506-32-1Relevant articles and documents
Liposomal Delivery of Diacylglycerol Lipase-Beta Inhibitors to Macrophages Dramatically Enhances Selectivity and Efficacy in Vivo
Shin, Myungsun,Snyder, Helena W.,Donvito, Giulia,Schurman, Lesley D.,Fox, Todd E.,Lichtman, Aron H.,Kester, Mark,Hsu, Ku-Lung
, p. 721 - 728 (2018)
Diacylglycerol lipase-beta (DAGLβ) hydrolyzes arachidonic acid (AA)-containing diacylglycerols to produce bioactive lipids including endocannabinoids and AA-derived eicosanoids involved in regulation of inflammatory signaling. Previously, we demonstrated that DAGLβ inactivation using the triazole urea inhibitor KT109 blocked macrophage inflammatory signaling and reversed allodynic responses of mice in inflammatory and neuropathic pain models. Here, we tested whether we could exploit the phagocytic capacity of macrophages to localize delivery of DAGLβ inhibitors to these cells in vivo using liposome encapsulated KT109. We used DAGLβ-tailored activity-based probes and chemical proteomic methods to measure potency and selectivity of liposomal KT109 in macrophages and tissues from treated mice. Surprisingly, delivery of ～5 μg of liposomal KT109 was sufficient to achieve ～80% inactivation of DAGLβ in macrophages with no apparent activity in other tissues in vivo. Our macrophage-targeted delivery resulted in a >100-fold enhancement in antinociceptive potency compared with free compound in a mouse inflammatory pain model. Our studies describe a novel anti-inflammatory strategy that is achieved by targeted in vivo delivery of DAGLβ inhibitors to macrophages.
Stable isotope liquid chromatography-tandem mass spectrometry assay for fatty acid amide hydrolase activity
Rakers, Christin,Zoerner, Alexander A.,Engeli, Stefan,Batkai, Sandor,Jordan, Jens,Tsikas, Dimitrios
, p. 699 - 705 (2012)
Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the hydrolysis of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) to arachidonic acid (AA) and ethanolamine (EA). Published FAAH activity assays mostly employ radiolabeled anandamide or synthetic fluorogenic substrates. We report a stable isotope liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for specific, sensitive, and high-throughput capable FAAH activity measurements. The assay uses AEA labeled with deuterium on the EA moiety (d4-AEA) as substrate and measures the specific reaction product tetradeutero-EA (d4-EA) and the internal standard 13C2-EA. Selected reaction monitoring of m/z 66 → m/z 48 (d4-EA) and m/z 64 → m/z 46 (13C2-EA) in the positive electrospray ionization mode after liquid chromatographic separation on a HILIC (hydrophilic interaction liquid chromatography) column is performed. The assay was developed and thoroughly validated using recombinant human FAAH (rhFAAH) and then was applied to human blood and dog liver samples. rhFAAH-catalyzed d4-AEA hydrolysis obeyed Michaelis-Menten kinetics (KM = 12.3 μM, Vmax = 27.6 nmol/min mg). Oleoyl oxazolopyridine (oloxa) was a potent, partial noncompetitive inhibitor of rhFAAH (IC50 = 24.3 nM). Substrate specificity of other fatty acid ethanolamides decreased with decreasing length, number of double bonds, and lipophilicity of the fatty acid skeleton. In human whole blood, we detected FAAH activity that was inhibited by oloxa.
A SIMPLE PROCESS FOR THE PURIFICATION OF ARACHIDONIC ACID
Corey, E. J.,Wright, Stephen W.
, p. 2729 - 2730 (1984)
Pure arachidonic acid (1) and docosahexanoic acid (3) are available by a two-step purification method from commercial 50percent acids with high efficiency.
Pig Liver Esterases Hydrolyze Endocannabinoids and Promote Inflammatory Response
Zhou, Qiongqiong,Yan, Bingfang,Sun, Wanying,Chen, Qi,Xiao, Qiling,Xiao, Yuncai,Wang, Xiliang,Shi, Deshi
, (2021/06/07)
Endocannabinoids are endogenous ligands of cannabinoid receptors and activation of these receptors has strong physiological and pathological significance. Structurally, endocannabinoids are esters (e.g., 2-arachidonoylglycerol, 2-AG) or amides (e.g., N-arachidonoylethanolamine, AEA). Hydrolysis of these compounds yields arachidonic acid (AA), a major precursor of proinflammatory mediators such as prostaglandin E2. Carboxylesterases are known to hydrolyze esters and amides with high efficiency. CES1, a human carboxylesterase, has been shown to hydrolyze 2-AG, and shares a high sequence identity with pig carboxylesterases: PLE1 and PLE6 (pig liver esterase). The present study was designed to test the hypothesis that PLE1 and PLE6 hydrolyze endocannabinoids and promote inflammatory response. Consistent with the hypothesis, purified PLE1 and PLE6 efficaciously hydrolyzed 2-AG and AEA. PLE6 was 40-fold and 3-fold as active as PLE1 towards 2-AG and AEA, respectively. In addition, both PLE1 and PLE6 were highly sensitive to bis(4-nitrophenyl) phosphate (BNPP), an aryl phosphodiester known to predominately inhibit carboxylesterases. Based on the study with BNPP, PLEs contributed to the hydrolysis of 2-AG by 53.4 to 88.4% among various organs and cells. Critically, exogenous addition or transfection of PLE6 increased the expression and secretion of proinflammatory cytokines in response to the immunostimulant lipopolysaccharide (LPS). This increase was recapitulated in cocultured alveolar macrophages and PLE6 transfected cells in transwells. Finally, BNPP reduced inflammation trigged by LPS accompanied by reduced formation of AA and proinflammatory mediators. These findings define an innovative connection: PLE-endocannabinoid-inflammation. This mechanistic connection signifies critical roles of carboxylesterases in pathophysiological processes related to the metabolism of endocannabinoids.
Effect of Site-Specific Peptide-Tag Labeling on the Biocatalytic Properties of Thermoalkalophilic Lipase from Geobacillus thermocatenulatus
Romero, Oscar,de las Rivas, Blanca,Lopez-Tejedor, David,Palomo, Jose M.
, p. 369 - 378 (2018/01/11)
Tailor-made peptides were investigated for site-specific tag labeling of Geobacillus thermocatenulatus lipase (GTL). GTL was first genetically modified by introducing a unique cysteine on the lid site of the enzyme to produce two variants (GTLσ-A193C and GTLσ-S196C). Chemical modification was performed by using a small library of cysteine-containing peptides. The synthesized peptide–lipase biocatalysts were highly stable, more active, more specific, and more selective toward different substrates than unmodified GTL. Very high enzyme thermostability of GTLσ-A193C modified with peptides Ac-Cys-Phe-Gly-Phe-Gly-Phe-CONH2 (1) and Ac-Cys-Phe-Phe-CONH2 (2) (>95 % activity after 24 h at 60 °C) was observed. The incorporation of 1 and 2 in GTLσ-S196C improved its catalytic activity in the hydrolysis of p-nitrophenyl butyrate by factors of three and greater than five, respectively. The specificity for short-chain versus long-chain esters was also strongly improved. The diacylglycerol activity of GTLσ-S196C was enhanced more than tenfold by the incorporation of 1 and more than threefold by modification of this variant with Ac-Cys-(Arg)7-CONH2 (6) in the hydrolysis of 1-stearoyl-2-arachidonoyl-sn-glycerol. The enantioselectivity of GTLσ-S196C increased for all formed bioconjugates, and the GTLσ-S196C–1 conjugate was the most active and selective in the hydrolysis of dimethylphenyl glutarate at pH 7 (72 % ee), also showing an inversion in the enzyme enantiopreference.
Synthesis of 14,15-EET from Arachidonic Acid Using Urea-Hydrogen Peroxide as the Oxidant
Xie, Fuchun,Li, Bingbing X.,Alkayed, Nabil J.,Xiao, Xiangshu
, p. 105 - 110 (2015/10/20)
14,15- Epoxyeicosatrienoic acid (14,15-EET) is an endogenous bioactive lipid with pharmacological benefits in multiple cardiovascular diseases. We describe here a practical synthesis of 14,15-EET from arachidonic acid using urea-hydrogen peroxide (UHP) as the oxidant.
Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons
Baggelaar, Marc P.,Chameau, Pascal J. P.,Kantae, Vasudev,Hummel, Jessica,Hsu, Ku-Lung,Janssen, Freek,Van Der Wel, Tom,Soethoudt, Marjolein,Deng, Hui,Den Dulk, Hans,Allarà, Marco,Florea, Bogdan I.,Di Marzo, Vincenzo,Wadman, Wytse J.,Kruse, Chris G.,Overkleeft, Herman S.,Hankemeier, Thomas,Werkman, Taco R.,Cravatt, Benjamin F.,Van Der Stelt, Mario
supporting information, p. 8851 - 8857 (2015/07/27)
Diacylglycerol lipase (DAGL)-α and -β are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific β-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that "on demand biosynthesis" of 2-AG is responsible for retrograde signaling.
Endocannabinoids anandamide and 2-arachidonoylglycerol are substrates for human CYP2J2 epoxygenase
McDougle, Daniel R.,Kambalyal, Amogh,Meling, Daryl D.,Das, Aditi
, p. 616 - 627 (2015/01/16)
The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The formation of the predominant AEA and 2-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane-bound cardiovascular epoxygenase.
Simultaneous determination of 2-arachidonoylglycerol, 1- arachidonoylglycerol and arachidonic acid in mouse brain tissue using liquid chromatography/tandem mass spectrometry
Zhang, Mei-Yi,Gao, Ying,Btesh, Joan,Kagan, Natasha,Kerns, Edward,Samad, Tarek A.,Chanda, Pranab K.
scheme or table, p. 167 - 177 (2010/08/06)
Endocannabinoids (ECs), such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), modulate a number of physiological processes, including pain, appetite and emotional state. Levels of ECs are tightly controlled by enzymatic biosynthesis and degradation in vivo. However, there is limited knowledge about the enzymes that terminate signaling of the major brain EC, 2-AG. Identification and quantification of 2-AG, 1-AG and arachidonic acid (AA) is important for studying the enzymatic hydrolysis of 2-AG. We have developed a sensitive and specific quantification method for simultaneous determination of 2-AG, 1-AG and AA from mouse brain and adipose tissues by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a simple brain sample preparationmethod. The separations were carried out based on reversed phase chromatography. Optimization of electrospray ionization conditions established the limits of detection (S/N=3) at 50, 25 and 65 fmol for 2-AG, 1-AG and AA, respectively. The methodswere selective, precise (%R.S.D.a range of 0.02-20, 0.01-10 and 0.05-50 ng/mg tissue for 2-AG, 1-AG and AA, respectively. The quantificationmethod was validated with consideration of thematrix effects and the mass spectrometry (MS) responses of the analytes and the deuterium labeled internal standard (IS). The developed methods were applied to study the hydrolysis of 2-AG from mouse brain extracts containingmembrane bound monoacylglycerol lipase (MAGL), and to measure the basal levels of 2-AG, 1-AG and AA in mouse brain and adipose tissues. Copyright
Radiosynthesis, in vitro and in vivo evaluation of 123I-labeled anandamide analogues for mapping brain FAAH
wyffels, Leonie,De Bruyne, Sylvie,Blanckaert, Peter,Lambert, Didier M.,De Vos, Filip
experimental part, p. 49 - 56 (2011/02/25)
Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids, including anandamide. This paper is the first report of the synthesis, [123I]-labeling and in vitro and in vivo evaluation of anandamide analogues as potential metabolic trapping radioligands for in vivo evaluation of brain FAAH. N-(2-Iodoethyl)linoleoylamide (2) and N-(2-iodoethyl)arachidonylamide (4) were synthesized with good yields (75% and 86%, respectively) in a two steps procedure starting from their respective acids. In vitro analyses, performed using recombinant rat FAAH and [3H]-anandamide, demonstrated interaction of 2 and 4 with FAAH (IC50 values of 5.78 μM and 3.14 μM, respectively). [123I]-2 and [123I]-4 were synthesized with radiochemical yields of 21% and 12%, respectively, and radiochemical purities were >90%. Biodistribution studies in mice demonstrated brain uptake for both tracers (maximum values of 1.23%ID/g at 3 min pi for [123I]-2 and 0.58%ID/g at 10 min pi for [123I]-4). However, stability studies demonstrated the sensitivity of both tracers to dehalogenation.
