True natural ligand for cannabinoid type-1 (CB1) receptors and the most abundant endogenous cannabinoid. 2-AG binds to the cannabinoid receptors (CB1 and CB2) and exhibits a variety of cannabimimetic activities in vitro and in vivo originally identified as the targets of Delta9-tetrahydocannabinol (-)-Delta9-tetrahydrocannabinol (THC), the psychoactive component of cannabis. Fatty acid amide hydrolase (FAAH) is the main AEA hydrolase, whereas a monoacylglycerol lipase (MAGL) is critical in degrading 2-AG. Monoacylglycerol lipase ABHD2 is a steroid-activating enzyme that hydrolyzes 2-arachidonoylglycerol. ABHD2 is expressed in sperm and in ovaries
11 moreTitle: 1-, 2- and 3-AG as substrates of the endocannabinoid enzymes and endogenous ligands of the cannabinoid receptor 1 Farah SI, Hilston S, Tran N, Zvonok N, Makriyannis A Ref: Biochemical & Biophysical Research Communications, 591:31, 2021 : PubMed
2-Arachidonoylglycerol (2-AG) is the most potent and abundant endocannabinoid that acts as a full agonist at the cannabinoid 1 (CB1) and 2 (CB2) receptors. It serves as a substrate for several serine hydrolases, including monoacylglycerol lipase (MGL), alpha/beta hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH). However, 2-AG's rapid conversion to 1-AG (the S stereoisomer) and 3-AG (the R stereoisomer) complicates in vivo signaling. Here, we present the interaction profiles of 2-AG and its isomerization products, 1- and 3-AG, with the endocannabinoid MGL, ABHD6 and FAAH enzymes as well as the CB1 receptor. The 1- and 3-AG enantiomers are less prone to isomerization, and their affinities to endocannabinoid enzymes and potencies at CB1 receptor are quite different compared to 2-AG. Although MGL is the principal hydrolytic enzyme of 2-AG, 3-AG (the R isomer) appears to be the best substrate for hMGL. Contrarily, 1-AG (the S isomer) demonstrates the worst substrate profile, indicating that the stereochemistry of 1(3)-monoacylglycerols is very important for MGL enzyme. On the other hand, both 1- and 3-AG (the sn1 monoacylglycerols) are efficiently hydrolyzed by hABHD6 without preference, while 2-AG (the sn2 monoacylglycerol) has the lowest rate of hydrolysis. FAAH, the principal hydrolytic enzyme for arachidonoylethanolamide (anandamide, AEA), catalyzes the hydrolysis of all three isomers with similar efficiencies. In a functional cAMP assay at CB1 receptor, all three isomers behaved as agonists, with 2-AG being the most potent, followed by 3-AG then 1-AG. The presented data provides stereochemical insights to design chemically stable AG analogs with preferential stability against enzymes of interest.
2-Arachidonoyl-glycerol (2-AG) is an endocannabinoid with anti-inflammatory properties. Blocking 2-AG hydrolysis to enhance CB2 signaling has proven effective in mouse models of inflammation. However, the expression of 2-AG lipases has never been thoroughly investigated in human leukocytes. Herein, we investigated the expression of seven 2-AG hydrolases by human blood leukocytes and alveolar macrophages (AMs) and found the following protein expression pattern: monoacylglycerol (MAG lipase; eosinophils, AMs, monocytes), carboxylesterase (CES1; monocytes, AMs), palmitoyl-protein thioesterase (PPT1; AMs), alpha/beta-hydrolase domain (ABHD6; mainly AMs), ABHD12 (all), ABHD16A (all), and LYPLA2 (lysophospholipase 2; monocytes, lymphocytes, AMs). We next found that all leukocytes could hydrolyze 2-AG and its metabolites derived from cyclooxygenase-2 (prostaglandin E2 -glycerol [PGE2 -G]) and the 15-lipoxygenase (15-hydroxy-eicosatetraenoyl-glycerol [15-HETE-G]). Neutrophils and eosinophils were consistently better at hydrolyzing 2-AG and its metabolites than monocytes and lymphocytes. Moreover, the efficacy of leukocytes to hydrolyze 2-AG and its metabolites was 2-AG >/= 15-HETE-G >> PGE2 -G for each leukocyte. Using the inhibitors methylarachidonoyl-fluorophosphonate (MAFP), 4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxyla te (JZL184), Palmostatin B, 4'-carbamoylbiphenyl-4-yl methyl(3-(pyridin-4-yl)benzyl)carbamate, N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4'-(aminocarbonyl)[1,1'-biphenyl]-4-yl ester carbamic acid (WWL70), 4'-[[[methyl[[3-(4-pyridinyl)phenyl]methyl]amino]carbonyl]oxy]-[1,1'-biphenyl]-4- carboxylic acid, ethyl ester (WWL113), tetrahydrolipstatin, and ML349, we could not pinpoint a specific hydrolase responsible for the hydrolysis of 2-AG, PGE2 -G, and 15-HETE-G by these leukocytes. Furthermore, JZL184, a selective MAG lipase inhibitor, blocked the hydrolysis of 2-AG, PGE2 -G, and 15-HETE-G by neutrophils and the hydrolysis of PGE2 -G and 15-HETE-G by lymphocytes, two cell types with limited/no MAG lipase. Using an activity-based protein profiling (ABPP) probe to label hydrolases in leukocytes, we found that they express many MAFP-sensitive hydrolases and an unknown JZL184-sensitive hydrolase of approximately 52 kDa. Altogether, our results indicate that human leukocytes are experts at hydrolyzing 2-AG and its metabolites via multiple lipases and probably via a yet-to-be characterized 52 kDa hydrolase. Blocking 2-AG hydrolysis in humans will likely abrogate the ability of human leukocytes to degrade 2-AG and its metabolites and increase their anti-inflammatory effects in vivo.
Exposure to chlorpyrifos (CPF) during the late preweanling period in rats inhibits the endocannabinoid metabolizing enzymes fatty acid hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resulting in accumulation of their respective substrates anandamide (AEA) and 2-arachidonylglycerol (2-AG). This occurs at 1.0mg/kg, but at a lower dosage (0.5mg/kg) only FAAH and AEA are affected with no measurable inhibition of either cholinesterase (ChE) or MAGL. The endocannabinoid system plays a vital role in nervous system development and may be an important developmental target for CPF. The endocannabinoid system plays an important role in the regulation of anxiety and, at higher dosages, developmental exposure to CPF alters anxiety-like behavior. However, it is not clear whether exposure to low dosages of CPF that do not inhibit ChE will cause any persistent effects on anxiety-like behavior. To determine if this occurs, 10-day old rat pups were exposed daily for 7 days to either corn oil or 0.5, 0.75, or 1.0mg/kg CPF by oral gavage. At 12h following the last CPF administration, 1.0mg/kg resulted in significant inhibition of FAAH, MAGL, and ChE, whereas 0.5 and 0.75mg/kg resulted in significant inhibition of only FAAH. AEA levels were significantly elevated in all three treatment groups as were palmitoylethanolamide and oleoylethanolamide, which are also substrates for FAAH. 2-AG levels were significantly elevated by 0.75 and 1.0mg/kg but not 0.5mg/kg. On day 25, the latency to emerge from a dark container into a highly illuminated novel open field was measured as an indicator of anxiety. All three CPF treatment groups spent significantly less time in the dark container prior to emerging as compared to the control group, suggesting a decreased level of anxiety. This demonstrates that repeated preweanling exposure to dosages of CPF that do not inhibit brain ChE can induce a decline in the level of anxiety that is detectable during the early postweanling period.
Carboxylesterase 2 (CES2/Ces2) proteins exert established roles in (pro)drug metabolism. Recently, human and murine CES2/Ces2c have been discovered as triglyceride (TG) hydrolases implicated in the development of obesity and fatty liver disease. The murine Ces2 family consists of seven homologous genes as opposed to a single CES2 gene in humans. However, the mechanistic role of Ces2 protein family members is not completely understood. In this study, we examined activities of all Ces2 members towards TGs, diglycerides (DGs) and monoglycerides (MGs) as substrate. Besides CES2/Ces2c, we measured significant TG hydrolytic activities for Ces2a, Ces2b, and Ces2e. Notably, these Ces2 members and CES2 efficiently hydrolyzed DGs and MGs and their activities even surpassed those measured for TG hydrolysis. The localization of CES2/Ces2c proteins at the ER may implicate a role of these lipases in lipid signaling pathways. We found divergent expression of Ces2 genes in the liver and intestine of mice on high fat diet, which could relate to changes in lipid signaling. Finally, we demonstrate reduced CES2 expression in the colon of patients with inflammatory bowel disease and a similar decline in Ces2 expression in the colon of a murine colitis model. Together, these results demonstrate that CES2/Ces2 members are highly efficient DG and MG hydrolases that may play an important role in liver and gut lipid signaling.
        
Title: 1-, 2- and 3-AG as substrates of the endocannabinoid enzymes and endogenous ligands of the cannabinoid receptor 1 Farah SI, Hilston S, Tran N, Zvonok N, Makriyannis A Ref: Biochemical & Biophysical Research Communications, 591:31, 2021 : PubMed
2-Arachidonoylglycerol (2-AG) is the most potent and abundant endocannabinoid that acts as a full agonist at the cannabinoid 1 (CB1) and 2 (CB2) receptors. It serves as a substrate for several serine hydrolases, including monoacylglycerol lipase (MGL), alpha/beta hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH). However, 2-AG's rapid conversion to 1-AG (the S stereoisomer) and 3-AG (the R stereoisomer) complicates in vivo signaling. Here, we present the interaction profiles of 2-AG and its isomerization products, 1- and 3-AG, with the endocannabinoid MGL, ABHD6 and FAAH enzymes as well as the CB1 receptor. The 1- and 3-AG enantiomers are less prone to isomerization, and their affinities to endocannabinoid enzymes and potencies at CB1 receptor are quite different compared to 2-AG. Although MGL is the principal hydrolytic enzyme of 2-AG, 3-AG (the R isomer) appears to be the best substrate for hMGL. Contrarily, 1-AG (the S isomer) demonstrates the worst substrate profile, indicating that the stereochemistry of 1(3)-monoacylglycerols is very important for MGL enzyme. On the other hand, both 1- and 3-AG (the sn1 monoacylglycerols) are efficiently hydrolyzed by hABHD6 without preference, while 2-AG (the sn2 monoacylglycerol) has the lowest rate of hydrolysis. FAAH, the principal hydrolytic enzyme for arachidonoylethanolamide (anandamide, AEA), catalyzes the hydrolysis of all three isomers with similar efficiencies. In a functional cAMP assay at CB1 receptor, all three isomers behaved as agonists, with 2-AG being the most potent, followed by 3-AG then 1-AG. The presented data provides stereochemical insights to design chemically stable AG analogs with preferential stability against enzymes of interest.
BACKGROUND: Endothelial dysfunction promotes atherogenesis, vascular inflammation, and thrombus formation. Reendothelialization after angioplasty is required in order to prevent stent failure. Previous studies have highlighted the role of 2-arachidonoylglycerol (2-AG) in murine experimental atherogenesis and in human coronary artery disease. However, the impact of 2-AG on endothelial repair and leukocyte-endothelial cell adhesion is still unknown. METHODS: Endothelial repair was studied in two treatment groups of wildtype mice following electrical injury of the common carotid artery. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184, which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received DMSO (vehicle). The effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability, leukocyte-endothelial cell adhesion, surface expression of adhesion molecules, and expression of endothelial NO synthase (NOS3) was studied in vitro. RESULTS: Elevated 2-AG levels significantly impaired reendothelialization in wildtype mice following electrical injury of the common carotid artery. In vitro, 2-AG significantly reduced viability of HCAEC. Additionally, 2-AG promoted adhesion of THP-1 monocytes to HCAEC following pre-treatment of the HCAEC with 2-AG. Adhesion molecules (E-selectin, ICAM-1 and VCAM-1) remained unchanged in arterial endothelial cells, whereas 2-AG suppressed the expression of NOS3 in HCAEC. CONCLUSION AND TRANSLATIONAL ASPECT: Elevated 2-AG levels hamper endothelial repair and HCAEC proliferation, while simultaneously facilitating leukocyte-endothelial cell adhesion. Given that 2-AG is elevated in patients with coronary artery disease and non-ST-segment elevation myocardial infarction, 2-AG might decrease reendothelialization after angioplasty and thus impact the clinical outcomes.
The endocannabinoid system has been shown to be a putative therapeutic target for retinal disease. Here, we aimed to investigate the ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) and novel inhibitors of its metabolic enzymes, alpha/beta-hydrolase domain-containing 6 (ABHD6) and monoacylglycerol lipase (MAGL), a) to protect the retina against excitotoxicity and b) the mechanisms involved in the neuroprotection. Sprague-Dawley rats, wild type and Akt2(-/-) C57BL/6 mice were intravitreally administered with phosphate-buffered saline or (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA). 2-AG was intravitreally co-administered with AMPA in the absence and presence of AM251 or AM630 (cannabinoid 1 and 2 receptor antagonists, respectively) or Wortmannin [Phosphoinositide 3-Kinase (PI3K)/Akt inhibitor]. Inhibitors of ABHD6 and dual ABHD6/MAGL (AM12100 and AM11920, respectively) were co-administered with AMPA intravitreally in rats. Immunohistochemistry was performed using antibodies raised against retinal neuronal markers (bNOS), microglia (Iba1) and macroglia (GFAP). TUNEL assay and real-time PCR were also employed. The CB2 receptor was expressed in rat retina (approx. 62% of CB1 expression). 2-AG attenuated the AMPA-induced increase in TUNEL(+) cells. 2-AG activation of both CB1 and CB2 receptors and the PI3K/Akt downstream signaling pathway, as substantiated by the use of Akt2(-/-) mice, afforded neuroprotection against AMPA excitotoxicity. AM12100 and AM11920 attenuated the AMPA-induced glia activation and produced a dose-dependent partial neuroprotection, with the dual inhibitor AM11920 being more efficacious. These results show that 2-AG has the pharmacological profile of a putative therapeutic for retinal diseases characterized by neurodegeneration and neuroinflammation, when administered exogenously or by the inhibition of its metabolic enzymes.
        
Title: Pseudomonas aeruginosa esterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization Bleffert F, Granzin J, Gohlke H, Batra-Safferling R, Jaeger KE, Kovacic F Ref: Acta Crystallographica F Struct Biol Commun, 75:270, 2019 : PubMed
The human membrane-bound alpha/beta-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence of n-octyl beta-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 A. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.
2-Arachidonoyl-glycerol (2-AG) is an endocannabinoid with anti-inflammatory properties. Blocking 2-AG hydrolysis to enhance CB2 signaling has proven effective in mouse models of inflammation. However, the expression of 2-AG lipases has never been thoroughly investigated in human leukocytes. Herein, we investigated the expression of seven 2-AG hydrolases by human blood leukocytes and alveolar macrophages (AMs) and found the following protein expression pattern: monoacylglycerol (MAG lipase; eosinophils, AMs, monocytes), carboxylesterase (CES1; monocytes, AMs), palmitoyl-protein thioesterase (PPT1; AMs), alpha/beta-hydrolase domain (ABHD6; mainly AMs), ABHD12 (all), ABHD16A (all), and LYPLA2 (lysophospholipase 2; monocytes, lymphocytes, AMs). We next found that all leukocytes could hydrolyze 2-AG and its metabolites derived from cyclooxygenase-2 (prostaglandin E2 -glycerol [PGE2 -G]) and the 15-lipoxygenase (15-hydroxy-eicosatetraenoyl-glycerol [15-HETE-G]). Neutrophils and eosinophils were consistently better at hydrolyzing 2-AG and its metabolites than monocytes and lymphocytes. Moreover, the efficacy of leukocytes to hydrolyze 2-AG and its metabolites was 2-AG >/= 15-HETE-G >> PGE2 -G for each leukocyte. Using the inhibitors methylarachidonoyl-fluorophosphonate (MAFP), 4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxyla te (JZL184), Palmostatin B, 4'-carbamoylbiphenyl-4-yl methyl(3-(pyridin-4-yl)benzyl)carbamate, N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4'-(aminocarbonyl)[1,1'-biphenyl]-4-yl ester carbamic acid (WWL70), 4'-[[[methyl[[3-(4-pyridinyl)phenyl]methyl]amino]carbonyl]oxy]-[1,1'-biphenyl]-4- carboxylic acid, ethyl ester (WWL113), tetrahydrolipstatin, and ML349, we could not pinpoint a specific hydrolase responsible for the hydrolysis of 2-AG, PGE2 -G, and 15-HETE-G by these leukocytes. Furthermore, JZL184, a selective MAG lipase inhibitor, blocked the hydrolysis of 2-AG, PGE2 -G, and 15-HETE-G by neutrophils and the hydrolysis of PGE2 -G and 15-HETE-G by lymphocytes, two cell types with limited/no MAG lipase. Using an activity-based protein profiling (ABPP) probe to label hydrolases in leukocytes, we found that they express many MAFP-sensitive hydrolases and an unknown JZL184-sensitive hydrolase of approximately 52 kDa. Altogether, our results indicate that human leukocytes are experts at hydrolyzing 2-AG and its metabolites via multiple lipases and probably via a yet-to-be characterized 52 kDa hydrolase. Blocking 2-AG hydrolysis in humans will likely abrogate the ability of human leukocytes to degrade 2-AG and its metabolites and increase their anti-inflammatory effects in vivo.
Exposure to chlorpyrifos (CPF) during the late preweanling period in rats inhibits the endocannabinoid metabolizing enzymes fatty acid hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resulting in accumulation of their respective substrates anandamide (AEA) and 2-arachidonylglycerol (2-AG). This occurs at 1.0mg/kg, but at a lower dosage (0.5mg/kg) only FAAH and AEA are affected with no measurable inhibition of either cholinesterase (ChE) or MAGL. The endocannabinoid system plays a vital role in nervous system development and may be an important developmental target for CPF. The endocannabinoid system plays an important role in the regulation of anxiety and, at higher dosages, developmental exposure to CPF alters anxiety-like behavior. However, it is not clear whether exposure to low dosages of CPF that do not inhibit ChE will cause any persistent effects on anxiety-like behavior. To determine if this occurs, 10-day old rat pups were exposed daily for 7 days to either corn oil or 0.5, 0.75, or 1.0mg/kg CPF by oral gavage. At 12h following the last CPF administration, 1.0mg/kg resulted in significant inhibition of FAAH, MAGL, and ChE, whereas 0.5 and 0.75mg/kg resulted in significant inhibition of only FAAH. AEA levels were significantly elevated in all three treatment groups as were palmitoylethanolamide and oleoylethanolamide, which are also substrates for FAAH. 2-AG levels were significantly elevated by 0.75 and 1.0mg/kg but not 0.5mg/kg. On day 25, the latency to emerge from a dark container into a highly illuminated novel open field was measured as an indicator of anxiety. All three CPF treatment groups spent significantly less time in the dark container prior to emerging as compared to the control group, suggesting a decreased level of anxiety. This demonstrates that repeated preweanling exposure to dosages of CPF that do not inhibit brain ChE can induce a decline in the level of anxiety that is detectable during the early postweanling period.
Steroids regulate cell proliferation, tissue development, and cell signaling via two pathways: a nuclear receptor mechanism and genome-independent signaling. Sperm activation, egg maturation, and steroid-induced anesthesia are executed via the latter pathway, the key components of which remain unknown. Here, we present characterization of the human sperm progesterone receptor that is conveyed by the orphan enzyme alpha/beta hydrolase domain-containing protein 2 (ABHD2). We show that ABHD2 is highly expressed in spermatozoa, binds progesterone, and acts as a progesterone-dependent lipid hydrolase by depleting the endocannabinoid 2-arachidonoylglycerol (2AG) from plasma membrane. The 2AG inhibits the sperm calcium channel (CatSper), and its removal leads to calcium influx via CatSper and ensures sperm activation. This study reveals that progesterone-activated endocannabinoid depletion by ABHD2 is a general mechanism by which progesterone exerts its genome-independent action and primes sperm for fertilization.
        
Title: Induction of endocannabinoid levels in juvenile rat brain following developmental chlorpyrifos exposure Carr RL, Adams AL, Kepler DR, Ward AB, Ross MK Ref: Toxicol Sci, 135:193, 2013 : PubMed
The endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) play vital roles during nervous system development. The degradation of 2-AG and AEA is mediated by monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively. These enzymes are inhibited following developmental chlorpyrifos (CPF) exposure. To investigate whether this inhibition is persistent or whether accumulation of endocannabinoids in the brain occurs, 10-day-old rat pups were orally exposed daily for 7 days to either corn oil or increasing dosages of CPF (1, 2.5, or 5mg/kg), and forebrains were collected at 4, 12, 24, and 48h following the last administration. All dosages inhibited cholinesterase (ChE), FAAH, and MAGL, and elevated AEA and 2-AG levels with the greatest effect occurring at 12h with ChE, FAAH, AEA, and 2-AG and at 4h with MAGL. With the high dosage, return to control levels occurred with 2-AG (48h) only. With the medium dosage, return to control levels occurred with MAGL, 2-AG, and AEA (48h) but not with ChE or FAAH. With the low dosage, return to control levels occurred with MAGL (12h), ChE and 2-AG (24h), and AEA (48h) but not with FAAH. With the lowest dosage, peak inhibition of FAAH (52%) is greater than that of ChE (24%) and that level of FAAH inhibition is sufficient to induce a persistent pattern of elevated AEA. It is possible that this pattern of elevation could alter the appropriate development of neuronal brain circuits.
        
Title: Inactivation of lipid glyceryl ester metabolism in human THP1 monocytes/macrophages by activated organophosphorus insecticides: role of carboxylesterases 1 and 2 Xie S, Borazjani A, Hatfield MJ, Edwards CC, Potter PM, Ross MK Ref: Chemical Research in Toxicology, 23:1890, 2010 : PubMed
Carboxylesterases (CES) have important roles in pesticide and drug metabolism and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad substrate specificity to hydrolyze endobiotics, such as cholesteryl esters and triacylglycerols. Here, we determined if two human CES isoforms, CES1 and CES2, hydrolyze the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide (AEA), and two prostaglandin glyceryl esters (PG-Gs), which are formed by COX-mediated oxygenation of 2AG. We show that recombinant CES1 and CES2 efficiently hydrolyze 2AG to arachidonic acid (AA) but not amide-containing AEA. Steady-state kinetic parameters for CES1- and CES2-mediated 2AG hydrolysis were, respectively, kcat, 59 and 43 min(-1); Km, 49 and 46 muM; and kcat/Km, 1.2 and 0.93 muM(-1) min(-1). kcat/Km values are comparable to published values for rat monoacylglycerol lipase (MAGL)-catalyzed 2AG hydrolysis. Furthermore, we show that CES1 and CES2 also efficiently hydrolyze PGE2-G and PGF2alpha-G. In addition, when cultured human THP1 macrophages were treated with exogenous 2AG or PG-G (10 muM, 1 h), significant quantities of AA or PGs were detected in the culture medium; however, the ability of macrophages to metabolize these compounds was inhibited (60-80%) following treatment with paraoxon, the toxic metabolite of the insecticide parathion. Incubation of THP1 cell lysates with small-molecule inhibitors targeting CES1 (thieno[3,2-e][1]benzothiophene-4,5-dione or JZL184) significantly reduced lipid glyceryl ester hydrolase activities (40-50% for 2AG and 80-95% for PG-Gs). Immunodepletion of CES1 also markedly reduced 2AG and PG-G hydrolase activities. These results suggested that CES1 is in part responsible for the hydrolysis of 2AG and PG-Gs in THP1 cells, although it did not rule out a role for other hydrolases, especially with regard to 2AG metabolism since a substantial portion of its hydrolysis was not inactivated by the inhibitors. An enzyme (Mr 31-32 kDa) of unknown function was detected by serine hydrolase activity profiling of THP1 cells and may be a candidate. Finally, the amounts of in situ generated 2AG and PG-Gs in macrophages were enhanced by treating the cells with bioactive metabolites of OP insecticides. Collectively, the results suggest that in addition to MAGL and fatty-acid amide hydrolase (FAAH), which have both been documented to terminate endocannabinoid signaling, CES may also have a role. Furthermore, since PG-Gs have been shown to possess biological activities in their own right, CES may represent an important enzyme class that regulates their in vivo levels.
        
Title: Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling Hoover HS, Blankman JL, Niessen S, Cravatt BF Ref: Bioorganic & Medicinal Chemistry Lett, 18:5838, 2008 : PubMed
The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacological studies using inhibitors of the 2-AG biosynthetic enzymes diacyglycerol lipase alpha and beta (DAGL-alpha/beta). Here, we show by competitive activity-based protein profiling that the DAGL-alpha/beta inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was observed for THL and RHC80267, suggesting that pharmacological effects observed with both agents may be viewed as good initial evidence for DAGL-dependent events.
        
Title: A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol Blankman JL, Simon GM, Cravatt BF Ref: Chemical Biology, 14:1347, 2007 : PubMed
Endogenous ligands for cannabinoid receptors ("endocannabinoids") include the lipid transmitters anandamide and 2-arachidonoylglycerol (2-AG). Endocannabinoids modulate a diverse set of physiological processes and are tightly regulated by enzymatic biosynthesis and degradation. Termination of anandamide signaling by fatty acid amide hydrolase (FAAH) is well characterized, but less is known about the inactivation of 2-AG, which can be hydrolyzed by multiple enzymes in vitro, including FAAH and monoacylglycerol lipase (MAGL). Here, we have taken a functional proteomic approach to comprehensively map 2-AG hydrolases in the mouse brain. Our data reveal that approximately 85% of brain 2-AG hydrolase activity can be ascribed to MAGL, and that the remaining 15% is mostly catalyzed by two uncharacterized enzymes, ABHD6 and ABHD12. Interestingly, MAGL, ABHD6, and ABHD12 display distinct subcellular distributions, suggesting that they may control different pools of 2-AG in the nervous system.
        
Title: Endothelium-dependent metabolism by endocannabinoid hydrolases and cyclooxygenases limits vasorelaxation to anandamide and 2-arachidonoylglycerol Ho WS, Randall MD Ref: British Journal of Pharmacology, 150:641, 2007 : PubMed
BACKGROUND AND PURPOSE: The endocannabinoids, N-arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG) are rapidly degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Whilst these lipid mediators are known to modulate vascular tone, the extent to which they are inactivated via local metabolism in the vasculature remains unclear. EXPERIMENTAL APPROACH: In rat isolated small mesenteric arteries, the regulatory role of FAAH, MGL and cyclooxygenase (COX) in relaxant responses to anandamide and 2-AG was evaluated by using inhibitors of these enzymes. Relaxations to non-hydrolysable analogues of endocannabinoids and arachidonic acid were also examined. KEY RESULTS: Relaxation to anandamide but not 2-AG was potentiated by the selective FAAH inhibitor, URB597 (1 microM). In contrast, MAFP (10 microM; an inhibitor of FAAH and MGL) enhanced responses to both anandamide and 2-AG. Inhibition of COX-1 by indomethacin (10 microM) potentiated relaxations to 2-AG, whereas inhibition of COX-2 by nimesulide (10 microM) potentiated anandamide-induced relaxation. With the exception of MAFP, effects of FAAH and COX inhibitors were dependent on the endothelium. Relaxation to methanandamide and noladin ether, the non-hydrolysable analogues of anandamide and 2-AG respectively, were insensitive to the enzyme inhibitors. CONCLUSION AND IMPLICATIONS: This study shows that local activity of FAAH, MGL and COX, which is present largely in the endothelium, limits the vasodilator action of endocannabinoids in rat small mesenteric arteries. Despite the differential roles played by these enzymes on relaxation to anandamide versus 2-AG, our results suggest that inhibitors of these enzymes enhance the vascular impact of endocannabinoids.
The pharmacology of monoacylglycerol lipase (MAGL) is not well understood. In consequence, the abilities of a series of analogues of 2-arachidonoylglycerol (2-AG) to inhibit cytosolic 2-oleoylglycerol and membrane-bound anandamide hydolysis by MAGL and fatty acid amide hydrolase (FAAH), respectively, have been investigated. 2-AG and its 1-regioisomer (1-AG) interacted with MAGL with similar affinities (IC(50) values 13 and 17 mum, respectively). Shorter homologues of 2-AG (2-linoleoylglycerol and 2-oleoylglycerol) had affinities for MAGL similar to 2-AG. This pattern was also seen when the arachidonoyl side chain of arachidonoyl trifluoromethylketone was replaced by an oleoyl side chain. Arachidonoyl serinol (IC(50) value 73 microM) was a weaker inhibitor of MAGL than 2-AG. The IC(50) values of noladin ether towards MAGL and FAAH were 36 and 3 microM, respectively. Arachidonoyl glycine interacted with FAAH (IC(50) value 4.9 microM) but only weakly interacted with MAGL (IC(50) value >100 microM). alpha-Methyl-1-AG had similar potencies towards MAGL and FAAH (IC(50) values of 11 and 33 microM, respectively). O-2203 (1-(20-cyano-16,16-dimethyl-eicosa-5,8,11,14-tetraenoyl) glycerol) and O-2204 (2-(20-hydroxy-16,16-dimethyl-eicosa-5,8,11,14-tetraenoyl) glycerol) were slightly less potent, but again affected both enzymes equally. alpha-Methyl-1-AG, O-2203 and O-2204 interacted only weakly with cannabinoid CB(1) receptors expressed in CHO cells (K(i) values 1.8, 3.7 and 3.2 microM, respectively, compared with 0.24 microM for 1-AG) and showed no evidence of central cannabinoid receptor activation in vivo at doses up to 30 mg kg(-1) i.v. It is concluded that compounds like alpha-Methyl-1-AG, O-2203 and O-2204 may be useful as leads for the discovery of selective MAGL inhibitors that lack direct effects upon cannabinoid receptors.