The endocannabinoid system has been shown to have a vast span of therapeutic applications due to its integral role in maintaining neurochemical homeostasis in the mammalian brain. A recently discovered endocannabinoid-degrading enzyme, alpha/beta hydrolase domain 6 enzyme (ABHD6), is an attractive new drug target and its inhibition can be applied to a number of disease pathologies including cancer, neurodegenerative disorders, and metabolic disorders. As it has only recently been discovered, little is known about ABHD6 and this has stunted the structure-based design of potent, specific inhibitors.This project focused on the biochemical, pharmacological, and mass spectrometric characterization of ABHD6 enzyme as a drug target. Cloning, E. coli-based expression, genetic engineering of soluble variation, and simple purification steps provided soluble, pure hABHD6 enzyme variants in enough quantities for these studies. A novel highthroughput biochemical screening assay was developed using optimized, coumarinbased fluorogenic substrates; additionally, the activity-based proteome profiling (ABPP) assay was adapted to screen recombinant endocannabinoid enzyme inhibitors for selectivity. Lead compounds showing potency moved forward to a more in-depth 8-point determination assay. Mutagenesis studies truncating the transmembrane domain (TM) of ABHD6 showed the TM is not necessary for catalysis. Ligand-assisted protein structure (LAPS) experiments performed using previously published, ABHD6 inhibitors WWL70 and AM6701 were the first to show proof of covalent mechanism for both inhibitors. These data were used to correctly dock the ligands onto a new homology model of ABHD6 to determine potential key interactions in the binding pocket.ii ACKNOWLEDGEMENTS

By use of osrylamide gel electmphoresis it has been Possible to identify of individual enzymes in crude extracts by protein bands (Steward and Barber I%4 Ann. N. Y. Acad. Sci. 121525