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Graduate Category: Physical and Life Sciences Degree Level: PhD Abstract ID# 1160 Proteomic Characterization of Recombinant Human α/β Hydrolase Domain 6 The biochemistry behind making a better Marijuana Christina Miyabe, Nikolai Zvonok, Alexander Zvonok and Alexandros Makriyannis Center for Drug Discovery (Northeastern University) Boston, MA Abstract: Background: Human α/β Hydrolase Domain 6 (hABHD6) is an endocannabinoidmetabolizing serine hydrolase that has been found to inactivate 2arachidonoylglycerol (2-AG), a potent agonist at both cannabinoid receptors. ABHD6 is tethered to the membrane post-synaptically, allowing it to control 2-AG concentrations at a unique subcellular localization as compared to its relative, monoacylglyerol lipase (MGL). Selective ABHD6 inhibitors have been shown to have therapeutic potential towards addiction, pain, inflammation, cancer, and metabolic, neuroinflammatory, and neurodegenerative disorders, but more structural data is needed to develop ABHD6selective inhibitors. Recombinant hABHD6 overexpressed in E. coli and purified by single step immobilized metal affinity chromatography is catalytically active and has comparable 2-AG hydrolysis parameters to the native enzyme. A novel highthroughput assay based on the fluorogenic substrate arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE) was developed and used to identify potent ABHD6 inhibitors. Matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was used to analyze hABHD6 by proteomic peptide fingerprinting and the identification covalent interactions between the active-site serine and potent inhibitors. AM6701, which has been shown to carbamylate the active-site serine of hMGL, acts through the same mechanism to inhibit ABHD6. The ABHD6-selective inhibitor WWL70 formed covalent attachment by SN2 addition; this is the first reported evidence for the molecular mechanism of action of WWL70. The further structural characterization of the binding pocket of ABHD6 is critical to identifying and developing more potent, selective inhibitors as new potential therapeutic agents. The psychoactive component of the marijuana plant Cannabis sativa, Δ9-tetrahydrocannabinol, was discovered and isolated in the 1960’s, while the endogenous ligands (endocannabinoids) and the receptors they interact with were not identified until much later in 1992. Since then, research has pushed towards understanding the underlying mechanism behind marijuana’s myriad of effects by better elucidating the function of the endogenous system it interacts with in an effort to design potent, selective therapeutic drugs that act upon the proteins in the system. References: arachidonic acid glycerol S148 2-AG catalytic triad transmembrane domain Results: Conclusion: Mass Spectrometry Protein Purification Human ABHD6 is a new, relatively unstudied endocannbinoid-metabolizing enzyme that has promising potential a therapeutic drug target. We have successfully genetically modified and purified an active variant of hABHD6 recombinantly expressed in the E. coli host system. We have also identified the mechanism of inhibition for two covalent inhibitors: AM6701 and WWL70. hABHD6 IC50 AM6701 +71 IC50 = 0.9 nM 5037- 1. Ahn, K., McKinney, M. K., and Cravatt, B.F. (2008) Chem. Rev. 108, 1687-1707. 2. Blankman, J. L., Simon, G. M., and Cravatt, B.F. (2007) Chem. Biol. 14, 1347-1356. 3. Saario, S. M., and Laitinen, J. T. (2007) Basic Clin. Pharmacol. Toxicol. 101, 287-293. +224 IC50 = 295 nM Acknowledgements: This work was supported by the NIH grant DA003801 (A.M.) and a PhD Candidate Fellowship Diversity Supplement from the National Institute on Drug Abuse. homology model of hABHD6 H306 D278 Digest protein with trypsin Analyze/identify specific peptides