Family Report for: Monoglyceridelipase_lysophospholip

Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates. The discovery and optimization of 3-substituted azetidine carbamate irreversible inhibitors of MAGL were aided by the generation of inhibitor-bound MAGL crystal structures. Compound 6, a highly efficient and selective MAGL inhibitor against recombinant enzyme and in a cellular context, was tested in vivo and shown to elevate central 2-AG levels at a 10 mg/kg dose.

Substantial challenges exist for investigating the cannabinoid receptor type 1 (CB1)-mediated discriminative stimulus effects of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA), compared with exogenous CB1 receptor agonists, such as Delta(9)-tetrahydrocannabinol (THC) and the synthetic cannabinoid CP55,940. Specifically, each endocannabinoid is rapidly degraded by the respective hydrolytic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Whereas MAGL inhibitors partially substitute for THC and fully substitute for CP55,940, FAAH inhibitors do not substitute for either drug. Interestingly, combined FAAH-MAGL inhibition results in full THC substitution, and the dual FAAH-MAGL inhibitor SA-57 serves as its own discriminative training stimulus. Because MAGL inhibitors fully substitute for SA-57, we tested whether the selective MAGL inhibitor MJN110 would serve as a training stimulus. Twelve of 13 C57BL/6J mice learned to discriminate MJN110 from vehicle, and the CB1 receptor antagonist rimonabant dose-dependently blocked its discriminative stimulus. CP55,940, SA-57, and another MAGL inhibitor JZL184, fully substituted for MJN110. In contrast, the FAAH inhibitor PF-3845 failed to substitute for the MJN110 discriminative stimulus, but produced a 1.6 (1.1-2.2; 95% confidence interval) leftward shift in the MJN110 dose-response curve. Inhibitors of other relevant enzymes (i.e., ABHD6, COX-2) and nicotine did not engender substitution. Diazepam partially substituted for MJN110, but rimonabant failed to block this partial effect. These findings suggest that MAGL normally throttles 2-AG stimulation of CB1 receptors to a magnitude insufficient to produce cannabimimetic subjective effects. Accordingly, inhibitors of this enzyme may release this endogenous brake producing effects akin to those produced by exogenously administered cannabinoids.

2-Arachidonoylglycerol plays a major role in endocannabinoid signaling, and is tightly regulated by the monoacylglycerol lipase (MAGL). Here we report the crystal structure of human MAGL. The protein crystallizes as a dimer, and despite structural homologies to haloperoxidases and esterases, it distinguishes itself by a wide and hydrophobic access to the catalytic site. An apolar helix covering the active site also gives structural insight into the amphitropic character of MAGL, and likely explains how MAGL interacts with membranes to recruit its substrate. Docking of 2-arachidonoylglycerol highlights a hydrophobic and a hydrophilic cavity that accommodate the lipid into the catalytic site. Moreover, we identified Cys201 as the crucial residue in MAGL inhibition by N-arachidonylmaleimide, a sulfhydryl-reactive compound. Beside the advance in the knowledge of endocannabinoids degradation routes, the structure of MAGL paves the way for future medicinal chemistry works aimed at the design of new drugs exploiting 2-arachidonoylglycerol transmission.

Phenotypic screening has identified small-molecule modulators of aging, but the mechanism of compound action often remains opaque due to the complexities of mapping protein targets in whole organisms. Here, we combine a library of covalent inhibitors with activity-based protein profiling to coordinately discover bioactive compounds and protein targets that extend lifespan in Caenorhabditis elegans. We identify JZL184-an inhibitor of the mammalian endocannabinoid (eCB) hydrolase monoacylglycerol lipase (MAGL or MGLL)-as a potent inducer of longevity, a result that was initially perplexing as C. elegans does not possess an MAGL ortholog. We instead identify FAAH-4 as a principal target of JZL184 and show that this enzyme, despite lacking homology with MAGL, performs the equivalent metabolic function of degrading eCB-related monoacylglycerides in C. elegans. Small-molecule phenotypic screening thus illuminates pure pharmacological connections marking convergent metabolic functions in distantly related organisms, implicating the FAAH-4/monoacylglyceride pathway as a regulator of lifespan in C. elegans.

Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates. The discovery and optimization of 3-substituted azetidine carbamate irreversible inhibitors of MAGL were aided by the generation of inhibitor-bound MAGL crystal structures. Compound 6, a highly efficient and selective MAGL inhibitor against recombinant enzyme and in a cellular context, was tested in vivo and shown to elevate central 2-AG levels at a 10 mg/kg dose.

Substantial challenges exist for investigating the cannabinoid receptor type 1 (CB1)-mediated discriminative stimulus effects of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA), compared with exogenous CB1 receptor agonists, such as Delta(9)-tetrahydrocannabinol (THC) and the synthetic cannabinoid CP55,940. Specifically, each endocannabinoid is rapidly degraded by the respective hydrolytic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Whereas MAGL inhibitors partially substitute for THC and fully substitute for CP55,940, FAAH inhibitors do not substitute for either drug. Interestingly, combined FAAH-MAGL inhibition results in full THC substitution, and the dual FAAH-MAGL inhibitor SA-57 serves as its own discriminative training stimulus. Because MAGL inhibitors fully substitute for SA-57, we tested whether the selective MAGL inhibitor MJN110 would serve as a training stimulus. Twelve of 13 C57BL/6J mice learned to discriminate MJN110 from vehicle, and the CB1 receptor antagonist rimonabant dose-dependently blocked its discriminative stimulus. CP55,940, SA-57, and another MAGL inhibitor JZL184, fully substituted for MJN110. In contrast, the FAAH inhibitor PF-3845 failed to substitute for the MJN110 discriminative stimulus, but produced a 1.6 (1.1-2.2; 95% confidence interval) leftward shift in the MJN110 dose-response curve. Inhibitors of other relevant enzymes (i.e., ABHD6, COX-2) and nicotine did not engender substitution. Diazepam partially substituted for MJN110, but rimonabant failed to block this partial effect. These findings suggest that MAGL normally throttles 2-AG stimulation of CB1 receptors to a magnitude insufficient to produce cannabimimetic subjective effects. Accordingly, inhibitors of this enzyme may release this endogenous brake producing effects akin to those produced by exogenously administered cannabinoids.

Monoglyceride lipases (MGLs) are a group of alpha/beta-hydrolases that catalyze the hydrolysis of monoglycerides (MGs) into free fatty acids and glycerol. This reaction serves different physiological functions, namely in the last step of phospholipid and triglyceride degradation, in mammalian endocannabinoid and arachidonic acid metabolism, and in detoxification processes in microbes. Previous crystal structures of MGLs from humans and bacteria revealed conformational plasticity in the cap region of this protein and gave insight into substrate binding. In this study, we present the structure of a MGL from Saccharomyces cerevisiae called Yju3p in its free form and in complex with a covalently bound substrate analog mimicking the tetrahedral intermediate of MG hydrolysis. These structures reveal a high conservation of the overall shape of the MGL cap region and also provide evidence for conformational changes in the cap of Yju3p. The complex structure reveals that, despite the high structural similarity, Yju3p seems to have an additional opening to the substrate binding pocket at a different position compared to human and bacterial MGL. Substrate specificities towards MGs with saturated and unsaturated alkyl chains of different lengths were tested and revealed highest activity towards MG containing a C18:1 fatty acid.

Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), 2-arachidonoyglycerol (2-AG). This study reports a potent covalent MAGL inhibitor, SAR127303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of 2-AG in mice. In vivo, SAR127303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR127303. Although inactive in acute seizure tests, repeated administration of SAR127303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that 2-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.

The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 A resolution. The crystal form was orthorhombic (space group P212121), with unit-cell parameters a = 77.2, b = 108.6, c = 167.7 A. The asymmetric unit contained four molecules with a solvent content of 46.4%.

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. CB(1) receptor antagonists enhanced basal IPSCs in CA1 pyramidal neurons in MAGL(-)/(-) mice, while the magnitude of DSI or CB(1) 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 CB(1) 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.

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is hydrolysed primarily by monoacylglycerol lipase (MAGL). Here, we investigated whether eCB-mediated retrograde synaptic depression in cerebellar slices was altered in MAGL knockout (MAGL(-/-)) mice. Depolarization-induced suppression of excitation (DSE) and metabotropic glutamate receptor (mGluR1)-mediated synaptic depression are mediated by 2-AG-induced activation of CB(1) receptors. We show that genetic deletion of MAGL prolonged DSE at parallel fibre (PF) or climbing fibre (CF) to Purkinje cell (PC) synapses. Likewise, mGluR1-mediated synaptic depression, induced either by high-frequency stimulation of PF or mGluR1 agonist DHPG, was prolonged in MAGL(-/-) mice. About 15% of 2-AG in the brain is hydrolysed by serine hydrolase alpha-beta-hydrolase domain 6 and 12 (ABHD6 and ABHD12). However, the selective ABHD6 inhibitor WWL123 had no significant effect on cerebellar DSE in MAGL(+/+) and (-/-) mice. The CB(1) receptor antagonist SR141716 significantly increased the amplitude of basal excitatory postsynaptic currents (EPSCs) in MAGL(-/-) mice but not in MAGL(+/+) mice. Conversely, the CB(1) agonist WIN55212 induced less depression of basal EPSCs in MAGL(-/-) mice than in MAGL(+/+) mice. These results provide genetic evidence that inactivation of 2-AG by MAGL determines the time course of eCB-mediated retrograde synaptic depression and that genetic deletion of MAGL causes tonic activation and consequential desensitization of CB(1) receptors.

Monoglyceride lipase (MGL) is a serine hydrolase that hydrolyses 2-arachidonoylglycerol (2-AG) into arachidonic acid and glycerol. 2-AG is an endogenous ligand of cannabinoid receptors, involved in various physiological processes in the brain. We present here the first crystal structure of human MGL in its apo form and in complex with the covalent inhibitor SAR629. MGL shares the classic fold of the alpha/beta hydrolase family but depicts an unusually large hydrophobic occluded tunnel with a highly flexible lid at its entry and the catalytic triad buried at its end. Structures reveal the configuration of the catalytic triad and the shape and nature of the binding site of 2-AG. The bound structure of SAR629 highlights the key interactions for productive binding with MGL. The shape of the tunnel suggests a high druggability of the protein and provides an attractive template for drug discovery.

Monoacylglycerols (MAGs) are short-lived intermediates of glycerolipid metabolism. Specific molecular species, such as 2-arachidonoylglycerol, which is a potent activator of cannabinoid receptors, may also function as lipid signaling molecules. In mammals, enzymes hydrolyzing MAG to glycerol and fatty acids, resembling the final step in lipolysis, or esterifying MAG to diacylglycerol, are well known; however, despite the high level of conservation of lipolysis, the corresponding activities in yeast have not been characterized yet. Here we provide evidence that the protein Yju3p functions as a potent MAG hydrolase in yeast. Cellular MAG hydrolase activity was decreased by more than 90% in extracts of Yju3p-deficient cells, indicating that Yju3p accounts for the vast majority of this activity in yeast. Loss of this activity was restored by heterologous expression of murine monoglyceride lipase (MGL). Since yju3Delta mutants accumulated MAG in vivo only at very low concentrations, we considered the possibility that MAGs are re-esterified into DAG by acyltransferases. Indeed, cellular MAG levels were further increased in mutant cells lacking Yju3p and Dga1p or Lro1p acyltransferase activities. In conclusion, our studies suggest that catabolic and anabolic reactions affect cellular MAG levels. Yju3p is the functional orthologue of mammalian MGL and is required for efficient degradation of MAG in yeast.

2-Arachidonoylglycerol plays a major role in endocannabinoid signaling, and is tightly regulated by the monoacylglycerol lipase (MAGL). Here we report the crystal structure of human MAGL. The protein crystallizes as a dimer, and despite structural homologies to haloperoxidases and esterases, it distinguishes itself by a wide and hydrophobic access to the catalytic site. An apolar helix covering the active site also gives structural insight into the amphitropic character of MAGL, and likely explains how MAGL interacts with membranes to recruit its substrate. Docking of 2-arachidonoylglycerol highlights a hydrophobic and a hydrophilic cavity that accommodate the lipid into the catalytic site. Moreover, we identified Cys201 as the crucial residue in MAGL inhibition by N-arachidonylmaleimide, a sulfhydryl-reactive compound. Beside the advance in the knowledge of endocannabinoids degradation routes, the structure of MAGL paves the way for future medicinal chemistry works aimed at the design of new drugs exploiting 2-arachidonoylglycerol transmission.

Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals. PAPERFLICK:

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.