Multimodal imaging provides rich biological information, which can be exploited to study drug activity, disease associated phenotypes, and pharmacological responses. Here we show discovery and validation of a new probe targeting the endocannabinoid alpha/beta-hydrolase domain 6 (ABHD6) enzyme by utilizing positron emission tomography (PET) and matrix-assisted laser desorption/ionization (MALDI) imaging. [(18)F]JZP-MA-11 as the first PET ligand for in vivo imaging of the ABHD6 is reported and specific uptake in ABHD6-rich peripheral tissues and major brain regions was demonstrated using PET. A proof-of-concept study in nonhuman primate confirmed brain uptake. In vivo pharmacological response upon ABHD6 inhibition was observed by MALDI imaging. These synergistic imaging efforts used to identify biological information cannot be obtained by a single imaging modality and hold promise for improving the understanding of ABHD6-mediated endocannabinoid metabolism in peripheral and central nervous system disorders.
ABHD11 (alpha/beta-hydrolase domain containing 11) is a non-annotated enzyme belonging to the family of metabolic serine hydrolases (mSHs). Its natural substrates and products are unknown. Using competitive activity-based protein profiling (ABPP) to identify novel inhibitors of human (h)ABHD11, three compounds from our chemical library exhibited low nanomolar potency towards hABHD11. Competitive ABPP of various proteomes revealed fatty acid amide hydrolase (FAAH) as the sole off-target among the mSHs. Our fluorescent activity assays designed for natural lipase substrates revealed no activity of hABHD11 towards mono- or diacylglycerols. A broader profiling using para-nitrophenyl (pNP)-linked substrates indicated no amidase/protease, phosphatase, sulfatase, phospholipase C or phosphodiesterase activity. Instead, hABHD11 readily utilized para-nitrophenyl butyrate (pNPC4), indicating lipase/esterase-type activity that could be exploited in inhibitor discovery. Additionally, a homology model was created based on the crystal structure of bacterial esterase YbfF. In contrast to YbfF, which reportedly hydrolyze long-chain acyl-CoA, hABHD11 did not utilize oleoyl-CoA or arachidonoyl-CoA. In conclusion, the present study reports the discovery of potent hABHD11 inhibitors with good selectivity among mSHs. We developed substrate-based activity assays for hABHD11 that could be further exploited in inhibitor discovery and created the first homology-based hABHD11 model, offering initial insights into the active site of this poorly characterized enzyme.
The endocannabinoid system remains an attractive molecular target for pharmacological intervention due to its roles in the central nervous system in learning, thinking, emotional function, regulation of food intake or pain sensation, as well as in the peripheral nervous system, where it modulates the action of cardiovascular, immune, metabolic or reproductive function. alpha/beta hydrolase domain containing 6 (ABHD6)-an enzyme forming part of the endocannabinoid system-is a newly discovered post-genomic protein acting as a 2-AG (2-arachidonoylglycerol) serine hydrolase. We have recently reported a series of 1,2,5-thiadiazole carbamates as potent and selective ABHD6 inhibitors. Here, we present comparative molecular field analysis (CoMFA) and molecular dynamics studies of these compounds. First, we performed a homology modeling study of ABHD6 based on the assumption that the catalytic triad of ABHD6 comprises Ser148-His306-Asp 278 and the oxyanion hole is formed by Met149 and Phe80. A total of 42 compounds was docked to the homology model using the Glide module from the Schrodinger suite of software and the selected docking poses were used for CoMFA alignment. A model with the following statistics was obtained: R (2) = 0.98, Q (2) = 0.55. In order to study the molecular interactions of the inhibitors with ABHD6 in detail, molecular dynamics was performed with the Desmond program. It was found that, during the simulations, the hydrogen bond between the inhibitor carbonyl group and the main chain of Phe80 is weakened, whereas a new hydrogen bond with the side chain of Ser148 is formed, facilitating the possible formation of a covalent bond. Graphical Abstract Left-right: Docking pose of 1 in the binding pocket of alpha/beta hydrolase domain containing 6 (ABHD6) selected for molecular alignment; CoMFA steric and electrostatic contour fields; changes in potential energy of the complex during simulations for the complex of 6 and ABHD6.
At present, inhibitors of alpha/beta-hydrolase domain 6 (ABHD6) are viewed as a promising approach to treat inflammation and metabolic disorders. This article describes the development of 1,2,5-thiadiazole carbamates as ABHD6 inhibitors. Altogether, 34 compounds were synthesized, and their inhibitory activity was tested using lysates of HEK293 cells transiently expressing human ABHD6 (hABHD6). Among the compound series, 4-morpholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate (JZP-430) potently and irreversibly inhibited hABHD6 (IC50 =44 nM) and showed approximately 230-fold selectivity over fatty acid amide hydrolase (FAAH) and lysosomal acid lipase (LAL), the main off-targets of related compounds. Additionally, activity-based protein profiling indicated that JZP-430 displays good selectivity among the serine hydrolases of the mouse brain membrane proteome. JZP-430 has been identified as a highly selective, irreversible inhibitor of hABHD6, which may provide a novel approach in the treatment of obesity and type II diabetes.
Compound 12a (JZP-361) acted as a potent and reversible inhibitor of human recombinant MAGL (hMAGL, IC50=46nM), and was found to have almost 150-fold higher selectivity over human recombinant fatty acid amide hydrolase (hFAAH, IC50=7.24muM) and 35-fold higher selectivity over human alpha/beta-hydrolase-6 (hABHD6, IC50=1.79muM). Additionally, compound 12a retained H1 antagonistic affinity (pA2=6.81) but did not show cannabinoid receptor activity, when tested at concentrations 10muM. Hence, compound 12a represents a novel dual-acting pharmacological tool possessing both MAGL-inhibitory and antihistaminergic activities.
This article describes our systematic approach to exploring the utility of the 1,3,4-oxadiazol-2-one scaffold in the development of ABHD6 inhibitors. Compound 3-(3-aminobenzyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (JZP-169, 52) was identified as a potent inhibitor of hABHD6, with an IC50 value of 216nM. This compound at 10muM concentration did not inhibit any other endocannabinoid hydrolases, such as FAAH, MAGL and ABHD12, or bind to the cannabinoid receptors (CB1 and CB2). Moreover, in competitive activity-based protein profiling (ABPP), compound 52 (JZP-169) at 10muM selectively targeted ABHD6 of the serine hydrolases of mouse brain membrane proteome. Reversibility studies indicated that compound 52 inhibited hABHD6 in an irreversible manner. Finally, homology modelling and molecular docking studies were used to gain insights into the binding of compound 52 to the active site of hABHD6.
The key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), are potential targets for various therapeutic applications. In this paper, we present more extensively the results of our previous work on piperazine and piperidine carboxamides and carbamates as FAAH and MAGL inhibitors. The best compounds of these series function as potent and selective MAGL/FAAH inhibitors or as dual FAAH/MAGL inhibitors at nanomolar concentrations. This study revealed that MAGL inhibitors should comprise leaving-groups with a conjugate acid pKa of 8-10, while diverse leaving groups are tolerated for FAAH inhibitors.
BACKGROUND: Human lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the alpha/beta-hydrolase domain (ABHD) containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Amino acid sequence comparison between seven mammalian BAT5 orthologs revealed that the overall primary structure was highly (>/=95%) conserved. Activity-based protein profiling (ABPP) confirmed successful generation of catalytically active human (h) and mouse (m) BAT5 in HEK293 cells, enabling further biochemical characterization. A sensitive fluorescent glycerol assay reported hBAT5-mediated hydrolysis of medium-chain saturated (C14ratio0), long-chain unsaturated (C18ratio1, C18ratio2, C20ratio4) monoacylglycerols (MAGs) and 15-deoxy-Delta12,14-prostaglandin J2-2-glycerol ester (15d-PGJ2-G). In contrast, hBAT5 possessed only marginal diacylglycerol (DAG), triacylglycerol (TAG), or lysophospholipase activity. The best MAG substrates were 1-linoleylglycerol (1-LG) and 15d-PGJ2-G, both exhibiting low-micromolar Km values. BAT5 had a neutral pH optimum and showed preference for the 1(3)- vs. 2-isomers of MAGs C18ratio1, C18ratio2 and C20ratio4. Inhibitor profiling revealed that beta-lactone-based lipase inhibitors were nanomolar inhibitors of hBAT5 activity (palmostatin B > tetrahydrolipstatin > ebelactone A). Moreover, the hormone-sensitive lipase inhibitor C7600 (5-methoxy-3-(4-phenoxyphenyl)-3H-[1], [3], [4]oxadiazol-2-one) was identified as a highly potent inhibitor (IC50 8.3 nM). Phenyl and benzyl substituted analogs of C7600 with increased BAT5 selectivity were synthesized and a preliminary SAR analysis was conducted to obtain initial insights into the active site dimensions. CONCLUSIONS/SIGNIFICANCE: This study provides an initial characterization of BAT5 activity, unveiling the biochemical and pharmacological properties with in vitro substrate preferences and inhibitor profiles. Utilization of glycerolipid substrates and sensitivity to lipase inhibitors suggest that BAT5 is a genuine lipase with preference for long-chain unsaturated MAGs and could in this capacity regulate glycerolipid metabolism in vivo as well. This preliminary SAR data should pave the way towards increasingly potent and BAT5-selective inhibitors.
Inhibition of the key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), has been proposed as potential mode of action for various therapeutic applications. Continuing our previous work, we take the first steps of structure-activity relationship exploration and show that 1,3,4-oxadiazol-2-ones can serve as scaffold for both selective FAAH and MAGL inhibitors, and also function as a dual FAAH/MAGL inhibitor at sub-micromolar IC50 values. Moreover, 10-fold selectivity against MAGL over FAAH was achieved with compound 3d (FAAH and MAGL IC50; 2.0 and 0.22muM). Lastly, enzyme and ligand features contributing to the potency and selectivity differences are analysed by molecular docking.
