Endogenous Cannabinoids: Structure and Metabolism

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1 REVIEW ARTICLE Journal of euroendocrinology 20 (Suppl. 1), 1 9 ª 2008 Blackwell ublishing Ltd. o claim to original works Endogenous Cannabinoids: Structure and Metabolism T. Bisogno Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio azionale delle Ricerche, ozzuoli, aples, Italy. Journal of euroendocrinology Correspondence to: Tiziana Bisogno, Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio azionale delle Ricerche, Via Campi Flegrei 34, ozzuoli, aples, Italy ( tbisogno@icmib.na.cnr.it). The finding of specific binding sites for D 9 -tetrahydrocannabinol, the psychoactive component of Cannabis sativa, has led to the discovery of the endocannabinoid system and has emphasised the physiological and pathological relevance of endocannabidoid lipid signalling. Subsequently, an increasing number of papers have been published on the biochemistry and pharmacology of endocannabinoids. An overview of the current understanding of structure and metabolism of the best studied endocannabinoids is provided, with a focus on the mechanisms responsible for their biosynthesis and inactivation. Key words: endocannabinoids, anandamide, 2-Arachidonoylglycerol. doi: /j x D 9 -Tetrahydrocannabinol (TC), the main psychoactive compound extracted from Cannabis sativa, is the most representative component of the mixture of therapeutic substances that are communally referred to as cannabinoids (1). It acts as an agonist at specific G-protein-coupled receptors, cannabinoid receptor subtypes CB 1 and CB 2 (2). The discovery of cannabinoid receptors has driven the investigation of the existence of their natural ligands and, soon after, endocannabinoids were identified. -arachidonoyl-ethanolamine (AEA, anandamide) and 2-arachidonoyl glycerol (2-AG) are the two most studied endocannabinoids. They are biosynthesised on demand by cleavage of their membrane lipid precursors -arachidonoyl-phosphatidylethanolamine (-ArE) and sn-1-acyl- 2-arachidonoylglycerols (DAGs) respectively, and then inactivated by intracellular hydrolysing enzymes. Cannabinoid receptors, endocannabinoids and enzymes that biosynthesise [-acyl-phosphatidylethanolamine-selective phospholipase D (AE-LD) and diacylglycerol lipase (DAGL)] and degrade [fatty acid amide hydrolase (FAA) and the monoacylglycerol lipases (MAGL)] AEA and 2-AG, respectively, are key components in the endocannabinoid system (3). Despite a large number of excellent studies having contributed to the current understanding of the endocannabinoid regulation and function, further efforts are necessary to fully comprehend this signalling system. ere, a review on the current knowledge on structure and metabolism of the best studied endogenous cannabinoids is presented. In particular, this review concentrates on the mechanisms responsible for the biosynthesis and inactivation of the endocannabinoids, as well as on the several inhibitors of their metabolism identified to date. Endocannabinoids and related -acyl-ethanolamines Endocannabinoids are defined as endogenous compounds capable of binding to and functionally activating cannabinoid receptors. At least five endocannabinoids have been identified to date (Fig. 1). Anandamide, the amide between arachidonic acid and ethanolamine, was the first endogenous ligand to be reported at the end of 1992 (4) and additional polyunsaturated ethanolamines that bind to cannabinoid receptors, homolinolenylethanolamide and docosatetraenylethanolamide, have been isolated in the brain. ther endocannabinoids, all derived from arachidonic acid, were identified. The identification of 2-AG, the ester of arachidonic acid with glycerol, isolated from canine gut and brain has also been reported (5, 6). Subsequently, the first endocannabinoid characterised by ether bond, 2-arachidonyl glyceryl ether or noladin ether, was isolated from porcine brain (7) and -arachidonoyldopamine (ADA), a selective CB 1 agonist and a potent agonist of vanilloid receptors, was discovered (8, 9). -arachidonoylethanolamine, the ester derivative of ethanolamine with arachidonic acid, with the same molecular weight as anandamide but with opposite orientation of the polar ethanolamine moiety, was named virodhamine from the Sanskrit word virodha, which means opposition (10). Endocannabinoid congeners and other -acyl-ethanolamines that do not activate cannabinoid receptors are generally classified as cannabimimetic compounds. In particular, palmitoylethanolamide exerts its antiinflammatory and analgesic activity, independently of cannabinoid receptors, by a mechanism that still remains a matter of debate (11). An autacoid local inflammation antagonism mechanism (ALIA),

2 2 T. Bisogno 2 Anandamide Virodhamine 2-Arachidonoyl-glycerol oladin ether -arachidonoyl-dopamine Fig. 1. Chemical structures of the endocannabinoids. an entourage effect on anadamide-mediated action or EA direct activation of both peroxisome-proliferator-activated receptor a (AR-a) and orphan G protein-coupled receptor GR55, have been reported. Stearoylethanolamide, another bioactive amide, has been suggested to activate a new binding site, which is not coupled to G protein (12) and its anorexic effect, associated with a reduction in liver stearoyl-coa desaturase-1 mra expression, has also been reported (13). Finally, oleoylethanolamide modulates feeding, body weight and lipid metabolism by activating AR-a (14) and excites peripheral vagal sensory nerves via vanilloid receptor vanilloid receptor-type 1 (TRV1) (15). In addition, oleoylethanolamide-mediated action by GR119 receptor has recently been reported (16). Although a route for the synthesis and inactivation of virhodamine, ADA and noladin ether has not been unequivocally demonstrated, the full characterisation of most of the proteins involved in AEA and 2-AG metabolism (i.e. of the enzymes responsible for their biosynthesis and degradation) is now available. Biosynthesis of endocannabinoids Unlike classical neurotransmitters and neuropeptides, endocannabinoids are not stored in intracellular compartments but are synthesised on demand (i.e. only when and where necessary) to be then immediately released from the cell via an as-yet-unidentified mechanism. The enhancement of intracellular Ca 2+ concentration, due to neuronal depolarisation or stimulation of metabotropic receptors coupled to G q 11 proteins, activates both AEA and 2-AG formation and release from the post-synaptic neurone to activate pre-synaptic cannabinoid receptors (3). Biosynthesis of AEA AEA is biosynthesised via a phospholipid-dependent pathway (Fig. 2) consisting of two steps of enzymatic reactions. The first is responsible for -ArE formation by the transfer of a fatty acyl chain from the sn-1 position of glycerophospholipids to the amino group of phosphatidylethanolamine. This process, however, might be catalysed by both an as yet unidentified calcium-dependent membrane-associated trans--acyltransferase (AT), which has not yet been cloned, and by a calcium-independent AT recently characterised as a rat lecithin retinol acyltransferase-like protein 1 (RL-1) (17). In a second step, AEA and phosphatidic acid are produced by the enzymatic hydrolysis of -ArE catalysed by a AE-selective phospholipase D that was only recently cloned (18). AE-LD is quite different from other LD enzymes; for example, it exhibits no selectivity for a particular fatty acid moiety on the sn-1, sn-2 and -position of AEs, and therefore is also responsible for the formation of other -acyl-ethanolamines (AEs). The amino acid sequence showed that this enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the b-lactamase fold (18). A close precursor product relationship between -ArE and AEA (19) and evidence that AE-LD, when overexpressed in cells, regulates the levels of AEs (20) have been reported. Although AEs are now well recognised as AE precursors, the existence of alternative and probably concomitant pathways for -ArE conversion to AEA have been proposed (21). In particular, tissues from AE-LD knockout mice are still capable of converting -ArE to AEA (22) and no considerable decrease of AEA basal levels have been reported in macrophages following gene-silencing of AE-LD enzyme (23). Recently, a phospholipase C-dependent pathway for -ArE conversion to AEA via formation of phospho-aea as a biosynthetic intermediate, and its dephosphorylation to AEA by the action of protein tyrosine phosphatase 22 (T22) has been reported (23). Furthermore, the phospholipase C phosphatase pathway appears to be responsible for LS-induced AEA formation in mouse macrophage cell line (23, 24). Additionally, 2-lyso-AE produced from -ArE by the action of a group IB secretory phospholipase A 2 might be converted to AEA via a selective lyso-phospholipase D (25). The predominant expression of sla2-ib in small intestine and stomach (25) did not account for the AEA formation observed in ª 2008 Blackwell ublishing Ltd, o claim to original works, Journal of euroendocrinology, 20 (Suppl. 1), 1 9

3 Endocannabinoids X 3 R 2 R 3 R 2 AT ( 2 ) LC ( 1 ) sla 2 _ R 3 _ R 3 Abh4 x 2 R 2 ( 3 ) AE-LD ( 4 ) lyso-ld hosphatase hosphodiesterase (5) Fig. 2. Major biosynthetic pathways for the endocannabinoid anandamide. Abh4, a b-hydrolase 4; lyso-ld, lyso-phospholipase D; sla 2, secretory phospholipase A 2, LC, phospholipase C; AE-LD, phospholipase D selective for -acyl-phosphatidylethanolamines; AT, trans--acyltransferase; (1) -arachidonoylphosphatidylethanolamine; (2) phospho-anandamide; (3) 2-lyso--arachidonoyl-phosphatidylethanolamine; (4) glycerol-phospho-anandamide; (5) anandamide. the brain of AE-LD knockout mice. Finally, in the mouse brain, a b-hydrolase 4 (Abh4) acts as phospholipase B lysophospholipase to catalyse the double-deacylation of AE, therefore generating the formation of a glycerol-phospho-aea intermediate, which is then converted into AEA by a specific phosphodiesterase (26). This potential redundancy in the biochemical pathways converting AEs into AEs has so far hindered the development of selective assays of AE-LD activity because this protein does not appear to be the only catalyst for AEA formation. owever, an understanding of the dominant function of each pathway responsible for AEA synthesis, depending on the stimuli, could suggest different targets for drug development in view of the evidence that changes of AEA levels are associated with certain diseases. Biosynthesis of 2-AG The most important biosynthetic precursors of 2-AG are the DAGs, produced from the hydrolysis of phosphoinositol bis-phosphate (I 2 ), catalysed by the I 2 -selective phospholipase C, or of phosphatidic acid (A), catalysed by a A phosphohydrolase (27 30). DAGs (Fig. 3) are then converted into 2-AG by sn-1 selective-dag lipases. Two sn-1 DAG lipase isozymes (DAGLa and DAGLb) have ª 2008 The Author. Journal Compilation ª 2008 Blackwell ublishing Ltd, Journal of euroendocrinology, 20 (Suppl. 1), 1 9

4 4 T. Bisogno hosphatidic acid C - R hospholipid C - R X A phosphohydrolase LC LA 1 CR CoA Arachidonoyl-CoenzymeA R X - Diacylglycerol sn-1 DAG lipase sn-1 lysophospholipid lyso-lc X - 2-Arachidonoyl-glycerol(2-AG) Lipase sn-1 lyso-a - X CR CR Fig. 3. Biosynthetic pathways for the endocannabinoid 2-arachidonoyl glycerol. A, hosphatidic acid; LC, phospholipase C; LA 1, phospholipase A 1 ; DAG, di-acyl-glycerol. been cloned and enzymatically characterised (31). They are mostly located in the plasma membrane, are stimulated by Ca 2+ and glutathione, appear to possess a catalytic triad typical of serine hydrolases, and do not exhibit strong selectivity for 2-arachidonate-containing DAGs. The distribution of the mra encoding for DAGLa correlates with the relative abundance of 2-AG in tissues and organs (29). The two enzymes exhibit a pattern of expression fitting with the proposed role of 2-AG as a mediator of neurite growth, during brain development, or as retrograde signal mediating depolarisation-induced suppression of neurotransmission and heterosynaptic plasticity, in the adult brain. Indeed, DAGLa is localised to the postsynaptic dendritic spines establishing synapses with CB 1 expressing axons (32, 33). Recently, a biosynthetic pathway responsible for 2-AG formation that does not require the sequential activation of LCb and DAGL was suggested (34). The hippocampal levels of anandamide, but not of 2-AG, are reduced in transgenic mice lacking G aq G a11 proteins, indicating that the basal biosynthesis of anandamide in this brain area is under the tonic control of receptors coupled to these G proteins, whereas 2-AG formation is not. n the other hand, in G aq G a11 null mice, the levels of anandamide, but not 2-AG, are still stimulated by kainic acid as much as in wild-type mice, thus suggesting that the stimulusinduced biosynthesis of 2-AG, but not of anandamide, does require G aq G a11 in order to occur (34). Endocannabinoid degradation Termination of AEA and 2-AG signalling appears to involve a twostep process that begins with transport across the plasma membrane, followed by enzymatic hydrolysis into arachidonic acid and ethanolamine or glycerol, respectively (Fig. 4) (35). It is still under investigation whether or not endocannabinoids are taken up by cells via a plasma membrane transporter (the putative endocannabinoid membrane transporter, EMT) that has not been isolated or cloned yet (36). Felder and co-workers, in a very recent paper focused their attention on the AEA reuptake mechanism (37). They reported four molecular models that account for the several hypotheses reported for AEA passage through the plasma membrane. In three of these models plasma membrane-associated FAA, passive diffusion driven by FAA and facilitative diffusion driven by FAA, the hydrolysis of AEA is the driving force of the process. In particular, FAA might bind to AEA from the extracellu- ª 2008 Blackwell ublishing Ltd, o claim to original works, Journal of euroendocrinology, 20 (Suppl. 1), 1 9

5 Endocannabinoids 5 Anandamide?? 2-Arachidonoyl-glycerol EMT FAA Anandamide 2-Arachidonoyl-glycerol MAGL Arachidonic acid Arachidonic acid Ethanolamine Glycerol Fig. 4. Mechanisms for -arachidonoyl-ethanolamine and 2-arachidonoyl glycerol inactivation. FAA, Fatty acid amide hydrolase; MAGL, monoacylglycerol lipase; EMT, endocannabinoid membrane transporter. lar space directly or, by regulating the gradient of concentration, might drive the AEA inward that can both diffuse passively, as lipophile compound, or be moved by a protein transporter or carrier molecule, respectively. The last model proposed endocytosis-mediated anandamide uptake is responsible for AEA sequestration, via a rapid endocytotic process, in caveolin-rich lipid rafts and for its subsequent release to be hydrolysed by FAA. In this model, the involvement of an auxiliary protein is not excluded. nce inside the cell, endocannabinoids are rapidly inactivated by an hydrolysing mechanism catalysed by two cloned enzymes. FAA is the oldest and best characterised enzyme involved in the degradation of both AEA and 2-AG, and its cloning, structural and kinetic properties, and distribution in the body and crystal structure have been described (38). FAA, which recognises other fatty acid primary amides, -acyltaurines and -acyl amino acids as a substrate is unusual among the amidase signature (AS) enzymes because it is an integral membrane enzyme. Site-directed mutagenesis and crystallographic X-ray identified an unusual serine-serine-lysine catalytic triad for FAA; moreover, the promoter region on the FAA gene has been studied. FAA is abundantly expressed throughout the central nervous system and FAA-positive neurones in the brain are found in proximity to nerve terminals that contain CB 1 cannabinoid receptors, supporting a prominent role for this enzyme in endocannabinoid deactivation (38, 39). The physiopathological relevance of FAA is becoming more and more evident as a result of studies utilising its chemical or genetic inactivation. Moreover, the functional proteomic discovery of a second membraneassociated AS enzyme in humans, which displays FAA activity, was reported (40). This enzyme, named FAA-2, exhibits only 20% homology with FAA, and is expressed in several mammalian species, but not in rodents. Evaluation of FAA-2 substrate selectivity, its tissue distribution and inhibitor sensitivity profile compared with the original FAA revealed that the two enzymes have similar but not identical characteristics. Although FAA can catalyse 2-AG hydrolysis, 2-AG levels, unlike those of AEA, are not increased in FAA knockout mice. This observation is in agreement with previ- ª 2008 The Author. Journal Compilation ª 2008 Blackwell ublishing Ltd, Journal of euroendocrinology, 20 (Suppl. 1), 1 9

6 6 T. Bisogno ously reported evidence on the existence of other enzymes catalysing 2-AG inactivation and different from FAA. Indeed, a MAGL inactive on AEA and having high homology with other human and mouse MAGLs, was cloned from the rat (41, 42). At the amino acid level, human and mouse MAGL are 84% identical, and mouse and rat MAGL are 92% identical. MAGL cloning, structural and catalytic properties have been described in a recent comprehensive review (43). In particular, site-directed mutagenesis identified the typical Ser-Asp-is triad of most serine hydrolases and, like most lipases, MAGL is also thought to contain a mobile helical segment, lid domain, covering the catalytic site. MAGL has not yet been crystallised. The observed complementary localisation in the rat brain for MAGL and FAA (i.e. pre-synaptic and post-synaptic, respectively) suggests different roles for the two endocannabinoids in the central nervous system (44). RA interference-mediated silencing of MAGL expression greatly enhances 2-AG, but not AEA, accumulation in ela cells, suggesting a primary role for MAGL in the degradation of 2-AG (45). The same authors also carried out immunodepletion experiments suggesting that MAGL accounts for only 50% of the total 2-AG-hydrolysing activity in soluble fractions of rat brain. Furthermore, evidence for the existence of a second MAGL activity that controls 2-AG levels in intact microglial cells has recently been provided (46). The novel MAGL lipase, mainly expressed in mitochondria and nuclei, is pharmacologically distinguishable from the cloned MAGL as well as from FAA. Inhibitors Considerable progress has been made in understanding the physiological functions of the endocannabinoids, and their corresponding potential pathological implications were recently discussed in comprehensive reviews and are not discussed here. It is becoming more and more evident that compounds able to modulate endocannabinoid metabolism might be used as potential therapeutic agents for the treatment of diseases where the endocannabinoid system is involved. In particular, an up-regulated or defective endocannabinoid system has been associated with controlling or, in some cases, contributing to pathological conditions. The use of specific inhibitors of both endocannabinoid degradation and biosynthesis acting as indirect agonist or indirect antagonist, respectively has also been reported. Since AEA and 2-AG biosynthetic enzymes have been identified only recently, little information on the development of selective inhibitors for these proteins is now available. owever, nonspecific inhibitors, such as drugs that alkylate serine, cysteine and histidine residues or BT, an inhibitor of Ca 2+ -independent LA 2, have been shown to inhibit the formation of AEA in cortical neurones (47). Regarding the formation of 2-AG, RC80267 [1,6- bis-(cyclohexyloximino-carbonylamino)-hexane], and tetrahydrolipstatin (TL), have been shown to inhibit DAGL-a at concentrations lower than those required to inhibit other lipases (31). Furthermore, two inhibitors of 2-AG biosynthesis have been developed so far, and -3841, with excellent selectivity for DAGL-a over the other proteins of the endocannabinoid system tested but, unfortunately, they are not suitable for systemic use in vivo (48). Although the existence and the molecular characterisation of EMT is still questioned, selective inhibitors of this process and their potential pharmacological applications have been reported. Endocannabinoid cellular uptake inhibitors exhibiting different degrees of potency, selectivity over other proteins of the endocannabinoid system and stability to enzymatic hydrolysis might be classified at least in two classes depending on their chemical structure. In particular, the first class includes long chain fatty acid derivatives such as amides or retroamides of arachidonic and oleic acid with amines containing an aromatic constituent. AM404, VDM-11, MDM-1 and -2, UCM707 and AM1172 belong to this class (49 53). The latter contains carbamate derivatives, which, lacking the long carbon chains, are supposed to be more selective (54, 55). The EMT inhibitors reported above have been successfully used in different animal models and have contributed to the investigation of the role of the endocannabinoid system in different pathological conditions of both the central and peripheral nervous system. Since FAA identification, much attention has been paid to the design and the synthesis of potent and selective inhibitors. Several classical types of serine hydrolase inhibitors have been reported to counteract endocannabinoid inactivation. In particular, the mechanism of inhibition of these compounds, including fluorophosphonates, trifluoromethyl ketones, the a-keto heterocycles L-135 and a-k 7, and the carbamates URB597, BMS-1, SA-47 and SA-72 (38, 39), appears to be due to the presence of an electrophile group capable of binding to the highly conserved nucleophile serine residue. ence, it is possible to foresee a potential inhibitory effect for these compounds on other serine hydrolyses (i.e. off-target enzymes). A proteomic approach, known as activity-based protein profiling, which allows the screening of inhibitors against multiple enzymes in parallel, has been successfully used to assess the selectivity of several FAA inhibitors. Interestingly, URB597 and L-135 have several off-targets, whereas SA-47 and SA-72 are exceptionally specific (56). o information is available regarding arachidonoyl-serotonin action on other serine hydrolyses, although its antagonism at the TRV1 receptor has been reported (57). Recently, Ahn et al. (58) reported the identification of piperidine piperazine urea compounds, F-750 and F-622, as potent and selective FAA inhibitors with no activity against other serine hydrolases in vitro or in vivo. FAA inhibitors that are efficacious in vivo have provided evidence demonstrating that the tonic control of FAA may have therapeutic value in the treatment of several disorders. Several compounds have been reported to inhibit 2-AG hydrolysis with potencies in the micromolar range but, unfortunately, they are not selective. In particular, MAGL is inhibited by nonspecific serine hydrolase inhibitors such as MAF, DSF, and diisopropyl fluorophosphates. Trifluoromethyl ketones and general lipase inhibitors such as U-73122, RC 80267, or TL were also found to inhibit 2-AG hydrolysis (43, 48). Different analogues of 2-AG, such as a-methyl-1-ag, and -2204, were tested as potential inhibitors of 2-oleoyl-glycerol hydrolysis but they lack selectivity for MAGL over FAA (59). Trifluoromethyl ketones functionalised as b-thioether with different lengths in the carbon atoms have been reported to inhibit MAGL in prostate cancer cells. URB602, a biphenyl carbamate compound lacking a long carbon chain, has recently been used in vivo to demonstrate the involvement of ª 2008 Blackwell ublishing Ltd, o claim to original works, Journal of euroendocrinology, 20 (Suppl. 1), 1 9

7 Endocannabinoids 7 2-AG in inflammatory nociception and stress-induced analgesia. owever, very recently, the ability of URB602 to discriminate between MAGL and FAA has been reinvestigated (60, 61). MAGL is sensitive to sulphydryl-specific compounds and, indeed, a maleimide derivative of arachidonic acid, -arachidonoylmaleimide was found to inhibit 2-AG hydrolysis in nanomolar range but, no date are available regarding its selectivity (62). Recently, tetraethylthiuram disulphide has been reported as a potent inhibitor of MAGL, even if its mechanism of inhibition needs to be further investigated (63). Conclusions Significant progress has been made in this research field. First came the discovery of the endocannabinoid receptors and, soon after, endocannabinoids were identified. Finally, the full characterisation of most of the proteins involved in AEA and 2-AG metabolism (i.e. enzymes responsible of their biosynthesis and degradation) has contributed to the discovery of a whole endogenous signalling system now known as the endocannabinoid system. Despite our understanding of the molecular composition of the endocannabinoid system, further information is required to fully understand the physiological meaning of this system. In particular, it is still necessary to: (i) assess the physiological role of virodhamine, ADA and noladin ether; (ii) determine their biosynthetic pathways; (iii) develop selective inhibitors of endocannabinoid biosynthesis; and (iv) clone the EMT, if such a protein does indeed exist. Conflicts of interest The author has declared no conflicts of interest. Received: 7 December 2007, accepted 1 February 2008 References 1 Gaoni Y, Mechoulam R. 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