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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.  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
8.  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
9.  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
10.  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.
Images
PMCID: PMC370636  PMID: 7204559
11.  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
12.  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
13.  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
14.  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
15.  N-Arachidonyl Maleimide Potentiates the Pharmacological and Biochemical Effects of the Endocannabinoid 2-Arachidonylglycerol through Inhibition of Monoacylglycerol Lipase 
Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with Δ9-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB1 receptor-mediated because they were attenuated (but not eliminated) by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl] and in CB1−/− mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5′-O-(3-[35S]thio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB1, non-CB2 cannabinoid receptors.
doi:10.1124/jpet.108.141382
PMCID: PMC2605346  PMID: 18682568
16.  The effects of obesity, diabetes and metabolic syndrome on the hydrolytic enzymes of the endocannabinoid system in animal and human adipocytes 
Background
Circulating endocannabinoid levels are increased in obesity and diabetes. We have shown that fatty acid amide hydrolase (FAAH, an endocannabinoid hydrolysing enzyme) in subcutaneous adipose tissue positively correlates with BMI in healthy volunteers. The aim of the present study was to investigate whether the hydrolytic enzymes of the endocannabinoid system are affected by diabetes or metabolic syndrome in obesity.
Methods
Using radiolabelled substrates, FAAH and monoacylglycerol lipase (MGL) activities were assessed in adipocytes from various adipose depots in Zucker rats (n = 22, subcutaneous abdominal, visceral and epididymal) and bariatric patients (n = 28, subcutaneous abdominal and omental).
Results
FAAH activity was significantly increased in adipocytes of obese (Zucker Fatty) compared to Zucker lean rats (P < 0.05) but was not raised in the Zucker Diabetic Fatty rats (ZDF). MGL activity was raised in both Zucker Fatty (P < 0.001-0.01) and ZDF rats (P < 0.05) and was positively correlated with body weight and plasma glucose levels (P < 0.01). In bariatric patients (BMI range 37–58 kg.m2), there was a trend for MGL activity to correlate positively with BMI, reaching significance when type 2 diabetic patients were removed. FAAH and MGL activities in obese humans were not correlated with blood pressure, skinfold thicknesses, fasting glucose, insulin, HbA1c, triglycerides or cholesterol levels.
Conclusions
FAAH in adipocytes is differentially altered in animal models of obesity and diabetes, while MGL activity is increased by both. However, in obese humans, FAAH or MGL activity in adipocytes is not affected by diabetes, dyslipidaemia or other markers of metabolic dysfunction. This suggests increased circulating levels of endocannabinoids are not a result of altered degradation in adipose tissue.
doi:10.1186/1476-511X-13-43
PMCID: PMC3995979  PMID: 24593280
Endocannabinoid; Adipose; Adipocyte; Obesity; Type 2 diabetes; Fatty acid amide hydrolase (FAAH); Monoacylglycerol lipase (MGL); Rat; Human
17.  Activity-Based Protein Profiling of Organophosphorus and Thiocarbamate Pesticides Reveals Multiple Serine Hydrolase Targets in Mouse Brain 
Organophosphorus (OP) and thiocarbamate (TC) agrochemicals are used worldwide as insecticides, herbicides, and fungicides, but their safety assessment in terms of potential off-targets remains incomplete. In this study, we used a chemoproteomic platform, termed activity-based protein profiling, to broadly define serine hydrolase targets in mouse brain of a panel of 29 OP and TC pesticides. Among the secondary targets identified, enzymes involved in degradation of endocannabinoid signaling lipids, monoacylglycerol lipase and fatty acid amide hydrolase, were inhibited by several OP and TC pesticides. Blockade of these two enzymes led to elevations in brain endocannabinoid levels and dysregulated brain arachidonate metabolism. Other secondary targets include enzymes thought to also play important roles in the nervous system and unannotated proteins. This study reveals a multitude of secondary targets for OP and TC pesticides and underscores the utility of chemoproteomic platforms in gaining insights into biochemical pathways that are perturbed by these toxicants.
doi:10.1021/jf101747r
PMCID: PMC3071868  PMID: 21341672
Activity-based protein profiling; cannabinoid; fatty acid amide hydrolase; monoacylglycerol lipase; organophosphorus; pesticides; serine hydrolase; thiocarbamate
18.  The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model 
Life sciences  2012;92(0):498-505.
Aim
The present study tested whether the selective monoacylglycerol lipase (MAGL) inhibitor JZL184 would reduce allodynia and paw edema in the carrageenan test.
Main methods
The anti-edematous and anti-allodynic effects of JZL184 were compared to those of PF-3845, an inhibitor of fatty acid amide hydrolase (FAAH), and diclofenac, a non-selective cyclooxygenase inhibitor. Cannabinoid receptor involvement in the anti-edematous and anti-allodynic effects of JZL184 was evaluated by administration of the respective CB1 and CB2 receptor antagonists rimonabant and SR144528 as well as with CB1(−/−) and CB2(−/−) mice. JZL184 (1.6, 4, 16, or 40 mg/kg) was administered for six days to assess tolerance.
Key findings
JZL184 administered before or after carrageenan significantly attenuated carrageenan-induced paw edema and mechanical allodynia. Complementary genetic and pharmacological approaches revealed that the anti-allodynic effects of JZL184 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, both the anti-edematous and anti-allodynic effects underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy.
Significance
These results suggest that the MAGL inhibitor JZL184 reduces inflammatory nociception through the activation of both CB1 and CB2 receptors, with no evidence of tolerance following repeated administration of low doses.
doi:10.1016/j.lfs.2012.06.020
PMCID: PMC3717616  PMID: 22749865
Carrageenan; Pain; Allodynia; Inflammation; 2-arachidonylglycerol (2-AG); Monoacylglycerol lipase (MAGL); Endogenous cannabinoid; Anandamide; Fatty acid amide hydrolase; CB1 receptor; CB2 receptor
19.  A Peptide Derived from G0/G1 Switch Gene 2 Acts as Noncompetitive Inhibitor of Adipose Triglyceride Lipase* 
The Journal of Biological Chemistry  2014;289(47):32559-32570.
Background: G0S2 acts as an endogenous inhibitor of ATGL.
Results: We designed a short peptide that is active in the nanomolar range. The G0S2-derived peptide inhibits ATGL activity in a noncompetitive manner.
Conclusion: G0S2-derived peptides act as potent and specific inhibitors of ATGL.
Significance: G0S2-derived peptides provide a novel tool to modulate ATGL activity.
The protein G0/G1 switch gene 2 (G0S2) is a small basic protein that functions as an endogenous inhibitor of adipose triglyceride lipase (ATGL), a key enzyme in intracellular lipolysis. In this study, we identified a short sequence covering residues Lys-20 to Ala-52 in G0S2 that is still fully capable of inhibiting mouse and human ATGL. We found that a synthetic peptide corresponding to this region inhibits ATGL in a noncompetitive manner in the nanomolar range. This peptide is highly selective for ATGL and does not inhibit other lipases, including hormone-sensitive lipase, monoacylglycerol lipase, lipoprotein lipase, and patatin domain-containing phospholipases 6 and 7. Because increased lipolysis is linked to the development of metabolic disorders, the inhibition of ATGL by G0S2-derived peptides may represent a novel therapeutic tool to modulate lipolysis.
doi:10.1074/jbc.M114.602599
PMCID: PMC4239610  PMID: 25258314
Adipose Triglyceride Lipase (ATGL); Enzyme Inhibitor; Lipid; Lipid Metabolism; Lipolysis; Peptides; Atglistatin; G0/G1 Switch Gene 2 (G0S2); hGW2052; Noncompetitive Inhibition
20.  Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis 
Extremophiles   2009;13(5):769-783.
Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90°C and between pH 6.5 and 10; the half-life of the enzymes at 75°C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53–2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.
doi:10.1007/s00792-009-0265-z
PMCID: PMC2757599  PMID: 19579003
Thermophiles and thermophilic enzymes; Anaerobic bacteria; Gene cloning and expression
21.  Effect of germinated brown rice extracts on pancreatic lipase, adipogenesis and lipolysis in 3T3-L1 adipocytes 
Background
This study investigated anti-obesity effects of seven different solvent (n-hexane, toluene, dicholoromethane, ethyl acetate, absolute methanol, 80% methanol and deionized water) extracts of germinated brown rice (GBR) on pancreatic lipase activity, adipogenesis and lipolysis in 3T3-L1 adipocytes.
Methods
GBR were extracted separately by employing different solvents with ultrasound-assisted. Pancreatic lipase activity was determined spectrophotometrically by measuring the hydrolysis of p-nitrophenyl butyrate (p-NPB) to p-nitrophenol at 405 nm. Adipogenesis and lipolysis were assayed in fully differentiated 3T3-L1 adipocytes by using Oil Red O staining and glycerol release measurement.
Results
GBR extract using hexane showed the highest inhibitory effect (13.58 ± 0.860%) at concentration of 200 μg/ml followed by hexane extract at 100 μg/ml (9.98 ± 1.048%) while ethyl acetate extract showed the lowest (2.62 ± 0.677%) at concentration of 200 μg/ml on pancreatic lipase activity. Water extract at 300 μg/ml showed 61.55 ± 3.824% of Oil Red O staining material (OROSM), a marker of adipogenesis. It significantly decrease (p < 0.05) lipid accumulation than control (OROSM = 100%), follow by ethyl acetate extract at 300 μg/ml (OROSM = 65.17 ± 3.131%). All the GBR extracts induced lipolysis with 1.22-1.83 fold of greater glycerol release than control.
Conclusions
GBR extracts especially the least polar and intermediate polar solvent extracts exhibited inhibitory effect on pancreatic lipase, decrease fat accumulation by adipocyte differentiation inhibition, and stimulate lipolysis on adipocytes. Therefore, GBR could be furthered study and developed as a functional food in helping the treatment and/or prevention of obesity.
doi:10.1186/1476-511X-13-169
PMCID: PMC4232653  PMID: 25367070
Obesity; Germinated brown rice; Pancreatic lipase; 3T3-L1 adipocytes
22.  High Extracellular Ca2+ Stimulates Ca2+-Activated Cl− Currents in Frog Parathyroid Cells through the Mediation of Arachidonic Acid Cascade 
PLoS ONE  2011;6(4):e19158.
Elevation of extracellular Ca2+ concentration induces intracellular Ca2+ signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca2+ pathways, but the direct mechanism responsible for the rise of intracellular Ca2+ concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca2+ signaling in frog parathyroid cells and show that Ca2+-activated Cl− channels are activated by intracellular Ca2+ increase through an inositol 1,4,5-trisphophate (IP3)-independent pathway. High extracellular Ca2+ induced an outwardly-rectifying conductance in a dose-dependent manner (EC50∼6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca2+-induced and Ca2+ dialysis-induced currents reversed at the equilibrium potential of Cl− and were inhibited by niflumic acid (a specific blocker of Ca2+-activated Cl− channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca2+-induced current, suggesting the change of intracellular Cl− concentration in a few minutes. Extracellular Ca2+-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca2+-induced current. IP3 dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG), arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE) dialysis increased the conductance identical to extracellular Ca2+-induced conductance. These results indicate that high extracellular Ca2+ raises intracellular Ca2+ concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl− conductance.
doi:10.1371/journal.pone.0019158
PMCID: PMC3084778  PMID: 21559478
23.  The modulation of pancreatic lipase activity by alginates 
Food Chemistry  2014;146(100):479-484.
Highlights
•Certain alginates are effective inhibitors of pancreatic lipase.•The level of lipase inhibition by alginates is related to the structure of the polymer.•High guluronate alginates achieve greater lipase inhibition than high mannuronate.•Alginates have the potential to be a well-tolerated obesity treatment.
Alginates are comprised of mannuronic (M) and guluronic acid (G) and have been shown to inhibit enzyme activity. Pancreatic lipase is important in dietary triacylglycerol breakdown; reducing pancreatic lipase activity would reduce triacylglycerol breakdown resulting in lower amounts being absorbed by the body.
Lipase activity in the presence of biopolymers was assessed by enzymatic assay using natural and synthetic substrates. Alginate inhibited pancreatic lipase by a maximum of 72.2% (±4.1) with synthetic substrate (DGGR) and 58.0% (±9.7) with natural substrate. High-G alginates from Laminaria hyperborea seaweed inhibited pancreatic lipase to a significantly higher degree than High-M alginates from Lessonia nigrescens, showing that inhibition was related to alginate structure.
High-G alginates are effective inhibitors of pancreatic lipase and are used in the food industry at low levels. They could be included at higher levels in foods without altering organoleptic qualities, potentially reduce the uptake of dietary triacylglycerol aiding in weight management.
doi:10.1016/j.foodchem.2013.09.075
PMCID: PMC4018670  PMID: 24176371
Alginate; Lipase; Inhibition; Obesity; Weight management
24.  Cytoplasmic Fungal Lipases Release Fungicides from Ultra-Deformable Vesicular Drug Carriers 
PLoS ONE  2012;7(5):e38181.
The Transfersome® is a lipid vesicle that contains membrane softeners, such as Tween 80, to make it ultra-deformable. This feature makes the Transfersome® an efficient carrier for delivery of therapeutic drugs across the skin barrier. It was reported that TDT 067 (a topical formulation of 15 mg/ml terbinafine in Transfersome® vesicles) has a much more potent antifungal activity in vitro compared with conventional terbinafine, which is a water-insoluble fungicide. Here we use ultra-structural studies and live imaging in a model fungus to describe the underlying mode of action. We show that terbinafine causes local collapse of the fungal endoplasmic reticulum, which was more efficient when terbinafine was delivered in Transfersome® vesicles (TFVs). When applied in liquid culture, fluorescently labeled TFVs rapidly entered the fungal cells (T1/2∼2 min). Entry was F-actin- and ATP-independent, indicating that it is a passive process. Ultra-structural studies showed that passage through the cell wall involves significant deformation of the vesicles, and depends on a high concentration of the surfactant Tween 80 in their membrane. Surprisingly, the TFVs collapsed into lipid droplets after entry into the cell and the terbinafine was released from their interior. With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs. Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome® degradation and neutralized the cytotoxic effect of Transfersome®-delivered terbinafine. These data suggest that (a) Transfersomes deliver the lipophilic fungicide Terbinafine to the fungal cell wall, (b) the membrane softener Tween 80 allows the passage of the Transfersomes into the fungal cell, and (c) fungal lipases digest the invading Transfersome® vesicles thereby releasing their cytotoxic content. As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.
doi:10.1371/journal.pone.0038181
PMCID: PMC3362563  PMID: 22666476
25.  Efficacy of Liposomal Bismuth-Ethanedithiol-Loaded Tobramycin after Intratracheal Administration in Rats with Pulmonary Pseudomonas aeruginosa Infection 
We sought to investigate alterations in quorum-sensing signal molecule N-acyl homoserine lactone secretion and in the release of Pseudomonas aeruginosa virulence factors, as well as the in vivo antimicrobial activity of bismuth-ethanedithiol incorporated into a liposome-loaded tobramycin formulation (LipoBiEDT-TOB) administered to rats chronically infected with P. aeruginosa. The quorum-sensing signal molecule N-acyl homoserine lactone was monitored by using a biosensor organism. P. aeruginosa virulence factors were assessed spectrophotometrically. An agar beads model of chronic Pseudomonas lung infection in rats was used to evaluate the efficacy of the liposomal formulation in the reduction of bacterial count. The levels of active tobramycin in the lungs and the kidneys were evaluated by microbiological assay. LipoBiEDT-TOB was effective in disrupting both quorum-sensing signal molecules N-3-oxo-dodeccanoylhomoserine lactone and N-butanoylhomoserine lactone, as well as significantly (P < 0.05) reducing lipase, chitinase, and protease production. At 24 h after 3 treatments, the CFU counts in lungs of animals treated with LipoBiEDT-TOB were of 3 log10 CFU/lung, comparated to 7.4 and 4.7 log10 CFU/lung, respectively, in untreated lungs and in lungs treated with free antibiotic. The antibiotic concentration after the last dose of LipoBiEDT-TOB was 25.1 μg/lung, while no tobramycin was detected in the kidneys. As for the free antibiotic, we found 6.5 μg/kidney but could not detect any tobramycin in the lungs. Taken together, LipoBiEDT-TOB reduced the production of quorum-sensing molecules and virulence factors and could highly improve the management of chronic pulmonary infection in cystic fibrosis patients.
doi:10.1128/AAC.01634-12
PMCID: PMC3535983  PMID: 23147741

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