Characterization of the α/β-hydrolase domain 6 (ABHD6) enzyme as a new target for drug discovery

Abstract

The serine α/β-hydrolase domain 6 (ABHD6) enzyme, a member of the endocannabinoid system, hydrolyzes the lipid signaling molecule 2-arachidonyl glycerol (2-AG) both in the CNS and periphery. Mounting evidence connects selective ABHD6 inhibition and positive outcomes for several diseases like metabolic disorders, inflammation, and neurodegenerative diseases. Therefore, ABHD6 becomes an attractive target for therapeutic intervention, yet, it is not well-characterized and structural data are lacking. Several hABHD6 constructs were developed for expression and purification of functional hABHD6 variants for biochemical characterization and structural studies. The hΔ29-4-ABHD6 was the most promising variant, without the first 29 amino acids and four aromatic residues (Y38YWY41) at the N-terminal end of the protein. It was expressed in BL21 E. coli cells under optimized conditions, and its activity was assessed using a fluorogenic and native substrates. The recombinant protein was successfully purified using immobilized metal affinity chromatography (IMAC) with and without detergent. The detergent-free purified hABHD6 was suitable for biophysical and biochemical characterization of the enzyme (structurally and functionally). This variant was also used to study interaction with different ABHD6 inhibitors in several biophysical and biochemical experiments. The active site of the hABHD6 and the mode of interaction of potent inhibitors were characterized using ligand-assisted protein structure approach and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The carbamate-based ABHD6 inhibitors have been confirmed to covalently interact and selectively carbamylate the enzyme active-site serine residue in MS studies. The stability of enzyme-inhibitor complexes were evaluated in protein NMR studies by calculating the residence time of covalent ABHD6 inhibitor. NMR experiments were able to discriminate between covalent and noncovalent inhibition. The onset of inhibition and extent of hydrolysis of ABHD6 inhibitors were assessed biochemically in extended preincubation time assay. The IC50 shift assay determined the inhibitors with time-dependent inhibition. The stability of the hΔ29-4-ABHD6 protein alone and with inhibitors was evaluated in thermal shift assay to identify the most suitable samples for x-ray structural studies. Samples of hΔ29-4-ABHD6 protein complexed with the candidate inhibitors were prepared for x-ray analysis. Collectively, the data of hABHD6-inhibitor interactions in LAPS, NMR and biochemical experiments provided a new direction in future design of the hABHD6 inhibitors. Obtaining the crystal structure of hABHD6 will significantly advance the drug discovery field by providing better understanding of the protein structure and identifying the key interactions between the ABHD6 enzyme and bound inhibitors.