(IC₅₀=2.84μM).SPR experiment proved that there was a significant interaction between the compound and MAGL.The result of molecular docking revealed that DC630-8 could form hydrogen bond orπ-πinteraction with several significant amino acid residues of MAGL.Furthermore,DC630-8 could significantly increase the level of 2-AG in RAW264.7 cells,and inhibit the m RNA expression of several inflammatory factors and chemokines after LPS stimulation.In another part of our work,we screened compounds using enzyme inhibition assay,we found that Y-WSG-0915 exhibited the most potent MAGL inhibitory activity(IC₅₀=47.62 n M),and the binding affinity between Y-WSG-0915and MAGL was strong as well（K_d=75.87 n M）.Later,our study has proved that Y-WSG-0915 is a reversible and mixed type inhibitor of MAGL.In this study,two novel scaffolds of MAGL small molecular inhibitors were obtained through two screening methods,which are expected to provide molecular probes for the study of MAGL-associated biological function and subsequent drug developments.