Biochemical and Pharmacological Characterization of Human a/ß Hydrolase Domain 6

Human α/β‐hydrolase domain‐containing 6 (ABHD6) is an uncharacterized endocannabinoid enzyme that inactivates the bioactive lipid‐signaling molecule 2‐arachidonoylglycerol, a potent agonist at both cannabinoid receptors. Inhibition of ABHD6 has therapeutic potential for addiction, pain, inflammation, cancer, and neurodegenerative/neuroinflammatory disorders. Human cDNA encoding native ABHD6 and N‐terminal transmembrane domain truncated variants were overexpressed in E. coli . Catalytic activities of all recombinant proteins were evaluated in a novel assay based on the fluorogenic substrate 7‐hydroxy‐6‐methoxy‐4‐methylcoumarin ester (AHMMCE). Intact and N‐terminal D37‐truncated ABHD6 showed similar K m and V max parameters, indicating that the TM domain is not essential for catalytic function. However, the N‐terminal D42‐truncated variant's ability to hydrolyze AHMMCE was significantly impaired, suggesting that residues between the 37 th and 42 nd amino acids may be essential for catalytic activity. Overexpressed ABHD6 enzyme was used in a novel high‐throughput fluorescent assay for the primary screening of potential inhibitors. Selectivity of identified inhibitors for ABHD6 over other serine hydrolases, is evaluated using affinity based protein profiling (ABPP) assay that has been optimized using the serine hydrolase specific covalent fluorescent probe FP‐rhodamine. Obtaining recombinant ABHD6 enzyme allows us characterize the binding pocket and assists in design of potent and selective ABHD6 inhibitors as novel analgesic and anti‐inflammatory agents. Acknowledgement: This work was supported by the NIDA grant DA003801 (A.M.)