The endocannabinoid 2-arachidonoylglycerol (2-AG) is a lipid mediator involved in various physiological processes. In response to neural activity, 2-AG is synthesized post-synaptically, then activates pre-synaptic cannabinoid CB1 receptors (CB1Rs) in a retrograde manner, resulting in transient and long-lasting reduction of neurotransmitter release. The signalling competence of 2-AG is tightly regulated by the balanced action between ‘on demand’ biosynthesis and degradation. We review recent research on monoacylglycerol lipase (MAGL), ABHD6 and ABHD12, three serine hydrolases that together account for approx. 99% of brain 2-AG hydrolase activity. MAGL is responsible for approx. 85% of 2-AG hydrolysis and colocalizes with CB1R in axon terminals. It is therefore ideally positioned to terminate 2-AG-CB1R signalling regardless of the source of this endocannabinoid. Its acute pharmacological inhibition leads to 2-AG accumulation and CB1R-mediated behavioural responses. Chronic MAGL inactivation results in 2-AG overload, desensitization of CB1R signalling and behavioural tolerance. ABHD6 accounts for approx. 4% of brain 2-AG hydrolase activity but in neurones it rivals MAGL in efficacy. Neuronal ABHD6 resides post-synaptically, often juxtaposed with CB1Rs, and its acute inhibition leads to activity-dependent accumulation of 2-AG. In cortical slices, selective ABHD6 blockade facilitates CB1R-dependent long-term synaptic depression. ABHD6 is therefore positioned to guard intracellular pools of 2-AG at the site of generation. ABHD12 is highly expressed in microglia and accounts for approx. 9% of total brain 2-AG hydrolysis. Mutations in ABHD12 gene are causally linked to a neurodegenerative disease called PHARC. Whether ABHD12 qualifies as a bona fide member to the endocannabinoid system remains to be established.

amide, ester, and thioester bonds. The majority of serine hydrolases contain an ␣ / ␤ -hydrolase domain (ABHD) fold and use a Ser-His-Asp (SHD) triad for catalysis. Although several members of the metabolic serine hydrolase family are relatively well defined, the majority remain poorly characterized with respect to their physiological substrates and functions.Members of the metabolic serine family are also intimately involved in the generation and degradation of the endocannabinoid 2-arachidonoylyglycerol (C20:4) (2-AG). In brain regions endowed with 2-AG signaling, "on demand" biosynthesis of 2-AG occurs through phospholipase C ␤ -catalyzed cleavage of the membrane phospholipid phoshatidylinositol bisphosphate to generate sn-2-arachidonoyl-containing diacylglycerol (DAG) species, which are subsequently hydrolyzed by sn -1-specifi c lipases (DAGL ␣ and DAGL ␤ ) to generate 2-AG ( 3 ). The endocannabinoids are involved in a broad range of (patho)physiological processes, including neurotransmission, appetite, nociception, addiction, infl ammation, peripheral metabolism, and reproduction ( 4-6 ). The biological actions of 2-AG are mediated via two G protein-coupled receptors (CB1R and CB2R) that show unique and tissue-specifi c distribution. CB1R is highly enriched in the brain.The major enzymatic route for 2-AG inactivation is via hydrolysis, generating arachidonic acid (AA) and glycerol. In the CNS, three serine hydrolases, namely monoacylglycerol lipase (MAGL) and the ␣ / ␤ -hydrolase domain Abstract In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG hydrolase. The postgenomic proteins ␣ / ␤ -hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fl uorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD12 in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD12 preferred the 1 (3)-and 2-isomers of arachidonoylglycerol. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D278-H306, ABHD12: S246-D333-H372) abolished enzymatic activity as well as labeling with the active site serine-directed fl uorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verifi ed because none of the mutants showed detectable expression. Inhibitor profi ling revealed striking potency differences between hABHD6 and hABHD12, a fi nding that, when combined with the substrate profi ling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD12, which currently lacks such inhibitors. The human serine hydrolases compris...

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1 Two endocannabinoids, arachidonoyl ethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) bind and activate G-protein-coupled cannabinoid receptors, but limited data exist on their relative ability to activate G-proteins. 2 Here we assess agonist potency and e cacy of various cannabinoids, including 2-AG, HU-310 (2-arachidonoyl glyceryl ether, a third putative endocannabinoid), HU-313 (another ether analogue of 2-AG), AEA, R-methanandamide (an enzymatically stable analogue of AEA), and CP-55,940 at rat brain CB 1 receptors using agonist-stimulated [ 35 S]-GTPgS binding to cerebellar membranes and whole brain sections. Degradation of endocannabinoids under experimental conditions was monitored by HPLC. 3 To enhance e cacy di erences, agonist dose-response curves were generated using increasing GDP concentrations. At 10 76 M GDP, all compounds, except HU-313, produced full agonists responses *2.5 fold over basal. The superior e cacy of 2-AG over all other compounds became evident by increasing GDP (10 75 and 10 74 M). 4 In membrane incubations, 2-AG was degraded by 85% whereas AEA and HU-310 were stable. Pretreatment of membranes with phenylmethylsulphonyl¯uoride inhibited 2-AG degradation, resulting in 2 fold increase in agonist potency. Such pretreatment had no e ect on AEA potency. 5 Responses in brain sections were otherwise consistent with membrane binding data, but 2-AG evoked only a weak signal in brain sections, apparently due to more extensive degradation. 6 These data establish that even under conditions of substantial degradation, 2-AG is a full e cacy agonist, clearly more potent than AEA, in mediating CB 1 receptor-dependent G-protein activity in native membranes.

Monoacylglycerol lipase (MAGL) terminates the signaling function of the endocannabinoid, 2-arachidonoylglycerol (2-AG). During 2-AG hydrolysis, MAGL liberates arachidonic acid, feeding the principal substrate for the neuroinflammatory prostaglandins. In cancer cells, MAGL redirects lipid stores toward protumorigenic signaling lipids. Thus MAGL inhibitors may have great therapeutic potential. Although potent and increasingly selective MAGL inhibitors have been described, their number is still limited. Here, we have characterized piperazine and piperidine triazole ureas that combine the high potency attributable to the triazole leaving group together with the bulky aromatic benzodioxolyl moiety required for selectivity, culminating in compound JJKK-048 that potently (IC50 < 0.4 nM) inhibited human and rodent MAGL. JJKK-048 displayed low cross-reactivity with other endocannabinoid targets. Activity-based protein profiling of mouse brain and human melanoma cell proteomes suggested high specificity also among the metabolic serine hydrolases.