Human Monoacylglycerol Lipase Research Articles

Identifying Potential Human Monoacylglycerol Lipase Inhibitors from the Phytoconstituents of Morinda Citrifolia L. Fruits by in silico Pharmacology and in vitro Study.

Human monoacylglycerol lipase (MGL) is accountable for the hydrolysis of 2-arachidonoylglycerol (2-AG), thus contributing pivotally to neuroprotection because 2-AG is the main source of arachidonic acid, the precursor of prostaglandins production. Inhibiting MGL reduces inflammatory damage in the ischemic brain and enhances cerebral blood flow. Plants have been reported for their neuroprotective effect, such as Morinda citrifolia on pentylenetetrazol (PTZ)-induced kindling seizures in mice, by reducing the seizures and restoring behavioral and biochemical changes, although the mechanism is not described. To evaluate the binding affinity and stability of phytoconstituents in M. citrifolia fruits toward human MGL (PDB ID 3PE6), compared to the known MGL inhibitors (JZL195 and ZYH). The in silico pharmacology study was validated by an in vitro study of the phytosterols and the ethanol extract of M. citrifolia fruits (EEMC) towards MGL. Initially, nine phytoconstituents of M. citrifolia fruits were docked to the catalytic pocket of human MGL (PDB ID: 3PE6), and compounds with the best affinity were subjected to a molecular dynamic (MD) simulation. The in vitro study was performed using the MGL inhibitor screening assay kit. The best binding affinity and stability toward human MGL were shown by stigmasterol and beta-sitosterol, and the MM-PBSA total binding energy of stigmasterol and beta-sitosterol to MGL is stronger than that of JZL195 and ZYH. Moreover, beta-sitosterol and EEMC inhibit MGL with an IC50 value of, respectively, 8.10μg/mL and 196.20μg/mL, while JZL195 shows an IC50 of 0.028μg/mL. Beta-sitosterol of Morinda citrifolia fruits may have the potential to protect human neurons by occupying the catalytic site of human MGL, thus competitively inhibiting the substrate of the enzyme. However, the inhibitory activity towards human MGL is lower than JZL195.

Lipases are differentially regulated by hormones to maintain free fatty acid homeostasis for insect brain development

BackgroundFree fatty acids (FFAs) play vital roles as energy sources and substrates in organisms; however, the molecular mechanism regulating the homeostasis of FFA levels in various circumstances, such as feeding and nonfeeding stages, is not fully clarified. Holometabolous insects digest dietary triglycerides (TAGs) during larval feeding stages and degrade stored TAGs in the fat body during metamorphosis after feeding cessation, which presents a suitable model for this study.ResultsThis study reported that two lipases are differentially regulated by hormones to maintain the homeostasis of FFA levels during the feeding and nonfeeding stages using the lepidopteran insect cotton bollworm Helicoverpa armigera as a model. Lipase member H-A-like (Lha-like), related to human pancreatic lipase (PTL), was abundantly expressed in the midgut during the feeding stage, while the monoacylglycerol lipase ABHD12-like (Abhd12-like), related to human monoacylglycerol lipase (MGL), was abundantly expressed in the fat body during the nonfeeding stage. Lha-like was upregulated by juvenile hormone (JH) via the JH intracellular receptor methoprene-tolerant 1 (MET1), and Abhd12-like was upregulated by 20-hydroxyecdysone (20E) via forkhead box O (FOXO) transcription factor. Knockdown of Lha-like decreased FFA levels in the hemolymph and reduced TAG levels in the fat body. Moreover, lipid droplets (LDs) were small, the brain morphology was abnormal, the size of the brain was small, and the larvae showed the phenotype of delayed pupation, small pupae, and delayed tissue remodeling. Knockdown of Abhd12-like decreased FFA levels in the hemolymph; however, TAG levels increased in the fat body, and LDs remained large. The development of the brain was arrested at the larval stage, and the larvae showed a delayed pupation phenotype and delayed tissue remodeling.ConclusionsThe differential regulation of lipases expression by different hormones determines FFAs homeostasis and different TAG levels in the fat body during the feeding larval growth and nonfeeding stages of metamorphosis in the insect. The homeostasis of FFAs supports insect growth, brain development, and metamorphosis.

Pristimerin, a triterpene that inhibits monoacylglycerol lipase activity, prevents the development of paclitaxel-induced allodynia in mice.

Background: Triterpenes such as euphol and pristimerin, which are plant secondary metabolites, were the first to be characterized as monoacylglycerol lipase (MAGL) inhibitors. MAGL inhibitors alleviate chemotherapy-induced neuropathic pain (CINP) in rodent models. Pristimerin has been shown to have additive anticancer activity with paclitaxel, a chemotherapeutic drug. However, the activity of pristimerin on CINP has not been evaluated. Objectives: The aims of this study were to evaluate whether various triterpenes had activity against recombinant human MAGL and MAGL activity in mouse tissues, and whether pristimerin could prevent development of paclitaxel-induced mechanical allodynia. Methods: The effects of four triterpenes betulinic acid, cucurbitacin B, euphol, and pristimerin on the activity human recombinant MAGL and MAGL activity of mice brain and paw skin tissues were evaluated using MAGL inhibitor screening and MAGL activity assay kits. The effects of treatment of female BALB/c mice with pristimerin intraperitoneally on the development of paclitaxel-induced mechanical allodynia were assessed using the dynamic plantar aesthesiometer and on nuclear factor-2 erythroid related factor-2 (Nrf2) gene expression in the paw skin were evaluated by real time polymerase chain reaction. Results: Pristimerin inhibited the human recombinant MAGL activity in a concentration-dependent manner like JZL-195, a MAGL inhibitor. Betulinic acid, cucurbitacin B and euphol inhibited human recombinant MAGL activity but their effects were not concentration dependent and were less to that of pristimerin. Pristimerin inhibited both mouse brain and paw skin MAGL activity in a concentration-dependent manner. Paclitaxel induced mechanical allodynia and increase in MAGL activity in the paw skin. Treatment with pristimerin prevented the development of paclitaxel-induced mechanical allodynia and the paclitaxel-induced increase in MAGL activity. Pristimerin significantly upregulated mRNA expression of Nrf2, a regulator of endogenous antioxidant defense. Conclusion: These results indicate that triterpenes inhibit human recombinant MAGL activity with varying degrees. Pristimerin inhibits both mouse brain and paw skin MAGL activity in a concentration-dependent manner, prevents both the development of paclitaxel-induced mechanical allodynia and the associated increase in MAGL activity in the paw skin, and might protect against paclitaxel-induced oxidative stress. Co-treatment with pristimerin and paclitaxel could be useful in the treatment of cancer and prevention of CINP.

Mass spectrometry-guided discovery of new analogs of bicyclic phosphotriester salinipostin and evaluation of their monoacylglycerol lipase inhibitory activity.

Natural products containing the highly unusual phosphotriester ring are known to be potent serine hydrolase inhibitors. The long-chain bicyclic enol-phosphotriester salinipostins (SPTs) from the marine actinomycete Salinispora have been identified as selective antimalarial agents. A potential regulatory function has been suggested for phosphotriesters based on their structural relationship with actinomycete signaling molecules and the prevalence of spt-like biosynthetic gene clusters across actinomycetes. In this study, we established a mass spectrometry-guided screening method for phosphotriesters focusing on their characteristic fragment ions. Applying this screening method to the SPT producer Salinispora tropica CNB-440, new SPT analogs (4-6) were discovered and their structures were elucidated by spectroscopic analyses. Previously known and herein-identified SPT analogs inhibited the activity of human monoacylglycerol lipase (MAGL), a key serine hydrolase in the endocannabinoid system, in the nanomolar range. Our method could be applied to the screening of phosphotriesters, potential serine hydrolase inhibitors and signaling molecules.

Identification of bioactive natural products using yeast：Application to monoacylglycerol lipase inhibitor extraction from Corydalis Rhizoma

Identification of bioactive principles from natural products is considered a challenging task in drug discovery. Recently, ligand fishing has been growing in interest as a sustainable strategy. In this study, a yeast-based drug discovery strategy was investigated to screen and fish active ingredients from natural products. Human monoacylglycerol lipase (MAGL) was first displayed on the cell wall of Pichia pastoris. The expression of MAGL on the cell surface was confirmed by immunofluorescence analysis. The activity toward 7-HCA which was consistent with free enzymes in solution. Recombinant yeast strains were used to screen the potential inhibitors from traditional Chinese medicines. Preliminary screening showed that the extracts of 12 herbs showed inhibition on MAGL activity, among which Corydalis Rhizoma had the highest inhibition rate of 60.66 ± 2.44%. Recombinant yeast fishing combined with HPLC-Q-TOF-MS/MS analysis was further used to identify the potential MAGL inhibitors. Finally, dehydrocorydaline (DHC) in Corydalis Rhizoma was determined as a ligand to MAGL with the IC50 value at 154.7 μΜ. Corydalis Rhizoma has been used as a pain reliever in TCM. Intraperitoneal injection of 7 mg kg− 1 DHC in chronic constriction injury model rats significantly attenuated the mechanical allodynia and thermal hyperalgesia. Meanwhile, 2-arachidonoylglycerol, the major MAGL substrate in the brain, was significantly increased both in the hippocampus and striatum. In conclusion, yeast-based ligand fishing combined with HPLC-Q-TOF-MS/MS is a powerful strategy for drug discovery in complex mixtures and DHC from Corydalis Rhizoma was confirmed with high inhibitory activity to MAGL either in vitro or in vivo .

A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase.

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

Analgesic and Anticancer Activity of Benzoxazole Clubbed 2-Pyrrolidinones as Novel Inhibitors of Monoacylglycerol Lipase.

Ten benzoxazole clubbed 2-pyrrolidinones (11–20) as human monoacylglycerol lipase inhibitors were designed on the criteria fulfilling the structural requirements and on the basis of previously reported inhibitors. The designed, synthesized, and characterized compounds (11–20) were screened against monoacylglycerol lipase (MAGL) in order to find potential inhibitors. Compounds 19 (4-NO2 derivative) and 20 (4-SO2NH2 derivative), with an IC50 value of 8.4 and 7.6 nM, were found most active, respectively. Both of them showed micromolar potency (IC50 value above 50 µM) against a close analogue, fatty acid amide hydrolase (FAAH), therefore considered as selective inhibitors of MAGL. Molecular docking studies of compounds 19 and 20 revealed that carbonyl of 2-pyrrolidinone moiety sited at the oxyanion hole of catalytic site of the enzyme stabilized with three hydrogen bonds (~2 Å) with Ala51, Met123, and Ser122, the amino acid residues responsible for the catalytic function of the enzyme. Remarkably, the physiochemical and pharmacokinetic properties of compounds 19 and 20, computed by QikProp, were found to be in the qualifying range as per the proposed guideline for good orally bioactive CNS drugs. In formalin-induced nociception test, compound 20 reduced the pain response in acute and late stages in a dose-dependent manner. They significantly demonstrated the reduction in pain response, having better potency than the positive control gabapentin (GBP), at 30 mg/kg dose. Compounds 19 and 20 were submitted to NCI, USA, for anticancer activity screening. Compounds 19 (NSC: 778839) and 20 (NSC: 778842) were found to have good anticancer activity on SNB-75 cell line of CNS cancer, exhibiting 35.49 and 31.88% growth inhibition (% GI), respectively.

Discovery of 8-prenylnaringenin from hop (Humulus lupulus L.) as a potent monoacylglycerol lipase inhibitor for treatments of neuroinflammation and Alzheimer's disease.

Monoacylglycerol lipase (MAGL), a serine hydrolase, converts endocannabinoid 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain and plays a bidirectional role in controlling nueroinflammation. MAGL, involved in Alzheimer's and Parkinson's diseases, is a promising target for treatment of neurodegenerative disorders. However, the irreversible inhibitors of MAGL lead to the desensitization of CB1 receptors further impairing the benefits associated with the indirect CB1 stimulation. Therefore, development of potent reversible inhibitors from natural products (NPs) and traditional chinese medicines (TCMs) are safer and free from adverse side effects and feasible to avoid drawbacks which irreversible inhibitors cause. Here, we employed pharmacophore-based screening of drug candidates coupled with molecular docking, biochemical assay and Ligplot analyses to identify and characterize inhibitors targeting human MAGL (hMAGL). The built pharmacophore model, Phar-MAGL successfully identified inhibitors NP-2 (IC50 = 9.5 ± 1.2 μM), NP-5 (IC50 = 14.5 ± 1.3 μM), and NP-3 (IC50 = 15.2 ± 1.4 μM), which apparently attenuated the activities of hMAGL in vitro. The evident activities of the identified inhibitors against hMAGL showed that the pharmacophore model, Phar-MAGL is reliable and efficient in screening inhibitors against hMAGL. Our study successfully identified a natrual product inhibitor, NP-2 (8-PN), from the plant Humulus lupulus L. (hops) and its positive effects in neurogenesis and neurodifferentiation along with the evident inhibitory potency against hMAGL revealed the potential for further optimizing and developing into drugs to treat neuroinflammation, Alzheimer's and Parkinson's diseases.

Conformational gating, dynamics and allostery in human monoacylglycerol lipase

Inhibition of human Monoacylglycerol Lipase (hMGL) offers a novel approach for treating neurological diseases. The design of inhibitors, targeting active-inactive conformational transitions of the enzyme, can be aided by understanding the interplay between structure and dynamics. Here, we report the effects of mutations within the catalytic triad on structure, conformational gating and dynamics of hMGL by combining kinetics, NMR, and HDX-MS data with metadynamics simulations. We found that point mutations alter delicate conformational equilibria between active and inactive states. HDX-MS reveals regions of the hMGL that become substantially more dynamic upon substitution of catalytic acid Asp-239 by alanine. These regions, located far from the catalytic triad, include not only loops but also rigid α-helixes and β-strands, suggesting their involvement in allosteric regulation as channels for long-range signal transmission. The results identify the existence of a preorganized global communication network comprising of tertiary (residue-residue contacts) and quaternary (rigid-body contacts) networks that mediate robust, rapid intraprotein signal transmission. Catalytic Asp-239 controls hMGL allosteric communications and may be considered as an essential residue for the integration and transmission of information to enzymes’ remote regions, in addition to its well-known role to facilitate Ser-122 activation. Our findings may assist in the identification of new druggable sites in hMGL.

Pyrrolidin-2-one linked benzofused heterocycles as novel small molecule monoacylglycerol lipase inhibitors and antinociceptive agents.

Eighteen pyrrolidin-2-one linked benzothiazole, and benzimidazole derivatives (10-27) were designed and synthesized. The structure of the compounds was confirmed by elemental and spectral (IR, 1 H-NMR and MS) data analysis. All the compounds were screened by human monoacylglycerol lipase (hMAGL) inhibition assay. Three benzimidazole compounds, 22 (4-Cl phenyl), 23 (3-Cl,4-F phenyl) and 25 (4-methoxy phenyl) were found to be the most potent, having an IC50 value of 8.6, 8.0 and 9.4nm, respectively. Among them, the halogen-substituted phenyl derivatives, compound 22 (4-Cl phenyl) and compound 23 (3-Cl,4-F phenyl), showed micromolar potency against fatty acid amide hydrolase (FAAH), having an IC50 value of 35 and 24µm, respectively. Benzimidazole derivative having 4-methoxyphenyl substitution (compound 25) was found to be a selective MAGL inhibitor (IC50 =9.4nm), with an IC50 value above 50µm against FAAH. In the formalin-induced nociception test, compound 25 showed a dose-dependent reduction of pain response in both acute and late phases. At 30mg/kg dose, it significantly reduced the pain response and showed greater potency than the reference drug gabapentin (GBP).

The Antimalarial Natural Product Salinipostin A Identifies Essential α/β Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites

Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/β serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

ω-Quinazolinonylalkyl aryl ureas as reversible inhibitors of monoacylglycerol lipase

The serine hydrolase monoacylglycerol lipase (MAGL) is involved in a plethora of pathological conditions, in particular pain and inflammation, various types of cancer, metabolic, neurological and cardiovascular disorders, and is therefore a promising target for drug development. Although a large number of irreversible-acting MAGL inhibitors have been discovered over the past years, there are only few compounds known so far which inhibit the enzyme in a reversible manner. Therefore, much effort is put into the development of novel chemical entities showing reversible inhibitory behavior, which is thought to cause less undesired side effects. To explore a wide range of chemical structures as MAGL binders, we have applied a virtual screening approach by docking small molecules into the crystal structure of human MAGL (hMAGL) and envisaged a library of 45 selected compounds which were then synthesized. Biochemical investigations included the determination of the inhibitory potency on hMAGL and two related hydrolases, i.e. human fatty acid amide hydrolase (hFAAH) and murine cholesterol esterase (mCEase). The most promising candidates from theses analyses, i.e. three ω-quinazolinonylalkyl aryl ureas bearing alkyl spacers of three to five methylene groups, exhibited IC50 values of 20-41 µM and reversible, detergent-insensitive behavior towards hMAGL. Among these compounds, the inhibitor 1-(3,5-bis(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)butyl)urea (96) was selected for further kinetic characterization, yielding a dissociation constant Ki = 15.4 µM and a mixed-type inhibition with a pronounced competitive component (α = 8.94). This mode of inhibition was further supported by a docking experiment, which suggested that the inhibitor occupies the substrate binding pocket of hMAGL.

Set-Up and Validation of a High Throughput Screening Method for Human Monoacylglycerol Lipase (MAGL) Based on a New Red Fluorescent Probe

Monoacylglycerol lipase (MAGL) is a serine hydrolase that has a key regulatory role in controlling the levels of 2-arachidonoylglycerol (2-AG), the main signaling molecule in the endocannabinoid system. Identification of selective modulators of MAGL enables both to provide new tools for investigating pathophysiological roles of 2-AG, and to discover new lead compounds for drug design. The development of sensitive and reliable methods is crucial to evaluate this modulatory activity. In the current study, we report readily synthesized long-wavelength putative fluorogenic substrates with different acylic side chains to find a new probe for MAGL activity. 7-Hydroxyresorufinyl octanoate proved to be the best substrate thanks to the highest rate of hydrolysis and the best Km and Vmax values. In addition, in silico evaluation of substrates interaction with the active site of MAGL confirms octanoate resorufine derivative as the molecule of choice. The well-known MAGL inhibitors URB602 and methyl arachidonylfluorophosphonate (MAFP) were used for the assay validation. The assay was highly reproducible with an overall average Z′ value of 0.86. The fast, sensitive and accurate method described in this study is suitable for low-cost high-throughput screening (HTS) of MAGL modulators and is a powerful new tool for studying MAGL activity.

The Antimalarial Natural Product Salinipostin a Identifies Essential α/β Serine Hydrolases Involved in Lipid Metabolism in <i>P. Falciparum</i> Parasites

Salinipostin A (Sal A) is a potent antimalarial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/β serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays high homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism and produces disorganized and stalled schizonts similar to Sal A. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

Design, synthesis, molecular modelling and in vitro cytotoxicity analysis of novel carbamate derivatives as inhibitors of Monoacylglycerol lipase

Monoacylglycerol lipase (MAGL) has an essential role in the catabolic pathway of the endocannabinoid 2-arachidonoylglycerol, which makes it a potential target for highly specific inhibitors for the treatment of a number of diseases. We designed and synthesized a series of carbamate analogues of URB602. We evaluated their inhibitory activity toward human MAGL in vitro both in cell culture and lysates. The target compounds exhibited moderate to excellent inhibitory activity against MAGL. The most promising compound 2b showed good inhibitory activity with IC50 value of 4.5 ± 0.70 μM reducing MAGL activity to 82% of controls at 10 μM compared to 66% for the parent compound URB602. Interestingly, compounds 2b and 2c induce cell death through the inhibition of MAGL. Molecular modelling approaches and docking studies, used to investigate inhibitory profiles, indicated that trifluoromethyl substitutions of the aryl group and the benzene ring present at the oxygen side of the carbamate molecule had a significant impact on the activity.

Effects of Distal Mutations on the Structure, Dynamics and Catalysis of Human Monoacylglycerol Lipase

An understanding of how conformational dynamics modulates function and catalysis of human monoacylglycerol lipase (hMGL), an important pharmaceutical target, can facilitate the development of novel ligands with potential therapeutic value. Here, we report the discovery and characterization of an allosteric, regulatory hMGL site comprised of residues Trp-289 and Leu-232 that reside over 18 Å away from the catalytic triad. These residues were identified as critical mediators of long-range communication and as important contributors to the integrity of the hMGL structure. Nonconservative replacements of Trp-289 or Leu-232 triggered concerted motions of structurally distinct regions with a significant conformational shift toward inactive states and dramatic loss in catalytic efficiency of the enzyme. Using a multimethod approach, we show that the dynamically relevant Trp-289 and Leu-232 residues serve as communication hubs within an allosteric protein network that controls signal propagation to the active site, and thus, regulates active-inactive interconversion of hMGL. Our findings provide new insights into the mechanism of allosteric regulation of lipase activity, in general, and may provide alternative drug design possibilities.

Rational Development of MAGL Inhibitors.

Hit identification and hit-to-lead optimization are key steps of the early drug discovery program. Starting from the X-ray crystal structure of the human monoacylglycerol lipase (hMAGL), we herein describe the computational and experimental procedures that we applied for identifying and optimizing a new active inhibitor of this target enzyme. A receptor-based virtual screening method is reported in details, together with enzymatic assays and a first round of hit optimization.

The role of human monoacylglycerol lipase (hMAGL) binding pocket in breakup of unsaturated phospholipid membranes

Human monoacylglycerol lipase (hMAGL) plays a key role in homeostatic tuning of the endocannabinoid signaling system and supports aggressive tumorogenesis, making this enzyme a promising therapeutic target. hMAGL features a membrane-associated lid domain that regulates entry of endocannabinoid lipid substrates into the hydrophobic channel accessing the active site, likely from the membrane bilayer. The present work applied simultaneous surface plasmon resonance and electrochemical impedance spectroscopy measurements to show that, in absence of the substrate, hMAGL can remove phospholipid molecules from the membrane and, thereby, disintegrate pre-formed, intact, tethered phospholipid bilayer membrane mimetics (tBLMs) composed of unsaturated phosphatidylcholines. To probe the mechanism of hMAGL-induced on tBLMs compromise, we investigated the effect of wild type and mutant hMAGLs and hMAGL rendered catalytically inactive, as a function of concentration and in the presence of chemically distinct active-site inhibitors. Our data show that hMAGL's lid domain and hydrophobic substrate-binding pocket play important roles in hMAGL-induced bilayer lipid mobilization, whereas hydrolytic activity of the enzyme does not appear to be a factor.

Lid domain plasticity and lipid flexibility modulate enzyme specificity in human monoacylglycerol lipase

Human monoacylglycerol lipase (MAGL) is a membrane-interacting enzyme that generates pro-inflammatory signaling molecules. For this reason, MAGL inhibition is a promising strategy to treat pain, cancer, and neuroinflammatory diseases. MAGL can hydrolyze monoacylglycerols bearing an acyl chain of different lengths and degrees of unsaturation, cleaving primarily the endocannabinoid 2-arachidonoylglycerol. Importantly, the enzymatic binding site of MAGL is confined by a 75-amino-acid-long, flexible cap domain, named ‘lid domain’, which is structurally similar to that found in several other lipases. However, it is unclear how lid domain plasticity affects catalysis in MAGL. By integrating extensive molecular dynamics simulations and free-energy calculations with mutagenesis and kinetic experiments, we here define a lid-domain-mediated mechanism for substrate selection and binding in MAGL catalysis. In particular, we clarify the key role of Phe159 and Ile179, two conserved residues within the lid domain, in regulating substrate specificity in MAGL. We conclude by proposing that other structurally related lipases may share this lid-domain-mediated mechanism for substrate specificity.

Free-energy studies reveal a possible mechanism for oxidation-dependent inhibition of MGL.

The function of monoacylglycerol lipase (MGL), a key actor in the hydrolytic deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2AG), is tightly controlled by the cell’s redox state: oxidative signals such as hydrogen peroxide suppress MGL activity in a reversible manner through sulfenylation of the peroxidatic cysteines, C201 and C208. Here, using as a starting point the crystal structures of human MGL (hMGL), we present evidence from molecular dynamics and metadynamics simulations along with high-resolution mass spectrometry studies indicating that sulfenylation of C201 and C208 alters the conformational equilibrium of the membrane-associated lid domain of MGL to favour closed conformations of the enzyme that do not permit the entry of substrate into the active site.