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1.  Quantitative spectrophotometric assay for staphylococcal lipase. 
We report the development of a specific spectrophotometric assay for the quantitative determination of lipase activity in Staphylococcus aureus. The assay is based on the rate of clearance of a tributyrin emulsion, and it can detect as little as 1.0 micrograms of purified Pseudomonas lipase per ml. By comparison with the reaction rates obtained with Pseudomonas lipase, we calculated that S. aureus PS54C and S6C produce approximately 15 and 60 micrograms of extracellular lipase per ml, respectively. Neither PS54, which is lysogenized with the converting bacteriophage L54a and is consequently lipase negative (Lip-), nor KS1905, a Lip- transpositional mutant of strain S6C, was positive in our spectrophotometric assay. The specificity of the spectrophotometric tributyrin assay was confirmed with a triolein plate assay; supernatants from S6C and PS54C hydrolyzed triolein, while supernatants from PS54 and KSI905 did not. In contrast to the results of the spectrophotometric tributyrin assay, all enzyme preparations tested (including commercially purified esterase) were positive when examined by a tributyrin plate assay. The lack of specificity in the tributyrin plate assay emphasizes the need to interpret the results of tributyrin lipolysis kinetically for assessing lipase activity in S. aureus.
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PMCID: PMC183012  PMID: 1444391
2.  Conformational Plasticity and Ligand Binding of Bacterial Monoacylglycerol Lipase* 
The Journal of Biological Chemistry  2013;288(43):31093-31104.
Background: Monoacylglycerol lipases hydrolyze monoacylglycerols into free fatty acids and glycerols.
Results: Crystal structures provide the structural basis for conformational plasticity and ligand binding of a monoacylglycerol lipase.
Conclusion: Hallmark features responsible for substrate binding and selectivity are conserved across species.
Significance: The first structures of a monoacylglycerol lipase in complex with substrate analogs are presented.
Monoacylglycerol lipases (MGLs) play an important role in lipid catabolism across all kingdoms of life by catalyzing the release of free fatty acids from monoacylglycerols. The three-dimensional structures of human and a bacterial MGL were determined only recently as the first members of this lipase family. In addition to the α/β-hydrolase core, they showed unexpected structural similarities even in the cap region. Nevertheless, the structural basis for substrate binding and conformational changes of MGLs is poorly understood. Here, we present a comprehensive study of five crystal structures of MGL from Bacillus sp. H257 in its free form and in complex with different substrate analogs and the natural substrate 1-lauroylglycerol. The occurrence of different conformations reveals a high degree of conformational plasticity of the cap region. We identify a specific residue, Ile-145, that might act as a gatekeeper restricting access to the binding site. Site-directed mutagenesis of Ile-145 leads to significantly reduced hydrolase activity. Bacterial MGLs in complex with 1-lauroylglycerol, myristoyl, palmitoyl, and stearoyl substrate analogs enable identification of the binding sites for the alkyl chain and the glycerol moiety of the natural ligand. They also provide snapshots of the hydrolytic reaction of a bacterial MGL at different stages. The alkyl chains are buried in a hydrophobic tunnel in an extended conformation. Binding of the glycerol moiety is mediated via Glu-156 and water molecules. Analysis of the structural features responsible for cap plasticity and the binding modes of the ligands suggests conservation of these features also in human MGL.
doi:10.1074/jbc.M113.491415
PMCID: PMC3829422  PMID: 24014019
Crystal Structure; Lipase; Lipids; Lipid Metabolism; Monoacylglycerol; Monoglyceride; X-ray Crystallography; MGL; Phosphonate Inhibitor; Substrate Analogs
3.  Molecular reorganization of endocannabinoid signalling in Alzheimer’s disease 
Brain  2011;134(4):1041-1060.
Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB1 cannabinoid receptors. Cognitive decline in Alzheimer’s disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimer’s disease. Comparative protein profiling and quantitative morphometry showed that overall CB1 cannabinoid receptor protein levels in the hippocampi of patients with Alzheimer’s disease remain unchanged relative to age-matched controls, and CB1 cannabinoid receptor-positive presynapses engulf amyloid-β-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase α and β isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimer’s (Braak stage VI), with ectopic sn-1-diacylglycerol lipase β expression found in microglia accumulating near senile plaques and apposing CB1 cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimer’s disease (Braak stage III). However, Alzheimer’s pathology differentially impacts serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase α/β-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB1 cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimer’s tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimer’s disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-β. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimer’s disease.
doi:10.1093/brain/awr046
PMCID: PMC3069704  PMID: 21459826
glia; human; neurodegeneration; retrograde signalling; synapse
4.  The complete digestion of human milk triacylglycerol in vitro requires gastric lipase, pancreatic colipase-dependent lipase, and bile salt-stimulated lipase. 
Journal of Clinical Investigation  1990;85(4):1221-1226.
Gastric lipase, pancreatic colipase-dependent lipase, and bile salt-stimulated lipase all have potential roles in digestion of human milk triacylglycerol. To reveal the function of each lipase, an in vitro study was carried out with purified lipases and cofactors, and with human milk as substrate. Conditions were chosen to resemble those of the physiologic environment in the gastrointestinal tract of breast-fed infants. Gastric lipase was unique in its ability to initiate hydrolysis of milk triacylglycerol. Activated bile salt-stimulated lipase could not on its own hydrolyze native milk fat globule triacylglycerol, whereas a limited hydrolysis by gastric lipase triggered hydrolysis by bile salt-stimulated lipase. Gastric lipase and colipase-dependent lipase, in combination, hydrolyzed about two thirds of total ester bonds, with monoacylglycerol and fatty acids being the end products. Addition of bile salt-stimulated lipase resulted in hydrolysis also of monoacylglycerol. When acting together with colipase-dependent lipase, bile salt-stimulated lipase contributed also to digestion of tri- and diacylglycerol. We conclude that digestion of human milk triacylglycerol depends on three lipases with unique, only partly overlapping, functions. Their concerted action results in complete digestion with free glycerol and fatty acids as final products.
PMCID: PMC296555  PMID: 2318975
5.  Pancreatic lipase-related protein 2 digests fats in human milk and formula in concert with gastric lipase and carboxyl ester lipase 
Pediatric research  2013;74(2):127-132.
INTRODUCTION
Dietary fats must be digested into fatty acids and monoacylglycerols prior to absorption. In adults, colipase-dependent pancreatic triglyceride lipase (PTL) contributes significantly to fat digestion. In newborn rodents and humans, the pancreas expresses low levels of PTL. In rodents, a homologue of PTL, pancreatic lipase related protein 2 (PLRP2) and carboxyl ester lipase (CEL) compensate for the lack of PTL. In human newborns, the role for PLRP2 in dietary fat digestion is unclear. To clarify the potential of human PLRP2 to influence dietary fat digestion in newborns, we determined PLRP2 activity against human milk and infant formula.
METHODS
The activity of purified recombinant PLRP2, gastric lipase and CEL against fats in human milk and formula was measured with each lipase alone and in combination with a standard pH-stat assay.
RESULTS
Colipase added to human milk stimulated fat digestion. PLRP2 and CEL had activity against human milk and formula. Pre-digestion with gastric lipase increased PLRP2 activity against both substrates. Together, CEL and PLRP2 activity was additive with formula and synergistic with human milk.
CONCLUSIONS
PLRP2 can digest fats in human milk and formula. PLRP2 acts in concert with CEL and gastric lipase to digest fats in human milk in vitro.
doi:10.1038/pr.2013.90
PMCID: PMC3737390  PMID: 23732775
6.  The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes 
Biochimica et Biophysica Acta  2012;1821(7):1012-1021.
Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2 Å and in complex with phenylmethylsulfonyl fluoride at 1.8 Å resolution. In both structures, bMGL adopts an α/β hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism.
Highlights
► First crystal structure of monoacylglycerol lipase from bacterial species. ► Small angle X-ray scattering shows that the protein is a monomer in solution. ► A large hydrophobic channel enables access of the substrate to the active site. ► Molecular dynamic simulations reveal open and closed states of the cap region. ► The cap architecture is conserved on a structural but not on a sequence level.
doi:10.1016/j.bbalip.2012.04.006
PMCID: PMC3790968  PMID: 22561231
MGL, monoacylglycerol lipase; bMGL, monoacylglycerol lipase from Bacillus sp. H257; hMGL, human monoacylglycerol lipase; PMSF, phenylmethylsulfonyl fluoride; SAXS, small angle X-ray scattering; RMSD, root mean square deviation; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; Monoacylglycerol lipase; Open conformation; Evolutionary conservation; X-ray crystallography; Molecular dynamics simulation; Small-angle X-ray scattering
7.  Inhibition of monoacylglycerol lipase by troglitazone, N-arachidonoyl dopamine and the irreversible inhibitor JZL184: comparison of two different assays 
British Journal of Pharmacology  2010;161(7):1512-1526.
BACKGROUND AND PURPOSE
Drugs used clinically usually have a primary mechanism of action, but additional effects on other biological targets can contribute to their effects. A potentially useful additional target is the endocannabinoid metabolizing enzyme monoacylglycerol lipase (MGL). We have screened a range of drugs for inhibition of MGL and compared the observed potencies using different MGL enzyme assays.
EXPERIMENTAL APPROACH
MGL activity was screened using recombinant human MGL (cell lysates and purified enzyme) with 4-nitrophenyl acetate (NPA) as substrate. 2-Oleolyglycerol metabolism by rat cerebellar cytosolic MGL and by recombinant MGL was also investigated.
KEY RESULTS
Among the 96 compounds screened in the NPA assay, troglitazone, CP55,940, N-arachidonoyl dopamine and AM404 inhibited NPA hydrolysis by the lysates with IC50 values of 1.1, 4.9, 0.78 and 3.1 µM, respectively. The potency for troglitazone is in the same range as its primary pharmacological activity, activation of peroxisome proliferator-activated receptor (PPAR) γ. Among PPARγ ligands, the potency order towards human MGL was troglitazone > ciglitazone > rosiglitazone > 15-deoxy-Δ12,14-prostaglandin J2≈ CAY 10415 > CAY 10514. In contrast to the time-dependent inhibitor JZL184, the potency of troglitazone was dependent upon the enzyme assay system used. Thus, troglitazone inhibited rat cytosolic 2-oleoylglycerol hydrolysis less potently (IC50 41 µM) than hydrolysis of NPA by the human MGL lysates.
CONCLUSIONS AND IMPLICATIONS
‘Hits’ in screening programmes for MGL inhibitors should be assessed in different MGL assays. Troglitazone may be a useful lead for the design of novel, dual action MGL inhibitors/PPARγ activators.
doi:10.1111/j.1476-5381.2010.00974.x
PMCID: PMC3010564  PMID: 20735405
monoacylglycerol lipase; cannabinoid; troglitazone; rosiglitazone; peroxisome proliferator-activated receptor γ
8.  Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1 
PLoS ONE  2013;8(12):e81618.
Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.
doi:10.1371/journal.pone.0081618
PMCID: PMC3847081  PMID: 24312564
9.  Spinal administration of the monoacylglycerol lipase inhibitor JZL184 produces robust inhibitory effects on nociceptive processing and the development of central sensitization in the rat 
British Journal of Pharmacology  2012;167(8):1609-1619.
Background and Purpose
The cannabinoid receptor-mediated analgesic effects of 2-arachidonoylglycerol (2-AG) are limited by monoacylglycerol lipase (MAGL). 4-nitrophenyl 4-[bis (1,3-benzodioxol-5-yl) (hydroxy) methyl] piperidine-1-carboxylate (JZL184) is a potent inhibitor of MAGL in the mouse, though potency is reportedly reduced in the rat. Here we have assessed the effects of spinal inhibition of MAGL with JZL184 on nociceptive processing in rats.
Experimental Approach
In vivo spinal electrophysiological assays in anaesthetized rats were used to determine the effects of spinal administration of JZL184 on spinal nociceptive processing in the presence and absence of hindpaw inflammation. Contributions of CB1 receptors to these effects was assessed with AM251. Inhibition of 2-oleoylglycerol hydrolytic activity and alterations of 2-AG in the spinal cord after JZL 184 were also assessed.
Key Results
Spinal JZL184 dose-dependently inhibited mechanically evoked responses of wide dynamic range (WDR) neurones in naïve anaesthetized rats, in part via the CB1 receptor. A single spinal administration of JZL184 abolished inflammation-induced expansion of the receptive fields of spinal WDR neurones. However, neither spinal nor systemic JZL184 altered levels of 2-AG, or 2-oleoylglycerol hydrolytic activity in the spinal cord, although JZL184 displayed robust inhibition of MAGL when incubated with spinal cord tissue in vitro.
Conclusions and Implications
JZL184 exerted robust anti-nociceptive effects at the level of the spinal cord in vivo and inhibited rat spinal cord MAGL activity in vitro. The discordance between in vivo and in vitro assays suggests that localized sites of action of JZL184 produce these profound functional inhibitory effects.
Linked Articles
This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8
doi:10.1111/j.1476-5381.2012.02179.x
PMCID: PMC3525864  PMID: 22924700
2-arachidonoylglycerol; endocannabinoid system; JZL184; monoacylglycerol lipase; pain; CB1 receptor
10.  Interaction of ligands for the peroxisome proliferator-activated receptor γ with the endocannabinoid system 
British Journal of Pharmacology  2007;151(8):1343-1351.
Background and purpose:
There is good evidence that agents interacting with the endocannabinoid system in the body can also interact with the peroxisome proliferator-activated receptor γ. The present study was designed to test whether the reverse is true, namely whether peroxisome proliferator-activated receptor γ ligands have direct effects upon the activity of the endocannabinoid metabolizing enzyme fatty acid amide hydrolase.
Experimental approach:
Fatty acid amide hydrolase activity was measured in rat brain homogenates, C6 glioma and RBL2H3 basophilic leukaemia cells. Cellular uptake of anandamide was also assessed in these cells.
Key results:
Peroxisome proliferator-activated receptor γ activators inhibited the metabolism of the endocannabinoid anandamide in rat brain homogenates with an order of potency MCC-555 > indomethacin ≈ ciglitazone ≈ 15-deoxy-Δ12,14-prostaglandin J2 ≈ pioglitazone > rosiglitazone > troglitazone. The antagonists BADGE, GW9662 and T0070907 were poor inhibitors of anandamide hydrolysis. The inhibition by ciglitazone was competitive and increased as the pH of the assay buffer was decreased; the Ki value at pH 6.0 was 17 μM. In intact C6 glioma cells assayed at pH 6.2, significant inhibition of anandamide hydrolysis was seen at 3 μM ciglitazone, whereas 100 μM was required to produce significant inhibition at pH 7.4. Ciglitazone also interacted with monoacylglycerol lipase as well as with cannabinoid CB1 and CB2 receptors.
Conclusions and implications:
Ciglitazone may be useful as a template for the design of novel dual action anti-inflammatory agents which are both inhibitors of fatty acid amide hydrolase and agonists at the peroxisome proliferator-activated receptor γ.
doi:10.1038/sj.bjp.0707352
PMCID: PMC2189817  PMID: 17592505
endocannabinoid; anandamide; fatty acid amide hydrolase; cannabinoid receptors; peroxisome proliferator-activated receptor γ; ciglitazone
11.  Biochemical and Pharmacological Characterization of the Human Lymphocyte Antigen B-Associated Transcript 5 (BAT5/ABHD16A) 
PLoS ONE  2014;9(10):e109869.
Background
Human lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the α/β-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 (C14∶0), long-chain unsaturated (C18∶1, C18∶2, C20∶4) monoacylglycerols (MAGs) and 15-deoxy-Δ12,14-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 C18∶1, C18∶2 and C20∶4. Inhibitor profiling revealed that β-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.
doi:10.1371/journal.pone.0109869
PMCID: PMC4188605  PMID: 25290914
12.  A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction▿ †  
Journal of Bacteriology  2010;192(18):4776-4785.
MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.
doi:10.1128/JB.00261-10
PMCID: PMC2937407  PMID: 20601476
13.  The inhibition of monoacylglycerol lipase by URB602 showed an anti-inflammatory and anti-nociceptive effect in a murine model of acute inflammation 
British Journal of Pharmacology  2007;152(5):787-794.
Background and purpose:
2-arachidonoylglycerol (2-AG) is an endocannabinoid whose hydrolysis is predominantly catalysed by the enzyme monoacylglycerol lipase (MAGL). The development of MAGL inhibitors could offer an opportunity to investigate the anti-inflammatory and anti-nociceptive role of 2-AG, which have not yet been elucidated. On these bases, URB602, a MAGL inhibitor, was tested in a murine model of inflammation/inflammatory pain.
Experimental approach:
Acute inflammation was induced by intraplantar injection of λ-carrageenan into mice. The highest dose to be employed has been selected performing the tetrad assays for cannabimimetic activity in mice. URB602 anti-inflammatory and anti-nociceptive efficacy (assessed by plethysmometer and plantar test, respectively) was evaluated both in a preventive regimen (drug administered 30 min before carrageenan) and in a therapeutic regimen (URB602 administered 30 min after carrageenan). To elucidate the cannabinoid receptor involvement, rimonabant and SR144528, CB1 and CB2 selective antagonists, respectively, were given 15 min before URB602.
Key results:
Systemic administration of URB602 elicited a dose-dependent anti-oedemigen and anti-nociceptive effect that was reversed exclusively by the CB2 receptor antagonist. The efficacy of URB602 persisted also when the compound was administered in a therapeutic regimen, suggesting the ability of URB602 to improve established disease.
Conclusions and implications:
The present report highlighted the ability of the selective MAGL inhibitor, URB602, to prevent and treat an acute inflammatory disease without producing adverse psychoactive effects. The data presented herein also contributed to clarify the physiological role of 2-AG in respect to inflammatory reactions, suggesting its protective role in the body.
doi:10.1038/sj.bjp.0707425
PMCID: PMC2190015  PMID: 17700715
2-arachidonoylglycerol; carrageenan; cannabinoid receptor; endocannabinoid system; inflammation; monoacylglycerol lipase; mouse tetrad; pain; URB602
14.  Lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis in vitro 
British Journal of Pharmacology  2006;150(2):186-191.
Background and purpose:
Two compounds, URB602 and URB754, have been reported in the literature to be selective inhibitors of monoacylglycerol lipase, although a recent study has questioned their ability to prevent 2-arachidonoyl hydrolysis by brain homogenates and cerebellar membranes. In the present study, the ability of these compounds to inhibit monoacylglycerol lipase and fatty acid amide hydrolase has been reinvestigated.
Experimental approach:
Homogenates and cell lines were incubated with test compounds and, thereafter, with either [3H]-2-oleoylglycerol or [3H]-anandamide. Labelled reaction products were separated from substrate using chloroform: methanol extraction.
Key results:
In cytosolic fractions from rat brain, URB602 and URB754 inhibited the hydrolysis of 2-oleoylglycerol with IC50 values of 25 and 48 μM, respectively. Anandamide hydrolysis by brain membranes was not sensitive to URB754, but was inhibited by URB602 (IC50 value 17 μM). Hydrolysis of 2-oleoylglycerol by human recombinant monoacylglycerol lipase was sensitive to URB602, but not URB754. The lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis was also observed for intact RBL2H3 basophilic leukaemia cells. C6 glioma expressed mRNA for monoacylglycerol lipase, and hydrolyzed 2-oleoylglycerol in a manner sensitive to inhibition by methyl arachidonoyl fluorophosphonate but not URB754 or URB597. MC3T3-E1 mouse osteoblastic cells, which did not express mRNA for monoacylglycerol lipase, hydrolyzed 2-oleoylglycerol in the presence of URB597, but the hydrolysis was less sensitive to methyl arachidonoyl fluorophosphonate than for C6 cells.
Conclusions and implications:
The data demonstrate that the compounds URB602 and URB754 do not behave as selective and/or potent inhibitors of monoacylglycerol lipase.
doi:10.1038/sj.bjp.0706971
PMCID: PMC2042901  PMID: 17143303
2-acylglycerol; anandamide; monoacylglycerol lipase; fatty acid amide hydrolase; URB597; URB602; URB754
15.  Phosphatidylinositol-specific Phospholipase C in Fetal Membranes and Uterine Decidua 
Journal of Clinical Investigation  1981;67(3):847-856.
An assay procedure was developed in which phosphatidyl[2-3H]inositol was employed as substrate for the measurement of phosphatidylinositol-specific phospholipase C activity. Employing this assay, phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua was identified and characterized. The specific activity of this enzyme in amnion (4.4 μmol × mg−1 protein × h−1) was three times that in uterine decidua and more than five times that in chorion laeve. No difference was found between the specific activity of phosphatidylinositol-specific phospholipase C in placental amnion and that in reflected amnion. The products of phosphatidylinositol hydrolysis in short-term incubations were stoichiometric amounts of diacylglycerol and inositol-1,2-cyclic-phosphate plus inositol-1-phosphate. After longer periods of incubation, monoacylglycerol also was detected. Diacylglycerol lipase activity also was demonstrated in these tissues. More than 90% of phosphatidylinositol-specific phospholipase C activity of amnion tissue was recovered in the 105,000-g supernatant fraction, and optimal enzymatic activity in vitro was observed at pH 6.5-7.5 in the presence of Ca2+ (8 mM) and mercaptoethanol (4 mM). Phosphatidylinositol-specific phospholipase C activity was stimulated by fatty acids in low concentrations, but was inhibited by lysophosphatidylcholine and a variety of detergents. No effect of labor on the specific activity of phosphatidylinositol-specific phospholipase C in either fetal membranes or uterine decidua could be detected. The finding of an active phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua is complementary to our previous finding of a selective loss of arachidonic acid from phosphatidylinositol of human fetal membranes during labor. The action of phosphatidylinositol-specific phospholipase C, coupled to diacylglycerol lipase action, could provide a mechanism for the release of arachidonic acid for prostaglandin biosynthesis during parturition.
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PMCID: PMC370636  PMID: 7204559
16.  Effects of cannabinoids and cannabinoid-enriched Cannabis extracts on TRP channels and endocannabinoid metabolic enzymes 
British Journal of Pharmacology  2011;163(7):1479-1494.
BACKGROUND AND PURPOSE
Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) interact with transient receptor potential (TRP) channels and enzymes of the endocannabinoid system.
EXPERIMENTAL APPROACH
The effects of 11 pure cannabinoids and botanical extracts [botanical drug substance (BDS)] from Cannabis varieties selected to contain a more abundant cannabinoid, on TRPV1, TRPV2, TRPM8, TRPA1, human recombinant diacylglycerol lipase α (DAGLα), rat brain fatty acid amide hydrolase (FAAH), COS cell monoacylglycerol lipase (MAGL), human recombinant N-acylethanolamine acid amide hydrolase (NAAA) and anandamide cellular uptake (ACU) by RBL-2H3 cells, were studied using fluorescence-based calcium assays in transfected cells and radiolabelled substrate-based enzymatic assays. Cannabinol (CBN), cannabichromene (CBC), the acids (CBDA, CBGA, THCA) and propyl homologues (CBDV, CBGV, THCV) of CBD, cannabigerol (CBG) and THC, and tetrahydrocannabivarin acid (THCVA) were also tested.
KEY RESULTS
CBD, CBG, CBGV and THCV stimulated and desensitized human TRPV1. CBC, CBD and CBN were potent rat TRPA1 agonists and desensitizers, but THCV-BDS was the most potent compound at this target. CBG-BDS and THCV-BDS were the most potent rat TRPM8 antagonists. All non-acid cannabinoids, except CBC and CBN, potently activated and desensitized rat TRPV2. CBDV and all the acids inhibited DAGLα. Some BDS, but not the pure compounds, inhibited MAGL. CBD was the only compound to inhibit FAAH, whereas the BDS of CBC > CBG > CBGV inhibited NAAA. CBC = CBG > CBD inhibited ACU, as did the BDS of THCVA, CBGV, CBDA and THCA, but the latter extracts were more potent inhibitors.
CONCLUSIONS AND IMPLICATIONS
These results are relevant to the analgesic, anti-inflammatory and anti-cancer effects of cannabinoids and Cannabis extracts.
LINKED ARTICLES
This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7
doi:10.1111/j.1476-5381.2010.01166.x
PMCID: PMC3165957  PMID: 21175579
cannabinoids; cannabis; botanical extract; TRP channel; TRPV1; TRPA1; TRPM8; endocannabinoid; monoacylglycerol lipase; diacylglycerol lipase
17.  A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects 
British Journal of Pharmacology  2013;169(4):784-793.
Background and Purpose
The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects.
Experimental Approach
Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice.
Key Results
Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLα, and selectivity (IC50 > 10 μM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥10 μM, and showed that this compound has only one major ‘off-target’, that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 μM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 μM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0–12 mg·kg−1, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight.
Conclusions and Implications
O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.
Linked Articles
This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8
doi:10.1111/bph.12013
PMCID: PMC3687659  PMID: 23072382
inhibitor; diacylglycerol; cannabinoid; 2-arachidonoylglycerol; serine lipase; anti-obesity effects
18.  Multiple Forms of Endocannabinoid and Endovanilloid Signaling Regulate the Tonic Control of GABA Release 
The Journal of Neuroscience  2015;35(27):10039-10057.
Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM251, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic 2-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and FAAH activity.
SIGNIFICANCE STATEMENT Tonic cannabinoid signaling plays a critical role in the regulation of synaptic transmission. However, the mechanistic details of how persistent CB1 cannabinoid receptor activity inhibits neurotransmitter release have remained elusive. Therefore, electrophysiological recordings, lipid measurements, and super-resolution imaging were combined to elucidate those signaling molecules and mechanisms that underlie tonic cannabinoid signaling. The findings indicate that constitutive CB1 activity has pivotal function in the tonic control of hippocampal GABA release. Moreover, the endocannabinoid 2-arachidonoylglycerol (2-AG) is continuously generated postsynaptically, but its synaptic effect is regulated strictly by presynaptic monoacylglycerol lipase activity. Finally, anandamide signaling antagonizes tonic 2-AG signaling via activation of postsynaptic transient receptor potential vanilloid TRPV1 receptors. This unexpected mechanistic diversity may be necessary to fine-tune GABA release probability under various physiological and pathophysiological conditions.
doi:10.1523/JNEUROSCI.4112-14.2015
PMCID: PMC4495235  PMID: 26157003
2-arachidonoylglycerol; endocannabinod; GABA; hippocampus; interneuron; TRPV1
19.  The activity of the endocannabinoid metabolising enzyme fatty acid amide hydrolase in subcutaneous adipocytes correlates with BMI in metabolically healthy humans 
Background
The endocannabinoid system (ECS) is a ubiquitously expressed signalling system, with involvement in lipid metabolism and obesity. There are reported changes in obesity of blood concentrations of the endocannabinoids anandamide (AEA) and 2-arachidonoylglcyerol (2-AG), and of adipose tissue expression levels of the two key catabolic enzymes of the ECS, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Surprisingly, however, the activities of these enzymes have not been assayed in conditions of increasing adiposity. The aim of the current study was to investigate whether FAAH and MGL activities in human subcutaneous adipocytes are affected by body mass index (BMI), or other markers of adiposity and metabolism.
Methods
Subcutaneous abdominal mature adipocytes, fasting blood samples and anthropometric measurements were obtained from 28 metabolically healthy subjects representing a range of BMIs. FAAH and MGL activities were assayed in mature adipocytes using radiolabelled substrates. Serum glucose, insulin and adipokines were determined using ELISAs.
Results
MGL activity showed no relationship with BMI or other adiposity indices, metabolic markers (fasting serum insulin or glucose) or serum adipokine levels (adiponectin, leptin or resistin). In contrast, FAAH activity in subcutaneous adipocytes correlated positively with BMI and waist circumference, but not with skinfold thickness, metabolic markers or serum adipokine levels.
Conclusions
In this study, novel evidence is provided that FAAH activity in subcutaneous mature adipocytes increases with BMI, whereas MGL activity does not. These findings support the hypothesis that some components of the ECS are upregulated with increasing adiposity in humans, and that AEA and 2-AG may be regulated differently.
doi:10.1186/1476-511X-10-129
PMCID: PMC3161878  PMID: 21813022
Endocannabinoid system; Fatty acid amide hydrolase; Monoacylglycerol lipase; Human; Adipocytes; BMI; Insulin; Glucose
20.  The influence of monoacylglycerol lipase inhibition upon the expression of epidermal growth factor receptor in human PC-3 prostate cancer cells 
BMC Research Notes  2014;7:441.
Background
It has been reported that direct activation of the cannabinoid CB1 receptor in epidermal growth factor (EGR)-stimulated PC-3 prostate cancer cells results in an anti-proliferative effect accompanied by a down-regulation of EGF receptors (EGFR). In the present study, we investigated whether similar effects are seen following inhibition of the endocannabinoid hydrolytic enzyme monoacylglycerol lipase (MGL).
Results
CB1 receptor expression levels were found to differ greatly between two experimental series conducted using PC-3 cells. The monoacylglycerol lipase inhibitor JZL184 increased levels of 2-arachidonoylglycerol in the PC-3 cells without producing changes in the levels of anandamide and related N-acylethanolamines. In the first series of experiments, JZL184 produced a small mitogenic effect for cells that had not been treated with EGF, whereas an anti-proliferative effect was seen for EGF-treated cells. An anti-proliferative effect for the EGF-treated cells was also seen with the CB receptor agonist CP55,940. In the second batch of cells, there was an interaction between JZL184 and CB1 receptor expression densities in linear regression analyses with EGFR expression as the dependent variable.
Conclusions
Inhibition of MGL by JZL184 can affect EGFR expression. However, the use in our hands of PC-3 cells as a model to investigate the therapeutic potential of MGL inhibitors and related compounds is compromised by their variability of CB1 receptor expression.
doi:10.1186/1756-0500-7-441
PMCID: PMC4109781  PMID: 25012825
Prostate cancer; Epidermal growth factor; Cannabinoid receptor; Monoacylglycerol lipase
21.  3D-QSAR study of benzotriazol-1-yl carboxamide scaffold as monoacylglycerol lipase inhibitors 
Purpose:
The purpose of this study is to build up the 3D pharmacophore of Monoacylglycerol lipase (MAGL) inhibitor and to provide the basis to design the novel and potent MAGL inhibitors.
Material and Method:
A 3D-QSAR study on benztriazol-1-yl carboxamide derivatives as monoacylglycerol lipase (MAGL) inhibitors was successfully performed by means of pharmacophore mapping using PHASE 3.5 module of Schrφdinger-9.4.
Result:
The 3D-QSAR obtained from APRRR-105 hypothesis was found to be statistically good with r2 = 0.9228 and q2 = 0.871, taking PLS factor 4. The statistical significance of the model was also confirmed by a high value of Fisher's ratio of 82.8 and a very low value of root-mean-square error (RMSE) 0.2564. Another parameter which signifies the model predictivity is Pearson R. Its value of 0.9512 showed that the correlation between predicted and observed activities for the test set compounds is excellent.
Conclusion:
The study suggested that one H-bond acceptor, one positive center, and proper positioning of hydrophobic groups near the distal aromatic ring C are the crucial determinants for MAGL inhibition. Thus, it can be assumed that the present QSAR analysis is enough to demonstrate MAGL inhibition with the help of APRRR-105 hypothesis and will be helpful in designing novel and potent MAGL inhibitors.
doi:10.4103/0975-7406.142957
PMCID: PMC4231386  PMID: 25400409
3D-QSAR; benztriazol-1-yl carboxamides; monoacylglycerol lipase
22.  Organophosphate-Sensitive Lipases Modulate Brain Lysophospholipids, Ether Lipids and Endocannabinoids 
Chemico-biological interactions  2008;175(1-3):355-364.
Lipases play key roles in nearly all cells and organisms. Potent and selective inhibitors help to elucidate their physiological functions and associated metabolic pathways. Organophosphorus (OP) compounds are best known for their anticholinesterase properties but selectivity for lipases and other targets can also be achieved through structural optimization. This review considers several lipid systems in brain modulated by highly OP-sensitive lipases. Neuropathy target esterase (NTE) hydrolyzes lysophosphatidylcholine as a preferred substrate. Gene deletion of NTE in mice is embryo lethal and the heterozygotes are hyperactive. NTE is very sensitive in vitro and in vivo to direct-acting OP delayed neurotoxicants and the related NTE-R is also inhibited in vivo. KIAA1363 hydrolyzes acetyl monoalkylglycerol ether of the platelet-activating factor de novo biosynthetic pathway and is a marker of cancer cell invasiveness. It is also a detoxifying enzyme that hydrolyzes chlorpyrifos oxon (CPO) and some other potent insecticide metabolites. Monoacylglycerol lipase and fatty acid amide hydrolase regulate endocannabinoid levels with roles in motility, pain and memory. Inhibition of these enzymes in mice by OPs, such as isopropyl dodecylfluorophosphonate (IDFP), leads to dramatic elevation of brain endocannabinoids and distinct cannabinoid-dependent behavior. Hormone-sensitive lipase that hydrolyzes cholesteryl esters and diacylglycerols is a newly-recognized in vivo CPO- and IDFP-target in brain. The OP chemotype can therefore be used in proteomic and metabolomic studies to further elucidate the biological function and toxicological significance of lipases in lipid metabolism. Only the first steps have been taken to achieve appropriate selective action for OP therapeutic agents.
doi:10.1016/j.cbi.2008.04.008
PMCID: PMC2582404  PMID: 18495101
acetylcholinesterase; brain; chemical warfare agent; fatty acid amide hydrolase; hormone-sensitive lipase; insecticide; KIAA1363; monoacylglycerol lipase; neuropathy target esterase; organophosphorus; serine hydrolase
23.  The antinociceptive triterpene β-amyrin inhibits 2-arachidonoylglycerol (2-AG) hydrolysis without directly targeting cannabinoid receptors 
British Journal of Pharmacology  2012;167(8):1596-1608.
BACKGROUND AND PURPOSE
Pharmacological activation of cannabinoid CB1 and CB2 receptors is a therapeutic strategy to treat chronic and inflammatory pain. It was recently reported that a mixture of natural triterpenes α- and β-amyrin bound selectively to CB1 receptors with a subnanomolar Ki value (133 pM). Orally administered α/β-amyrin inhibited inflammatory and persistent neuropathic pain in mice through both CB1 and CB2 receptors. Here, we investigated effects of amyrins on the major components of the endocannabinoid system.
EXPERIMENTAL APPROACH
We measured CB receptor binding interactions of α- and β-amyrin in validated binding assays using hCB1 and hCB2 transfected CHO-K1 cells. Effects on endocannabinoid transport in U937 cells and breakdown using homogenates of BV2 cells and pig brain, as well as purified enzymes, were also studied.
KEY RESULTS
There was no binding of either α- or β-amyrin to hCB receptors in our assays (Ki > 10 µM). The triterpene β-amyrin potently inhibited 2-arachidonoyl glycerol (2-AG) hydrolysis in pig brain homogenates, but not that of anandamide. Although β-amyrin only weakly inhibited purified human monoacylglycerol lipase (MAGL), it also inhibited α,β-hydrolases and more potently inhibited 2-AG breakdown than α-amyrin and the MAGL inhibitor pristimerin in BV2 cell and pig brain homogenates.
CONCLUSIONS AND IMPLICATIONS
We propose that β-amyrin exerts its analgesic and anti-inflammatory pharmacological effects via indirect cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid 2-AG without interacting directly with CB receptors. Triterpenoids appear to offer a very broad and largely unexplored scaffold for inhibitors of the enzymic degradation of 2-AG.
LINKED ARTICLES
This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8
doi:10.1111/j.1476-5381.2012.02059.x
PMCID: PMC3525863  PMID: 22646533
amyrin; 2-AG; CB1 receptor; endocannabinoid system; hydrolases; monoacyl glycerol lipase; pristimerin; triterpene
24.  A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors 
British Journal of Pharmacology  2009;157(6):974-983.
Background and purpose:
Monoacylglycerol lipase (MGL) is a presynaptic serine hydrolase that inactivates the endocannabinoid neurotransmitter, 2-arachidonoyl-sn-glycerol. Recent studies suggest that cysteine residues proximal to the enzyme active site are important for MGL function. In the present study, we characterize the role of cysteines in MGL function and identify a series of cysteine-reactive agents that inhibit MGL activity with nanomolar potencies by interacting with cysteine residue 208.
Experimental approach:
A series of cysteine traps were screened for the ability to inhibit MGL in vitro. Rapid dilution assays were performed to determine reversibility of inhibition. Molecular modelling and site-directed mutagenesis were utilized to identify cysteine residues targeted by the inhibitors.
Key results:
The screening revealed that 2-octyl-4-isothiazolin-3-one (octhilinone) inhibited purified rat recombinant MGL (IC50= 88 ± 12 nM) through a partially reversible mechanism. Initial structure–activity relationship studies showed that substitution of the n-octyl group of octhilinone with a more lipophilic oleoyl group increased inhibitor potency (IC50= 43 ± 8 nM), while substitution with a methyl group produced the opposite effect (IC50= 239 ± 68 nM). The inhibitory potency of octhilinone was selectively decreased by mutating cysteine 208 in MGL to glycine (IC50; wild-type, 151 ± 17 nM; C208G, 722 ± 74 nM), but not by mutation of other cysteine residues (C32, C55, C201, C208 and C242).
Conclusions and implications:
The results indicated that cysteine 208 plays an important role in MGL function and identified a novel class of isothiazolinone-based MGL inhibitors with nanomolar potency in vitro.
doi:10.1111/j.1476-5381.2009.00276.x
PMCID: PMC2737656  PMID: 19486005
monoacylglycerol lipase; 2-arachidonoylglycerol; endocannabinoids
25.  The FAAH inhibitor URB597 efficiently reduces tyrosine hydroxylase expression through CB1- and FAAH-independent mechanisms 
British Journal of Pharmacology  2013;169(4):794-807.
Background
Anandamide and 2-arachidonoylglycerol are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between ‘on demand’ generation and enzymatic degradation [by fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase]. Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis.
Experimental Approach
We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabelled substrate hydrolysis and endogenous endocannabinoids.
Key Results
Anandamide decreased TH promoter activity in N1E115 cells through CB1 receptor activation. Unexpectedly, URB597 reduced TH expression (pEC50 = 8.7 ± 0.2) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB1 receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain.
Conclusions
While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB1 and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when interpreting the data.
Linked Articles
This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8
doi:10.1111/j.1476-5381.2012.02208.x
PMCID: PMC3687660  PMID: 22970888
endocannabinoid; carbamate; off-target; dopamine; tyrosine hydroxylase promoter; CB1-independent mechanism; Abn-CBD; cannabidiol; MAPK; PF750

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