Development of terphenyl-2-methyloxazol-5(4H)-one derivatives as selective reversible MAGL inhibitors

Monoacylglycerol lipase is a serine hydrolase that plays a major role in the degradation of the endocannabinoid neurotransmitter 2-arachidonoylglycerol. A wide number of MAGL inhibitors are reported in literature; however, many of them are characterised by an irreversible mechanism of action and this behavior determines an unwanted chronic MAGL inactivation, which acquires a functional antagonism of the endocannabinoid system. The possible use of reversible MAGL inhibitors has only recently been explored, due to the lack of known compounds possessing efficient reversible inhibitory activities. In this work, we report a new series of terphenyl-2-methyloxazol-5(4H)-one derivatives characterised by a reversible MAGL-inhibition mechanism. Among them, compound 20b showed to be a potent MAGL reversible inhibitor (IC₅₀?=?348?nM) with a good MAGL/FAAH selectivity. Furthermore, this compound showed antiproliferative activities against two different cancer cell lines that overexpress MAGL.     

Influence of the degree of unsaturation of the acyl side chain upon the interaction of analogues of 1-arachidonoylglycerol with monoacylglycerol lipase and fatty acid amide hydrolase

Little is known as to the structural requirements of the acyl side chain for interaction of acylglycerols with monoacylglycerol lipase (MAGL), the enzyme chiefly responsible for the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain. In the present study, a series of twelve analogues of 1-AG (the more stable regioisomer of 2-AG) were investigated with respect to their ability to inhibit the metabolism of 2-oleoylglycerol by cytosolic and membrane-bound MAGL. In addition, the ability of the compounds to inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) was investigated. For cytosolic MAGL, compounds with 20 carbon atoms in the acyl chain and 2-5 unsaturated bonds inhibited the hydrolysis of 2-oleoylglycerol with similar potencies (IC50 values in the range 5.1-8.2 microM), whereas the two compounds with a single unsaturated bond were less potent (IC50 values 19 and 21 microM). The fully saturated analogue 1-monoarachidin did not inhibit the enzyme, whereas the lower side chain analogues 1-monopalmitin and 1-monomyristin inhibited the enzyme with IC50 values of 12 and 32 microM, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC50 value 4.5 microM). Introduction of an alpha-methyl group for the C20:4, C20:3, and C22:4 compounds did not affect potency in a consistent manner. For the FAAH and the membrane-bound MAGL, there was no obvious relationship between the degree of unsaturation of the acyl side chain and the ability to inhibit the enzymes. It is concluded that increasing the number of unsaturated bonds on the acyl side chain of 1-AG from 1 to 5 has little effect on the affinity of acylglycerols for cytosolic MAGL.     

Piperazine and piperidine carboxamides and carbamates as inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)

The key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), are potential targets for various therapeutic applications. In this paper, we present more extensively the results of our previous work on piperazine and piperidine carboxamides and carbamates as FAAH and MAGL inhibitors. The best compounds of these series function as potent and selective MAGL/FAAH inhibitors or as dual FAAH/MAGL inhibitors at nanomolar concentrations. This study revealed that MAGL inhibitors should comprise leaving-groups with a conjugate acid pK_a of 8–10, while diverse leaving groups are tolerated for FAAH inhibitors.     

The Macamide N-3-Methoxybenzyl-Linoleamide Is a Time-Dependent Fatty Acid Amide Hydrolase (FAAH) Inhibitor

The Peruvian plant Lepidium meyenii (Maca) has been shown to possess neuroprotective activity both in vitro and in vivo [1–3]. Previous studies have also demonstrated the activity of the pentane extract and its macamides, the most representative lipophilic constituents of Maca, in the endocannabinoid system as fatty acid amide hydrolase (FAAH) inhibitors. One of the most active macamides, N-3-methoxybenzyl-linoleamide [4, 5], was studied to determine its mechanism of interaction with FAAH and whether it has inhibitory activity on mono-acyl glycerol lipase (MAGL), the second enzyme responsible for endocannabinoid degradation [6]. Macamide concentrations from 1 to 100 μM were tested using FAAH and MAGL inhibitor assay methods and showed no effect on MAGL. Tests with other conditions were performed in order to characterize the inhibitory mechanism of FAAH inhibition. N-3-methoxybenzyl-linoleamide displayed significant time-dependent and dose-dependent FAAH inhibitory activity. The mechanism of inhibition was most likely irreversible or slowly reversible. These results suggest the potential application of macamides isolated from Maca as FAAH inhibitors, as they might act on the central nervous system to provide analgesic, anti-inflammatory, or neuroprotective effects, by modulating the release of neurotransmitters.     

Critical enzymes involved in endocannabinoid metabolism

Investigations of the pathways involved in the metabolism of endocannabinoids have grown exponentially in recent years following the discovery of cannabinoid receptors (CB) and their endogenous ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The in vivo biosynthesis of AEA has been shown to occur through several pathways mediated by N-acylphosphatidylethanolamide-phospholipase D (NAPE-PLD), a secretory PLA(2) and PLC. 2-AG, a second endocannabinoid is generated through the action of selective enzymes such as phosphatidic acid phsophohydrolase, diacylglycerol lipase (DAGL), phosphoinositide-specific PLC (PI-PLC) and lyso-PLC. A putative membrane transporter or facilitated diffusion is involved in the cellular uptake or release of endocannabinoids. AEA is metabolized by fatty acid amidohydrolase (FAAH) and 2-AG is metabolized by both FAAH and monoacylglycerol lipase (MAGL). The author presents an integrative overview of current research on the enzymes involved in the metabolism of endocannabinoids and discusses possible therapeutic interventions for various diseases, including addiction.     

Differential regulation of endocannabinoid synthesis and degradation in the uterus during embryo implantation

Preimplantation embryo development to the blastocyst stage and uterine differentiation to the receptive state are prerequisites for embryo implantation. Burgeoning evidence suggests that endocannabinoid signaling is critical to early pregnancy events. Anandamide (N-arachidonoylethanolamine) and 2-AG (2-arachidonoylglycerol) are two major endocannabinoids that bind to and activate G-protein coupled cannabinoid receptors CB1 and CB2. We have previously shown that a physiological tone of anandamide is critical to preimplantation events in mice, since either silencing or amplification of anandamide signaling causes retarded development and oviductal retention of embryos via CB1, leading to deferred implantation and compromised pregnancy outcome. Whether 2-AG, which also influences many biological functions, has any effects on early pregnancy remains unknown. Furthermore, mechanisms by which differential uterine endocannabinoid gradients are established under changing pregnancy state is not clearly understood. We show here that 2-AG is present at levels one order of magnitude higher than those of anandamide in the mouse uterus, but with similar patterns as anandamide, i.e. lower levels at implantation sites and higher at interimplantation sites. We also provide evidence that region- and stage-specific uterine expression of N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), and sn-1-diacylglycerol (DAG) lipase alpha (DAGLalpha) and monoacylglycerol lipase (MAGL) for synthesis and hydrolysis of anandamide and 2-AG, respectively, creates endocannabinoid gradients conducive to implantation. Our genetic evidence suggests that FAAH is the major degrading enzyme for anandamide, whereas COX-2, MAGL and to some extent COX-1 participate in metabolizing 2-AG in the pregnant uterus. The results suggest that aberrant functioning of these pathways impacting uterine anandamide and/or 2-AG levels would compromise pregnancy outcome.     

Identification and characterization of a new reversible MAGL inhibitor

Monoacylglycerol lipase is a serine hydrolase that play a major role in the degradation of 2-arachidonoylglycerol, an endocannabinoid neurotransmitter implicated in several physiological processes. Recent studies have shown the possible role of MAGL inhibitors as anti-inflammatory, anti-nociceptive and anti-cancer agents. The use of irreversible MAGL inhibitors determined an unwanted chronic MAGL inactivation, which acquires a functional antagonism function of the endocannabinoid system. However, the application of reversible MAGL inhibitors has not yet been explored, mainly due to the scarcity of known compounds possessing efficient reversible inhibitory activities. In this study we reported the first virtual screening analysis for the identification of reversible MAGL inhibitors. Among the screened compounds, the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) is a promising reversible MAGL inhibitor lead (K_i = 8.6 μM), which may be used for the future development of a new class of MAGL inhibitors. Furthermore, the results demonstrate the validity of the methodologies that we followed, encouraging additional screenings of other commercial databases.     

The discovery of diazetidinyl diamides as potent and reversible inhibitors of monoacylglycerol lipase (MAGL)

Monoacylglycerol lipase (MAGL) has emerged as an attractive drug target because of its important role in regulating the endocannabinoid 2-arachidonoylglycerol (2-AG) and its hydrolysis product arachidonic acid (AA) in the brain. Herein, we report the discovery of a novel series of diazetidinyl diamide compounds 6 and 10 as potent reversible MAGL inhibitors. In addition to demonstrating potent MAGL inhibitory activity in the enzyme assay, the thiazole substituted diazetidinyl diamides 6d–l and compounds 10 were also effective at increasing 2-AG levels in a brain 2-AG accumulation assay in homogenized rat brain. Furthermore, selected compounds have been shown to achieve good brain penetration after oral administration in an animal study.     

4-Aryliden-2-methyloxazol-5(4H)-one as a new scaffold for selective reversible MAGL inhibitors

This study reports on a preliminary structure–activity relationship exploration of 4-aryliden-2-methyloxazol-5(4H)-one-based compounds as MAGL/FAAH inhibitors. Our results highlight that this scaffold may serve for the development of selective MAGL inhibitors. A 69-fold selectivity against MAGL over FAAH was achieved for compound 16b (MAGL and FAAH IC₅₀?=?1.6 and 111?µM, respectively). Furthermore, the best compound behaved as a reversible ligand and showed promising antiproliferative activity in cancer cells.     

Docking based virtual screening and molecular dynamics study to identify potential monoacylglycerol lipase inhibitors

Monoacylglycerol lipase (MAGL) is one of the key enzymes of the endocannabinoid system (ECS). It hydrolyzes one of the major endocannabinoid, 2-arachidonoylglycerol (2-AG), an endogenous full agonist at G protein coupled cannabinoid receptors CB1 and CB2. Numerous studies showed that MGL inhibitors are potentially useful for the treatment of pain, inflammation, cancer and CNS disorders. These provocative findings suggested that pharmacological inhibition of MAGL function may confer significant therapeutic benefits. In this study, we presented hybrid ligand and structure-based approaches to obtain a novel set of virtual leads as MAGL inhibitors. The constraints used in this study, were Glide score, binding free energy estimates and ADME properties to screen the ZINC database, containing approximately 21 million compounds. A total of seven virtual hits were obtained, which showed significant binding affinity towards MAGL protein. Ligand, ZINC24092691 was employed in complex form with the protein MAGL, for molecular dynamics simulation study, because of its excellent glide score, binding free energy and ADME properties. The RMSD of ZINC24092691 was observed to stay at 0.1 nm (1 Å) in most of the trajectories, which further confirmed its ability to inhibit the protein MAGL. The hits were then evaluated for their ability to inhibit human MAGL. The compound ZINC24092691 displayed the noteworthy inhibitory activity reducing MAGL activity to 21.15% at 100 nM concentration, with an IC₅₀ value of 10 nM.     

Methylation and acetylation of 15-hydroxyanandamide modulate its interaction with the endocannabinoid system

The biological activity of endocannabinoids like anandamide (AEA) and 2-arachidonoylglycerol (2-AG) is subjected in vivo to a “metabolic control”, exerted mainly by catabolic enzymes. AEA is inactivated by fatty acid amide hydrolase (FAAH), that is inhibited competitively by hydroxyanandamides (HAEAs) generated from AEA by lipoxygenase activity. Among these derivatives, 15-HAEA has been shown to be an effective (K_i ∼0.6 μM) FAAH inhibitor, that blocks also type-1 cannabinoid receptor (CB1R) but not other components of the “endocannabinoid system (ECS)”, like the AEA transporter (AMT) or CB2R. Here, we extended the study of the effect of 15-HAEA on the AEA synthetase (NAPE-PLD) and the AEA-binding vanilloid receptor (TRPV1), showing that 15-HAEA activates the former (up to ∼140% of controls) and inhibits the latter protein (down to ∼70%). We also show that 15-HAEA halves the synthesis of 2-AG and almost doubles the transport of this compound across the membrane. In addition, we synthesized methyl and acetyl derivatives of 15-HAEA (15-MeOAEA and 15-AcOAEA, respectively), in order to check their ability to modulate FAAH and the other ECS elements. In fact, methylation and acetylation are common biochemical reactions in the cellular environment. We show that 15-MeOAEA, unlike 15-AcOAEA, is still a powerful competitive inhibitor of FAAH (K_i ∼0.7 μM), and that both derivatives have negligible interactions with the other proteins of ECS. Therefore, 15-MeOAEA is a FAAH inhibitor more selective than 15-HAEA. Further molecular dynamics analysis gave clues to the molecular requirements for the interaction of 15-HAEA and 15-MeOAEA with FAAH.     

Therapeutic potential of monoacylglycerol lipase inhibitors

Marijuana and aspirin have been used for millennia to treat a wide range of maladies including pain and inflammation. Both cannabinoids, like marijuana, that exert anti-inflammatory action through stimulating cannabinoid receptors, and cyclooxygenase (COX) inhibitors, like aspirin, that suppress pro-inflammatory eicosanoid production have shown beneficial outcomes in mouse models of neurodegenerative diseases and cancer. Both cannabinoids and COX inhibitors, however, have untoward effects that discourage their chronic usage, including cognitive deficits and gastrointestinal toxicity, respectively. Recent studies have uncovered that the serine hydrolase monoacylglycerol lipase (MAGL) links the endocannabinoid and eicosanoid systems together through hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) to provide the major arachidonic acid (AA) precursor pools for pro-inflammatory eicosanoid synthesis in specific tissues. Studies in recent years have shown that MAGL inhibitors elicit anti-nociceptive, anxiolytic, and anti-emetic responses and attenuate precipitated withdrawal symptoms in addiction paradigms through enhancing endocannabinoid signaling. MAGL inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. In cancer, MAGL inhibitors have been shown to have anti-cancer properties not only through modulating the endocannabinoid–eicosanoid network, but also by controlling fatty acid release for the synthesis of protumorigenic signaling lipids. Thus, MAGL serves as a critical node in simultaneously coordinating multiple lipid signaling pathways in both physiological and disease contexts. This review will discuss the diverse (patho)physiological roles of MAGL and the therapeutic potential of MAGL inhibitors in treating a vast array of complex human diseases.     

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

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

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

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

Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase

Fatty acid amidohydrolase, a membrane-bound enzyme found in a variety of mammalian cells, is responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide. In an earlier study we reported that Tetrahymena pyriformis was able to secrete a FAAH-like activity in starvation medium (Karava V., Fasia L., Siafaka-Kapadai A., FEBS Lett. 508 (2001) 327-331). In this study the endocannabinoid anandamide, was found to be metabolized by T. pyriformis homogenate by the action of a FAAH-like enzyme, in a time- and concentration-dependent manner. The main metabolic products of [3H]anandamide hydrolysis were [3H]arachidonic acid and ethanolamine. Amidohydrolase activity was maximal at pH 9-10, it was inhibited by phenylmethylsulfonyl fluoride and arachidonyltrifluoromethyl ketone and was Ca2+ and Mg(2+)-independent. Kinetic experiments demonstrated that the enzyme had an apparent K(m) of 2.5 microM and V(max) of 20.6 nmol/min mg. Subcellular fractionation of T. pyriformis homogenate showed that the activity was present in every subcellular fraction with highest specific activity in the microsomal as well as in non-microsomal membrane fraction. Immunoblot analysis of selected subcellular fractions, using an anti-FAAH polyclonal antibody, revealed the presence of an immunoreactive protein with a molecular mass approximately 66 kDa similar to the molecular mass of the mammalian enzyme. In conclusion, this study demonstrates that a FAAH similar to the mammalian enzyme is present in a unicellular eukaryote, indicating the importance of FAAH activity throughout evolution. It also supports the notion that Tetrahymena species may be a suitable model for metabolic studies on endocannabinoids, as well as for the study of drugs targeted towards FAAH.     

Occurrence and possible biological role of the endocannabinoid system in the sea squirt Ciona intestinalis

A cannabinoid receptor orthologue (CiCBR) has been described in the sea squirt Ciona intestinalis. Here we report that CiCBR mRNA expression is highest in cerebral ganglion, branchial pharynx, heart and testis of C. intestinalis, and that this organism also contains cannabinoid receptor ligands and some of the enzymes for ligand biosynthesis and inactivation. Using liquid chromatography-mass spectrometry, the endocannabinoid anandamide was found in all tissues analysed (0.063-5.423 pmol/mg of lipid extract), with the highest concentrations being found in brain and heart. The endocannabinoid 2-arachidonoylglycerol (2-AG) was fivefold more abundant than anandamide, and was most abundant in stomach and intestine and least abundant in heart and ovaries (2.677-50.607 pmol/mg of lipid extract). Using phylogenomic analysis, we identified orthologues of several endocannabinoid synthesizing and degrading enzymes. In particular, we identified and partly sequenced a fatty acid amide hydrolase (FAAH) orthologue, showing 44% identity with human FAAH and containing nearly all the amino acids necessary for a functional FAAH enzyme. Ciona intestinalis also contained specific binding sites for cannabinoid receptor ligands, and an amidase enzyme with pH-dependency and subcellular/tissue distribution similar to mammalian FAAHs. Finally, a typical C. intestinalis behavioural response, siphon reopening after closure induced by mechanical stimulation, was inhibited by the cannabinoid receptor agonist HU-210, and this effect was significantly attenuated by mammalian cannabinoid receptor antagonists.     

Endocannabinoids,hormone-cytokine networks and human fertility

Anandamide (N -arachidonoylethanolamine, AEA) is a major endocannabinoid, shown to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here, we review the roles of AEA, of the AEA-binding cannabinoid (CB) receptors, of the selective AEA membrane transporter (AMT), and of the AEA-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), in human gestation. In particular, we discuss the interplay between the endocannabinoid system and the hormone-cytokine array involved in the control of human pregnancy, showing that the endocannabinoids take part in the immunological adaptation occurring during early pregnancy. In this line, we discuss the critical role of FAAH in human peripheral lymphocytes, showing that the expression of this enzyme is regulated by progesterone, Th1 and Th2 cytokines, which also regulate fertility. Moreover, we show that AEA and the other endocannabinoid, 2-arachidonoylglycerol, inhibit the release of the fertility-promoting cytokine leukemia inhibitory factor from human lymphocytes. Taken together, low FAAH and consistently high blood levels of AEA, but not CB receptors or AMT, can be early (<8 weeks of gestation) markers of spontaneous abortion, potentially useful as diagnostic tools for large-scale, routine monitoring of gestation in humans.     

Conformational requirements for endocannabinoid interaction with the cannabinoid receptors, the anandamide transporter and fatty acid amidohydrolase

Anandamide (N-arachidonoylethanolamine) has been identified as an endogenous ligand of the G-protein coupled cannabinoid CB(1) receptor. Recent studies have postulated the existence of carrier-mediated anandamide transport which is involved in the termination of the biological effects of anandamide. A membrane bound amidohydrolase (fatty acid amide hydrolase, FAAH), located intracellulary, hydrolyzes and inactivates anandamide and other endogenous cannabinoids such as 2-arachidonoylglycerol (2-AG). Structure-activity relationships (SARs) for endocannabinoid interaction with the CB receptors, the anandamide transporter and FAAH are currently emerging in the literature. This review considers the divergences between these SARs and focuses upon the conformational implications for endocannabinoid recognition at each of these biological targets.     

Ulcerative Colitis Induces Changes on the Expression of the Endocannabinoid System in the Human Colonic Tissue

Background

Recent studies suggest potential roles of the endocannabinoid system in gastrointestinal inflammation. Although cannabinoid CB₂ receptor expression is increased in inflammatory disorders, the presence and function of the remaining proteins of the endocannabinoid system in the colonic tissue is not well characterized.

Methodology

Cannabinoid CB₁ and CB₂ receptors, the enzymes for endocannabinoid biosynthesis DAGLα, DAGLβ and NAPE-PLD, and the endocannabinoid-degradating enzymes FAAH and MAGL were analysed in both acute untreated active ulcerative pancolitis and treated quiescent patients in comparison with healthy human colonic tissue by immunocytochemistry. Analyses were carried out according to clinical criteria, taking into account the severity at onset and treatment received.

Principal Findings

Western blot and immunocytochemistry indicated that the endocannabinoid system is present in the colonic tissue, but it shows a differential distribution in epithelium, lamina propria, smooth muscle and enteric plexi. Quantification of epithelial immunoreactivity showed an increase of CB₂ receptor, DAGLα and MAGL expression, mainly in mild and moderate pancolitis patients. In contrast, NAPE-PLD expression decreased in moderate and severe pancolitis patients. During quiescent pancolitis, CB₁, CB₂ and DAGLα expression dropped, while NAPE-PLD expression rose, mainly in patients treated with 5-ASA or 5-ASA+corticosteroids. The number of immune cells containing MAGL and FAAH in the lamina propria increased in acute pancolitis patients, but dropped after treatment.

Conclusions

Endocannabinoids signaling pathway, through CB₂ receptor, may reduce colitis-associated inflammation suggesting a potential drugable target for the treatment of inflammatory bowel diseases.     

Development of the first potent and specific inhibitors of endocannabinoid biosynthesis

Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases alpha and beta (DAGLalpha and beta) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLalpha to screen new synthetic substances as DAGLalpha inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLalpha and MAGL activities were assessed by using sn-1-[14C]-oleoyl-2-arachidonoyl-glycerol and 2-[3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat) (IC50 approximately 60 nM), the most potent inhibitors of DAGLalpha were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50 = 500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50 = 160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl-O-p-nitro-phenyl oleylphosphonate] inhibited both DAGLalpha and MAGL with similar potencies (IC50 = 0.8-0.1 and 3.7-3.2 microM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.