The endocannabinoid signaling system regulates diverse physiologic processes and has attracted considerable attention as a potential pharmaceutical target for treating diseases, such as pain, ...anxiety/depression, and metabolic disorders. The principal ligands of the endocannabinoid system are the lipid transmitters N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), which activate the two major cannabinoid receptors, CB1 and CB2. Anandamide and 2-AG signaling pathways in the nervous system are terminated by enzymatic hydrolysis mediated primarily by the serine hydrolases fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. In this review, we will discuss the development of FAAH and MAGL inhibitors and their pharmacological application to investigate the function of anandamide and 2-AG signaling pathways in preclinical models of neurobehavioral processes, such as pain, anxiety, and addiction. We will place emphasis on how these studies are beginning to discern the different roles played by anandamide and 2-AG in the nervous system and the resulting implications for advancing endocannabinoid hydrolase inhibitors as next-generation therapeutics.
The serine hydrolase α/β-hydrolase domain 6 (ABHD6) hydrolyzes the most abundant endocannabinoid (eCB) in the brain, 2-arachidonoylglycerol (2-AG), and controls its availability at cannabinoid ...receptors. We show that ABHD6 inhibition decreases pentylenetetrazole (PTZ)-induced generalized tonic-clonic and myoclonic seizure incidence and severity. This effect is retained in Cnr1−/− or Cnr2−/− mice, but blocked by addition of a subconvulsive dose of picrotoxin, suggesting the involvement of GABAA receptors. ABHD6 inhibition also blocked spontaneous seizures in R6/2 mice, a genetic model of juvenile Huntington’s disease known to exhibit dysregulated eCB signaling. ABHD6 blockade retained its antiepileptic activity over chronic dosing and was not associated with psychomotor or cognitive effects. While the etiology of seizures in R6/2 mice remains unsolved, involvement of the hippocampus is suggested by interictal epileptic discharges, increased expression of vGLUT1 but not vGAT, and reduced Neuropeptide Y (NPY) expression. We conclude that ABHD6 inhibition may represent a novel antiepileptic strategy.
•ABHD6 blockade by WWL123 controls PTZ-induced seizures without psychomotor effects•The effect of WWL123is retained in Cnr1−/− and Cnr2−/− mice but blocked by picrotoxin•WWL123 also blocks spontaneous seizures in the R6/2 mouse model of juvenile HD•R6/2 hippocampi feature electrographic abnormalities and profound loss of NPY
Cannabinoids reduce seizure incidence but also induce psychotropic side effects. Here Naydenov and Horne et al. show that seizure incidence is controlled by inhibition of ABHD6, a novel endocannabinoid-degrading enzyme, without producing psychotropic side effects.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The serine hydrolase monoacylglycerol lipase (MGLL) converts the endogenous cannabinoid receptor agonist 2-arachidonoylglycerol (2-AG) and other monoacylglycerols into fatty acids and glycerol. ...Genetic or pharmacological inactivation of MGLL leads to elevation in 2-AG in the central nervous system and corresponding reductions in arachidonic acid and eicosanoids, producing antinociceptive, anxiolytic, and antineuroinflammatory effects without inducing the full spectrum of psychoactive effects of direct cannabinoid receptor agonists. Here, we report the optimization of hexafluoroisopropyl carbamate-based irreversible inhibitors of MGLL, culminating in a highly potent, selective, and orally available, CNS-penetrant MGLL inhibitor, 28 (ABX-1431). Activity-based protein profiling experiments verify the exquisite selectivity of 28 for MGLL versus other members of the serine hydrolase class. In vivo, 28 inhibits MGLL activity in rodent brain (ED50 = 0.5–1.4 mg/kg), increases brain 2-AG concentrations, and suppresses pain behavior in the rat formalin pain model. ABX-1431 (28) is currently under evaluation in human clinical trials.
The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB1 cannabinoid receptors on neurons and CB2 cannabinoid receptors on microglia. ...Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase alpha-beta-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB2-mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activity-dependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB1-dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Advances in human genetics are leading to the discovery of new disease-causing mutations at a remarkable rate. Many such mutations, however, occur in genes that encode for proteins of unknown ...function, which limits our molecular understanding of, and ability to devise treatments for, human disease. Here, we use untargeted metabolomics combined with a genetic mouse model to determine that the poorly characterized serine hydrolase α/β-hydrolase domain-containing (ABHD)12, mutations in which cause the human neurodegenerative disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract), is a principal lysophosphatidylserine (LPS) lipase in the mammalian brain. ABHD12 ⁻/⁻ mice display massive increases in a rare set of very long chain LPS lipids that have been previously reported as Toll-like receptor 2 activators. We confirm that recombinant ABHD12 protein exhibits robust LPS lipase activity, which is also substantially reduced in ABHD12 ⁻/⁻ brain tissue. Notably, elevations in brain LPS lipids in ABHD12 ⁻/⁻ mice occur early in life (2–6 mo) and are followed by age-dependent increases in microglial activation and auditory and motor defects that resemble the behavioral phenotypes of human PHARC patients. Taken together, our data provide a molecular model for PHARC, where disruption of ABHD12 causes deregulated LPS metabolism and the accumulation of proinflammatory lipids that promote microglial and neurobehavioral abnormalities.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue ...click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue.
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•Tissue clearing greatly improves click chemistry labeling in mammalian tissues•CATCH enables brain-wide, subcellular in situ imaging of drug-target engagement•CATCH allows cellular drug-target identification and validation•CATCH quantitively determines on- and off-target in vivo drug binding
Clearing-assisted tissue click chemistry (CATCH) allows for the in vivo visualization of target-bound drug molecules and enables the identification of drug distribution and engagement in mammalian tissue.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Endocannabinoid (eCB) signaling is tightly regulated by eCB biosynthetic and degradative enzymes. The eCB 2-arachidonoylglycerol (2-AG) is hydrolyzed primarily by monoacylglycerol lipase (MAGL). ...Here, we investigated whether eCB signaling, synaptic function, and learning behavior were altered in MAGL knock-out mice. We report that MAGL⁻/⁻ mice exhibited prolonged depolarization-induced suppression of inhibition (DSI) in hippocampal CA1 pyramidal neurons, providing genetic evidence that the inactivation of 2-AG by MAGL determines the time course of the eCB-mediated retrograde synaptic depression. CB₁ receptor antagonists enhanced basal IPSCs in CA1 pyramidal neurons in MAGL⁻/⁻ mice, while the magnitude of DSI or CB₁ receptor agonist-induced depression of IPSCs was decreased in MAGL⁻/⁻ mice. These results suggest that 2-AG elevations in MAGL⁻/⁻ mice cause tonic activation and partial desensitization of CB₁ receptors. Genetic deletion of MAGL selectively enhanced theta burst stimulation (TBS)-induced long-term potentiation (LTP) in the CA1 region of hippocampal slices but had no significant effect on LTP induced by high-frequency stimulation or long-term depression induced by low-frequency stimulation. The enhancement of TBS-LTP in MAGL⁻/⁻ mice appears to be mediated by 2-AG-induced suppression of GABA(A) receptor-mediated inhibition. MAGL⁻/⁻ mice exhibited enhanced learning as shown by improved performance in novel object recognition and Morris water maze. These results indicate that genetic deletion of MAGL causes profound changes in eCB signaling, long-term synaptic plasticity, and learning behavior.
Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. We found that a similar form of ...functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and functional antagonism of the brain cannabinoid system, respectively.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat ...diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (>80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The endocannabinoid 2-arachidonoylglycerol (2-AG) is a retrograde lipid messenger that modulates synaptic function, neurophysiology, and behavior. 2-AG signaling is terminated by enzymatic ...hydrolysis—a reaction that is principally performed by monoacylglycerol lipase (MAGL). MAGL is broadly expressed throughout the nervous system, and the contributions of different brain cell types to the regulation of 2-AG activity in vivo remain poorly understood. Here, we genetically dissect the cellular anatomy of MAGL-mediated 2-AG metabolism in the brain and show that neurons and astrocytes coordinately regulate 2-AG content and endocannabinoid-dependent forms of synaptic plasticity and behavior. We also find that astrocytic MAGL is mainly responsible for converting 2-AG to neuroinflammatory prostaglandins via a mechanism that may involve transcellular shuttling of lipid substrates. Astrocytic-neuronal interplay thus provides distributed oversight of 2-AG metabolism and function and, through doing so, protects the nervous system from excessive CB1 receptor activation and promotes endocannabinoid crosstalk with other lipid transmitter systems.
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•Genetic mouse models reveal cellular specificity of 2-AG metabolism•Astrocytes and neurons collaborate to terminate endocannabinoid signaling•Coordinated astrocytic-neuronal metabolism protects against CB1R desensitization•Astrocytes couple 2-AG hydrolysis to neuroinflammatory prostaglandin production
The endocannabinoid 2-arachidonoylglycerol (2-AG) is a retrograde lipid messenger that broadly modulates brain synapses, neurophysiology, and behavior. Viader et al. show that endocannabinoid signaling is regulated by the cooperative, transcellular metabolism of 2-AG, which is shuttled between neurons and astrocytes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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