Inhibitor Report for: URB602

Monoglyceride lipase (MGL), the main enzyme responsible for the hydrolytic deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG), is an intracellular serine hydrolase that plays critical roles in many physiological and pathological processes, such as pain, inflammation, neuroprotection and cancer. The crystal structures of MGL that are currently available provide valuable information about how this enzyme might function and interact with site-directed small-molecule inhibitors. On the other hand, its conformational equilibria and the contribution of regulatory cysteine residues present within the substrate-binding pocket or on protein surface remain open issues. Several classes of MGL inhibitors have been developed, from early reversible ones, such as URB602 and pristimerin, to carbamoylating agents that react with the catalytic serine, such as JZL184 and more recent O-hexafluoroisopropyl carbamates. Other inhibitors that modulate MGL activity by interacting with conserved regulatory cysteines act through mechanisms that deserve to be more thoroughly investigated.

BACKGROUND: The endocannabinoids are emerging as natural brain protective substances that exert potentially beneficial effects in several neurological disorders by virtue of their hypothermic, immunomodulatory, vascular, antioxidant, and antiapoptotic actions. This study was undertaken to assess whether preventing the deactivation of the endocannabinoid 2-arachidonoylglycerol (2-AG) with the monoacylglycerol lipase (MAGL) inhibitor URB602 can provide neuroprotective effects in hypoxia-ischemia (HI)-induced brain injury. METHODS: URB602 was administered into the right lateral ventricle 30min before 7-day-old pup rats were subjected to HI. The neuroprotective effect was evaluated on postnatal day (PN) 14 or at adulthood (PN80) using behavioral and histological analyses. Activated caspase-3 expression and propidium iodide labeling were assessed as indexes of apoptotic and necrotic cell death, respectively. RESULTS: Pretreatment with URB602 reduced apoptotic and necrotic cell death, as well as the infarct volume measured at PN14. At adulthood, URB602-treated HI animals performed better at the T-maze and the Morris maze, and also showed a significant reduction of brain damage. CONCLUSION: These results demonstrate that a pretreatment with URB602 significantly reduces brain damage and improves functional outcome, indicating that endocannabinoid-degrading enzymes may represent an important target for neuroprotection in neonatal ischemic brain injury.

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid (endocannabinoid) lipid whose functions remain poorly understood. Guindon and colleagues report the novel finding that exogenous application of 2-AG induces peripheral antinociceptive effects that are mediated, at least in part, by actions at peripheral cannabinoid CB(2) receptors. URB602, a recently described inhibitor of monoacylglycerol lipase, an enzyme that catalyzes 2-AG hydrolysis in vivo, also induced peripheral antinociceptive effects and enhanced the actions of 2-AG. Peripheral analgesic mechanisms represent promising therapeutic targets for suppressing pain in the absence of unwanted central nervous system side-effects (e.g. psychoactivity) associated with activation of central CB(1) receptors. The therapeutic potential of inhibitors of 2-AG deactivation for the treatment of inflammatory pain is discussed.

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+/-0.70muM reducing MAGL activity to 82% of controls at 10muM 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.

BACKGROUND: Clinical reports suggest that rather than directly driving cocaine use, stress may create a biological context within which other triggers for drug use become more potent. We hypothesize that stress-induced increases in corticosterone "set the stage" for relapse by promoting endocannabinoid-induced attenuation of inhibitory transmission in the prelimbic cortex (PL). METHODS: We have established a rat model for these stage-setting effects of stress. In this model, neither a stressor (electric footshock) nor stress-level corticosterone treatment alone reinstates cocaine seeking following self-administration and extinction, but each treatment potentiates reinstatement in response to an otherwise subthreshold cocaine priming dose (2.5 mg/kg, intraperitoneal). The contributions of endocannabinoid signaling in the PL to the effects of stress-level corticosterone on PL neurotransmission and cocaine seeking were determined using intra-PL microinfusions. Endocannabinoid-dependent effects of corticosterone on inhibitory synaptic transmission in the rat PL were determined using whole-cell recordings in layer V pyramidal neurons. RESULTS: Corticosterone application attenuated inhibitory synaptic transmission in the PL via cannabinoid receptor type 1 (CB1R)- and 2-arachidonoylglycerol-dependent inhibition of gamma-aminobutyric acid release without altering postsynaptic responses. The ability of systemic stress-level corticosterone treatment to potentiate cocaine-primed reinstatement was recapitulated by intra-PL injection of corticosterone, the CB1R agonist WIN 55,212-2, or the monoacylglycerol lipase inhibitor URB602. Corticosterone effects on reinstatement were attenuated by intra-PL injections of either the CB1R antagonist, AM251, or the diacylglycerol lipase inhibitor, DO34. CONCLUSIONS: These findings suggest that stress-induced increases in corticosterone promote cocaine seeking by mobilizing 2-arachidonoylglycerol in the PL, resulting in CB1R-mediated attenuation of inhibitory transmission in this brain region.

2-Arachidonoylglycerol and anandamide are the main endocannabinoids, which act through cannabinoid type-1 and type-2 receptors. Among its many functions, anandamide modulates anxiety-like behaviors in the ventromedial prefrontal cortex. The role of 2-arachidonoylglycerol in this region, however, has remained unclear. Here, we verified whether intra- ventromedial prefrontal cortex injection of 2-arachidonoylglycerol or URB602, a monoacylglycerol lipase inhibitor (responsible for 2-arachidonoylglycerol hydrolysis), induce anxiolytic-like effects in Wistar rats. Since activation of metabotropic glutamate receptor type 5 promotes diacylglycerol lipase-alpha-mediated 2-arachidonoylglycerol synthesis, we also verified if the blockade of this receptor impairs the anxiolytic-like effect induced by URB 602. 2-Arachidonoylglycerol reduced anxiety-like response in rats exposed to the Elevated Plus Maze test, an effect mimicked by URB602. Cannabinoid type-1 and type-2 receptor antagonists prevented these effects. The pre-treatment with an ineffective dose of MPEP, a metabotropic glutamate receptor type 5 antagonist, also attenuated the anxiolytic-like effect of URB602. Moreover, immunofluorescence microscopy revealed co-expression of metabotropic glutamate receptor type 5 and diacylglycerol lipase-alpha in several neurons in slices from the ventromedial prefrontal cortex. Altogether, our results implicate 2-arachidonoylglycerol and both cannabinoid receptors on anxiety-related behaviors mediated by ventromedial prefrontal cortex. Further, these data support a role for the coupling between metabotropic glutamate receptor type 5 activation and 2-arachidonoylglycerol signalling as a mechanism modulating aversive responses.

Background Drugs that modulate endocannabinoid signalling are effective in reducing nociception in animal models of pain and may be of value in the treatment of migraine. Methods We investigated the anti-nociceptive effects of inhibition of monoacylglycerol lipase (MGL), a key enzyme in the hydrolysis of the 2-arachidonoylglycerol, in a rat model of migraine based on nitroglycerin (NTG) administration. We evaluated c-fos expression in specific brain areas and nociceptive behavior in trigeminal and extra-trigeminal body areas. Results URB602, a reversible MGL inhibitor, did not show any analgesic effect in the tail flick test, but it inhibited NTG-induced hyperalgesia in both the tail flick test and the formalin test applied to the hind paw or to the orofacial area. Quite unexpectedly, URB602 potentiated formalin-induced hyperalgesia in the trigeminal area when used alone. The latter result was also confirmed using a structurally distinct, irreversible MGL inhibitor, JZL184. URB602 did not induce neuronal activation in the area of interest, but significantly reduced the NTG-induced neuronal activation in the ventrolateral column of the periaqueductal grey and the nucleus trigeminalis caudalis. Conclusions These findings support the hypothesis that modulation of the endocannabinoid system may be a valuable approach for the treatment of migraine. The topographically segregated effect of MGL inhibition in trigeminal/extra-trigeminal areas calls for further mechanistic research.

Colorectal cancer (CRC) is a major health problem in Western countries. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) exerts antiproliferative actions in a number of tumoral cell lines, including CRC cells. Monoacylglycerol lipase (MAGL), a serine hydrolase that inactivates 2-AG, is highly expressed in aggressive human cancer cells. Here, we investigated the role of MAGL in experimental colon carcinogenesis. The role of MAGL was assessed in vivo by using the xenograft and the azoxymethane models of colon carcinogenesis; MAGL expression was evaluated by RT-PCR and immunohistochemistry; 2-AG levels were measured by liquid chromatography mass spectrometry; angiogenesis was evaluated in tumor tissues [by microvessel counting and by investigating the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) proteins] as well as in human umbilical vein endothelial cells (HUVEC); cyclin D1 was evaluated by RT-PCR. MAGL and 2-AG were strongly expressed in tumor tissues. The MAGL inhibitor URB602 reduced xenograft tumor volume, this effect being associated to down-regulation of VEGF and FGF-2, reduction in the number of vessels and down-regulation of cyclin D1. In HUVEC, URB602 exerted a direct antiangiogenic effect by inhibiting FGF-2 induced proliferation and migration, and by modulating pro/anti-angiogenic agents. In experiments aiming at investigating the role of MAGL in chemoprevention, URB602 attenuated azoxymethane-induced preneoplastic lesions, polyps and tumors. MAGL, possibly through modulation of angiogenesis, plays a pivotal role in experimental colon carcinogenesis. Pharmacological inhibition of MAGL could represent an innovative therapeutic approach to reduce colorectal tumor progression.

Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured beta-cells and islets. In this report we have determined effects of increasing intracellular MAG in the beta-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca2+ to glucose and depolarization but to a lesser degree suggesting that altered Ca2+ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.

Monoglyceride lipase (MGL), the main enzyme responsible for the hydrolytic deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG), is an intracellular serine hydrolase that plays critical roles in many physiological and pathological processes, such as pain, inflammation, neuroprotection and cancer. The crystal structures of MGL that are currently available provide valuable information about how this enzyme might function and interact with site-directed small-molecule inhibitors. On the other hand, its conformational equilibria and the contribution of regulatory cysteine residues present within the substrate-binding pocket or on protein surface remain open issues. Several classes of MGL inhibitors have been developed, from early reversible ones, such as URB602 and pristimerin, to carbamoylating agents that react with the catalytic serine, such as JZL184 and more recent O-hexafluoroisopropyl carbamates. Other inhibitors that modulate MGL activity by interacting with conserved regulatory cysteines act through mechanisms that deserve to be more thoroughly investigated.

AIMS: URB602 is a selective inhibitor of monoacylglycerol lipase (MAGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl glycerol (2-AG). It has been described that URB602 significantly enhances depolarization-induced increases in 2-AG. A high level of homocysteine (Hcy) is a modifiable risk factor for developing Alzheimer's disease (AD). The aim of this study was to investigate the protective effects of URB602 on Hcy-induced impairments underlying its cellular and molecular mechanism in primary cultured caudate nucleus (CN) neurons. MAIN
METHODS: The expressions of cyclooxygenase-2 (COX-2), ERK1/2, NF-kappaB and IkappaB-alpha as well as cleaved caspase-3 and p-Bcl-2 in Hcy-, URB602 or SR1 (a selective inhibitor of CB1 receptor)-treated primary cultured neurons in CN were measured by immunoblotting technique and neurotoxicity assays were performed by using Hoechst staining. KEY FINDINGS: The MAGL inhibitor URB602 exerted a neuroprotective effect on Hcy-induced impairment through suppression of cyclooxygenase-2 (COX-2) elevation and ERK1/2 and NF-kappaB phosphorylation as well as suppressions of IkappaB-alpha degradation in a CB1 receptor-dependent way. Moreover, anti-neuronal impairments of URB602 were mediated by modulating down-regulation of cleaved caspase-3 expression and up-regulation of p-Bcl-2 expression in a CB1 receptor-dependent manner in primary cultured CN neurons. SIGNIFICANCE: These data suggest that the MAGL inhibitor is a promising therapeutic target for some neurodegenerative disorders, such as AD, via the COX-2 signaling pathway.

Human monoacylglycerol lipase (MAGL), a soluble serine hydrolase that belongs to the alpha/beta hydrolase fold superfamily, regulates 2-arachidonoyl glycerol level in the endocannabinoid system, which is implicated in a number of severe diseases, and therefore, inhibition of MAGL activity is crucial in the treatment of these diseases. We have synthesized a red fluorogenic substrate, 7-hydroxyresorufinyl-arachidonate (7-HRA), for a new MAGL assay. This assay is simple, sensitive, and reliable and useful for identifying compounds that modulate MAGL activity. In addition, the assay emits red fluorescence, which can significantly reduce interference due to compound fluorescence and dust or lint, all of which fluoresce in the blue wavelength. MAGL catalyzes the hydrolysis of 7-HRA to generate arachidonic acid and a highly red fluorescent resorufin, excitation at 571 nm and emission at 588 nm, with a Km of 0.87 muM and Vmax of 26 nmol min(-1) mg protein(-1). The known MAGL inhibitors URB602, methyl arachidonyl fluorophosphonate, and JZL184 were used to validate the test assay. The assay was highly reproducible with an overall average Z' value of 0.80. This new red fluorogenic substrate and the resulting enzyme assay could be used in high-throughput screening to identify and develop new potential MAGL inhibitors.

BACKGROUND: The endocannabinoids are emerging as natural brain protective substances that exert potentially beneficial effects in several neurological disorders by virtue of their hypothermic, immunomodulatory, vascular, antioxidant, and antiapoptotic actions. This study was undertaken to assess whether preventing the deactivation of the endocannabinoid 2-arachidonoylglycerol (2-AG) with the monoacylglycerol lipase (MAGL) inhibitor URB602 can provide neuroprotective effects in hypoxia-ischemia (HI)-induced brain injury. METHODS: URB602 was administered into the right lateral ventricle 30min before 7-day-old pup rats were subjected to HI. The neuroprotective effect was evaluated on postnatal day (PN) 14 or at adulthood (PN80) using behavioral and histological analyses. Activated caspase-3 expression and propidium iodide labeling were assessed as indexes of apoptotic and necrotic cell death, respectively. RESULTS: Pretreatment with URB602 reduced apoptotic and necrotic cell death, as well as the infarct volume measured at PN14. At adulthood, URB602-treated HI animals performed better at the T-maze and the Morris maze, and also showed a significant reduction of brain damage. CONCLUSION: These results demonstrate that a pretreatment with URB602 significantly reduces brain damage and improves functional outcome, indicating that endocannabinoid-degrading enzymes may represent an important target for neuroprotection in neonatal ischemic brain injury.

The Transfersome(R) is a lipid vesicle that contains membrane softeners, such as Tween 80, to make it ultra-deformable. This feature makes the Transfersome(R) an efficient carrier for delivery of therapeutic drugs across the skin barrier. It was reported that TDT 067 (a topical formulation of 15 mg/ml terbinafine in Transfersome(R) vesicles) has a much more potent antifungal activity in vitro compared with conventional terbinafine, which is a water-insoluble fungicide. Here we use ultra-structural studies and live imaging in a model fungus to describe the underlying mode of action. We show that terbinafine causes local collapse of the fungal endoplasmic reticulum, which was more efficient when terbinafine was delivered in Transfersome(R) vesicles (TFVs). When applied in liquid culture, fluorescently labeled TFVs rapidly entered the fungal cells (T(1/2)~2 min). Entry was F-actin- and ATP-independent, indicating that it is a passive process. Ultra-structural studies showed that passage through the cell wall involves significant deformation of the vesicles, and depends on a high concentration of the surfactant Tween 80 in their membrane. Surprisingly, the TFVs collapsed into lipid droplets after entry into the cell and the terbinafine was released from their interior. With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs. Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome(R) degradation and neutralized the cytotoxic effect of Transfersome(R)-delivered terbinafine. These data suggest that (a) Transfersomes deliver the lipophilic fungicide Terbinafine to the fungal cell wall, (b) the membrane softener Tween 80 allows the passage of the Transfersomes into the fungal cell, and (c) fungal lipases digest the invading Transfersome(R) vesicles thereby releasing their cytotoxic content. As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.

The present experiments employed in vivo microdialysis to characterize the effects of commonly used endocannabinoid clearance inhibitors on basal and depolarization-induced alterations in interstitial endocannabinoid levels in the nucleus accumbens of rat brain. Compounds targeting the putative endocannabinoid transporter and hydrolytic enzymes (FAAH and MAGL) were compared. The transporter inhibitor AM404 modestly enhanced depolarization-induced increases in 2-arachidonoyl glycerol (2-AG) levels but did not alter levels of N-arachidonoyl-ethanolamide (anandamide, AEA). The transport inhibitor UCM707 did not alter dialysate levels of either endocannabinoid. The FAAH inhibitors URB597 and PF-3845 robustly increased AEA levels during depolarization without altering 2-AG levels. The MAGL inhibitor URB602 significantly enhanced depolarization-induced increases in 2-AG, but did not alter AEA levels. In contrast, the MAGL inhibitor JZL184 did not alter 2-AG or AEA levels under any condition tested. Finally, the dual FAAH/MAGL inhibitor JZL195 significantly enhanced depolarization-induced increases in both AEA and 2-AG levels. In contrast to the present observations in rats, prior work in mice has demonstrated a robust JZL184-induced enhancement of depolarization-induced increases in dialysate 2-AG. Thus, to further investigate species differences, additional tests with JZL184, PF-3845, and JZL195 were performed in mice. Consistent with prior reports, JZL184 significantly enhanced depolarization-induced increases in 2-AG without altering AEA levels. PF-3845 and JZL195 produced profiles in mouse dialysates comparable to those observed in rats. These findings confirm that interstitial endocannabinoid levels in the brain can be selectively manipulated by endocannabinoid clearance inhibitors. While PF-3845 and JZL195 produce similar effects in both rats and mice, substantial species differences in JZL184 efficacy are evident, which is consistent with previous studies.

While endocannabinoid modulation of both GABAergic and glutamatergic synaptic transmission and plasticity has been extensively investigated, our understanding of the role of endocannabinoids in protecting neurons from harmful insults remains limited. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous ligand and a full agonist for cannabinoid receptors, exhibits anti-inflammatory and neuroprotective effects via a CB1 receptor (CB1R)-mediated mechanism. However, it is still not clear whether 2-AG is also able to protect neurons from beta-amyloid (Abeta)-induced neurodegeneration. Here, we demonstrate that exogenous application of 2-AG significantly protected hippocampal neurons in culture against Abeta-induced neurodegeneration and apoptosis. This neuroprotective effect was blocked by SR141716 (SR-1), a selective CB1R antagonist, but not by SR144528 (SR-2), a selective CB2R antagonist, or capsazepine (CAP), a selective transient receptor potential cation channels, subfamily V, member 1 (TRPV1) receptor antagonist. To determine whether endogenous 2-AG is capable of protecting neurons from Abeta insults, hippocampal neurons in culture were treated with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), the enzyme hydrolyzing 2-AG. MAGL inhibition that elevates endogenous levels of 2-AG also significantly reduced Abeta-induced neurodegeneration and apoptosis. The 2-AG-produced neuroprotective effects appear to be mediated via CB1R-dependent suppression of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and nuclear factor-kappaB (NF-kappaB) phosphorylation and cyclooxygenase-2 (COX-2) expression. Our results suggest that elevation of endogenous 2-AG by inhibiting its hydrolysis has potential as a novel efficacious therapeutic approach for preventing, ameliorating or treating Alzheimer's disease.

BACKGROUND AND PURPOSE The endocannabinoid 2-arachidonoylglycerol (2-AG) is degraded primarily by monoacylglycerol lipase (MGL). We compared peripheral antinociceptive effects of JZL184, a novel irreversible MGL inhibitor, with the reversible MGL-preferring inhibitor URB602 and exogenous 2-AG in rats. EXPERIMENTAL APPROACH Nociception in the formalin test was assessed in groups receiving dorsal paw injections of vehicle, JZL184 (0.001-300 microg), URB602 (0.001-600 microg), 2-AG (ED(50)), 2-AG + JZL184 (at their ED(50)), 2-AG + URB602 (at their ED(50)), AM251 (80 microg), AM251 + JZL184 (10 microg), AM630 (25 microg) or AM630 + JZL184 (10 microg). Effects of MGL inhibitors on endocannabinoid accumulation and on activities of endocannabinoid-metabolizing enzymes were assessed. KEY RESULTS Intra-paw administration of JZL184, URB602 and 2-AG suppressed early and late phases of formalin pain. JZL184 and URB602 acted through a common mechanism. JZL184 (ED(50) Phase 1: 0.06 +/- 0.028; Phase 2: 0.03 +/- 0.011 microg) produced greater antinociception than URB602 (ED(50) Phase 1: 120 +/- 51.3; Phase 2: 66 +/- 23.9 microg) or 2-AG. Both MGL inhibitors produced additive antinociceptive effects when combined with 2-AG. Antinociceptive effects of JZL184, like those of URB602, were blocked by cannabinoid receptor 1 (CB(1)) and cannabinoid receptor 2 (CB(2)) antagonists. JZL184 suppressed MGL but not fatty-acid amide hydrolase or N-arachidonoyl-phosphatidylethanolamine phospholipase D activities ex vivo. URB602 increased hind paw 2-AG without altering anandamide levels. CONCLUSIONS AND IMPLICATIONS MGL inhibitors suppressed formalin-induced pain through peripheral CB(1) and CB(2) receptor mechanisms. MGL inhibition increased paw skin 2-AG accumulation to mediate these effects. MGL represents a target for the treatment of inflammatory pain.

We have synthesised an extensive series of URB602 analogues as inhibitors of monoacylglycerol lipase (MAGL), which is the major enzyme responsible for metabolising the endocannabinoid 2-arachidonylglycerol. The recently identified crystal structure of MAGL was used in the design strategy and revealed three possible binding sites for URB602 and the proposed analogues. A test series of carbamate analogues were docked into the identified sites to predict the most favourable binding location. The synthesised analogues of URB602 explored the biological effects of isosteric replacement, ring size and substitution, para substitution of the biphenyl moiety and the incorporation of a bicyclic element. The compounds were tested for their ability to inhibit human MAGL. The carbamate analogue 16 displayed the most significant inhibitory activity, reducing MAGL activity to 26% of controls at 100 muM compared to 73% for the parent compound URB602.

The comparative effects of atropine and the indirect cannabinomimetics URB597 (a fatty acid amide hydrolase inhibitor) and URB602 (a monoacylglycerol lipase inhibitor) on functional and neurobehavioral endpoints following acute diisopropylfluorophosphate intoxication were studied. Male Sprague-Dawley rats were treated with vehicle or DFP (2.5mg/kg, sc), immediately post-treated with either vehicle, atropine (16mg/kg), URB597 (3mg/kg), URB602 (10mg/kg) or a combination of URB597 and URB602, and functional signs of toxicity as well as nocturnal motor activity were measured daily for seven consecutive days. Performance in the elevated plus maze (for anxiety-like behavior) and the forced swimming test (for depression-like behavior) was measured at days 6-8 and 27-29 after dosing. Twenty-four hours after dosing, DFP markedly reduced cholinesterase activity in selected brain regions and peripheral tissues (diaphragm and plasma). Substantial recovery of cholinesterase activity was noted at both 8 and 29days after dosing but significant inhibition was still noted in some brain regions at the latest time-point. DFP elicited body weight reductions and typical signs of cholinergic toxicity, and reduced nocturnal ambulation and rearing. Atropine and the cannabinomimetics (alone and in combination) partially attenuated DFP-induced functional signs of toxicity. None of the post-treatments reversed the DFP-induced reduction in ambulation or rearing, however. No significant treatment-related effects on elevated plus maze performance were noted. DFP-treated rats exhibited decreased swimming and increased immobility in the forced swimming test at both time-points. None of the post-treatments had any effect on DFP-induced changes in immobility or swimming at day 8. At day 29, atropine and the combination of URB597/URB602 significantly blocked DFP-induced changes in immobility, while URB597 and the combination reversed DFP-induced changes in swimming. The results suggest that early blockade of muscarinic receptors and enhancement of eCB signaling can attenuate both acute and delayed effects elicited by DFP.

The endogenous cannabinoid system plays an important role in the regulation of gastrointestinal function in health and disease. Endocannabinoid levels are regulated by catabolic enzymes. Here, we describe the presence and localization of monoacylglycerol lipase (MGL), the major enzyme responsible for the degradation of 2-arachidonoylglycerol. We used molecular, biochemical, immunohistochemical, and functional assays to characterize the distribution and activity of MGL. MGL mRNA was present in rat ileum throughout the wall of the gut. MGL protein was distributed in the muscle and mucosal layers of the ileum and in the duodenum, proximal colon, and distal colon. We observed MGL expression in nerve cell bodies and nerve fibers of the enteric nervous system. There was extensive colocalization of MGL with PGP 9.5 and calretinin-immunoreactive neurons, but not with nitric oxide synthase. MGL was also present in the epithelium and was highly expressed in the small intestine. Enzyme activity levels were highest in the duodenum and decreased along the gut with lowest levels in the distal colon. We observed both soluble and membrane-associated enzyme activities. The MGL inhibitor URB602 significantly inhibited whole gut transit in mice, an action that was abolished in cannabinoid 1 receptor-deficient mice. In conclusion, MGL is localized in the enteric nervous system where endocannabinoids regulate intestinal motility. MGL is highly expressed in the epithelium, where this enzyme may have digestive or other functions yet to be determined.

Diisopropylfluorophosphate (DFP) elicits cholinergic toxicity by inhibiting acetylcholinesterase, leading to accumulation of the neurotransmitter acetylcholine and excessive stimulation of cholinergic receptors throughout the body. Endocannabinoids inhibit the release of neurotransmitters including acetylcholine via a widely distributed retrograde signaling pathway. Endocannabinoid signaling is therefore a potential therapeutic target for the management of OP poisoning. We first evaluated the relative in vitro and in vivo (2.5mg/kg, sc) effects of DFP on cholinesterase, fatty acid amide hydrolase (FAAH, an endocannabinoid degrading enzyme), monoacylglycerol lipase (MAGL, another endocannabinoid degrading enzyme) and cannabinoid receptor (CB1) binding in rat hippocampus. The effects of WIN 55212-2 (cannabinoid receptor agonist, 1.5mg/kg), URB597 (FAAH inhibitor, 3mg/kg), URB602 (MAGL inhibitor, 10mg/kg) or AM404 (endocannabinoid uptake inhibitor, 10mg/kg) on DFP toxicity were then examined. Adult male rats were given either peanut oil or DFP followed immediately by vehicle or one of the four cannabinomimetic drugs. Functional signs of toxicity were evaluated for 24h and then rats were sacrificed for neurochemical measurements. DFP inhibited cholinesterase, FAAH, MAGL and CB1 receptor binding in vitro in a concentration-dependent manner, with highest and lowest potency against cholinesterase and FAAH, respectively. In vivo, DFP inhibited hippocampal cholinesterase (89%) and FAAH (42%), but had no significant effect on MAGL or CB1 binding. Rats treated with DFP alone showed typical signs of cholinergic toxicity including involuntary movements and excessive secretions (SLUD signs). WIN 55212-2, URB597, URB602 and AM404 all significantly reduced involuntary movements following DFP exposure in a time-dependent manner, and most (URB597, URB602 and AM404) also significantly reduced DFP-induced SLUD signs. These results suggest that enhancing endocannabinoid signaling can attenuate the acute toxicity of DFP and provide rationale for further investigations on the role of endocannabinoids in cholinergic toxicity.

A novel fluorescence-based assay of monoacylglycerol lipase (MAGL) activity that is simple, sensitive, and amenable to the screening of small molecule inhibitors is described. Purified recombinant human MAGL protein and 7-hydroxycoumarinyl-arachidonate (7-HCA), a fluorogenic substrate for MAGL, were employed in the assay. MAGL protein catalyzes the hydrolysis of 7-HCA to generate arachidonic acid and the highly fluorescent 7-hydroxyl coumarin (7-HC). Release of 7-HC was measured using a fluorometer. MAGL protein catalyzed the hydrolysis of 7-HCA with an apparent K(m) of 9.8 microM and V(max) of 1.7 mmol/min/mg of protein. The assay is specific for MAGL as assay buffer alone or heat-denatured MAGL protein had no significant activity against 7-HCA. Furthermore, MAGL activity was inhibited in a dose-dependent manner by the specific inhibitor URB602 as well as N-arachidonyl maleimide with 50% inhibitory concentration values of 3.1 microM and 155 nM, respectively. The assay was further optimized under different conditions, including pH range and bovine serum albumin protein and dimethyl sulfoxide concentrations. The assay was found to be reproducible, having Z' values ranging from 0.7 to 0.9, and is therefore suitable for high-throughput screening.

BACKGROUND AND PURPOSE: 2-arachidonoylglycerol (2-AG) is an endocannabinoid whose hydrolysis is predominantly catalysed by the enzyme monoacylglycerol lipase (MAGL). The development of MAGL inhibitors could offer an opportunity to investigate the anti-inflammatory and anti-nociceptive role of 2-AG, which have not yet been elucidated. On these bases, URB602, a MAGL inhibitor, was tested in a murine model of inflammation/inflammatory pain. EXPERIMENTAL APPROACH: Acute inflammation was induced by intraplantar injection of lambda-carrageenan into mice. The highest dose to be employed has been selected performing the tetrad assays for cannabimimetic activity in mice. URB602 anti-inflammatory and anti-nociceptive efficacy (assessed by plethysmometer and plantar test, respectively) was evaluated both in a preventive regimen (drug administered 30 min before carrageenan) and in a therapeutic regimen (URB602 administered 30 min after carrageenan). To elucidate the cannabinoid receptor involvement, rimonabant and SR144528, CB1 and CB2 selective antagonists, respectively, were given 15 min before URB602. KEY
RESULTS: Systemic administration of URB602 elicited a dose-dependent anti-oedemigen and anti-nociceptive effect that was reversed exclusively by the CB2 receptor antagonist. The efficacy of URB602 persisted also when the compound was administered in a therapeutic regimen, suggesting the ability of URB602 to improve established disease. CONCLUSIONS AND IMPLICATIONS: The present report highlighted the ability of the selective MAGL inhibitor, URB602, to prevent and treat an acute inflammatory disease without producing adverse psychoactive effects. The data presented herein also contributed to clarify the physiological role of 2-AG in respect to inflammatory reactions, suggesting its protective role in the body.

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid (endocannabinoid) lipid whose functions remain poorly understood. Guindon and colleagues report the novel finding that exogenous application of 2-AG induces peripheral antinociceptive effects that are mediated, at least in part, by actions at peripheral cannabinoid CB(2) receptors. URB602, a recently described inhibitor of monoacylglycerol lipase, an enzyme that catalyzes 2-AG hydrolysis in vivo, also induced peripheral antinociceptive effects and enhanced the actions of 2-AG. Peripheral analgesic mechanisms represent promising therapeutic targets for suppressing pain in the absence of unwanted central nervous system side-effects (e.g. psychoactivity) associated with activation of central CB(1) receptors. The therapeutic potential of inhibitors of 2-AG deactivation for the treatment of inflammatory pain is discussed.

The N-aryl carbamate URB602 (biphenyl-3-ylcarbamic acid cyclohexyl ester) is an inhibitor of monoacylglycerol lipase (MGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Here, we investigated the mechanism by which URB602 inhibits purified recombinant rat MGL by using a combination of biochemical and structure-activity relationship (SAR) approaches. We found that URB602 weakly inhibits recombinant MGL (IC(50) = 223 +/- 63 microM) through a rapid and noncompetitive mechanism. Dialysis experiments and SAR analyses suggest that URB602 acts through a partially reversible mechanism rather than by irreversible carbamoylation of MGL. Finally, URB602 (100 microM) elevates 2-AG levels in hippocampal slice cultures without affecting levels of other endocannabinoid-related substances. Thus, URB602 may provide a useful tool by which to investigate the physiological roles of 2-AG and explore the potential interest of MGL as a therapeutic target.

The endocannabinoids (eCBs) anandamide and 2-arachidonoyl glycerol (2-AG) are inactivated by a two-step mechanism. First, they are carried into cells, and then anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). Here we provide evidence for a previously undescribed MGL activity expressed by microglial cells. We found that the mouse microglial cell line BV-2 does not express MGL mRNA and yet efficiently hydrolyzes 2-AG. URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) reduces this hydrolysis by 50%, suggesting the involvement of FAAH. The remaining activity is blocked by classic MGL inhibitors [[1,1-biphenyl]-3-yl-carbamic acid, cyclohexyl ester (URB602) and MAFP (methylarachidonyl fluorophosphate)] and is unaffected by inhibitors of COXs (cyclooxygenases), LOXs (lipooxygenases), and DGLs (diacylglycerol lipases), indicating the involvement of a novel MGL activity. Accordingly, URB602 leads to selective accumulation of 2-AG in intact BV-2 cells. Although MGL expressed in neurons is equally distributed between the cytosolic, mitochondrial, and nuclear fractions, the novel MGL activity expressed by BV-2 cells is enriched in mitochondrial and nuclear fractions. A screen for novel inhibitors of eCB hydrolysis identified several compounds that differentially block MGL, FAAH, and the novel MGL activity. Finally, we provide evidence for expression of the novel MGL by mouse primary microglia in culture. Our results suggest the presence of a novel, pharmacologically distinct, MGL activity that controls 2-AG levels in microglia.

BACKGROUND AND PURPOSE: Two compounds, URB602 and URB754, have been reported in the literature to be selective inhibitors of monoacylglycerol lipase, although a recent study has questioned their ability to prevent 2-arachidonoyl hydrolysis by brain homogenates and cerebellar membranes. In the present study, the ability of these compounds to inhibit monoacylglycerol lipase and fatty acid amide hydrolase has been reinvestigated. EXPERIMENTAL APPROACH: Homogenates and cell lines were incubated with test compounds and, thereafter, with either [(3)H]-2-oleoylglycerol or [(3)H]-anandamide. Labelled reaction products were separated from substrate using chloroform: methanol extraction. KEY RESULTS: In cytosolic fractions from rat brain, URB602 and URB754 inhibited the hydrolysis of 2-oleoylglycerol with IC(50) values of 25 and 48 microM, respectively. Anandamide hydrolysis by brain membranes was not sensitive to URB754, but was inhibited by URB602 (IC(50) value 17 microM). Hydrolysis of 2-oleoylglycerol by human recombinant monoacylglycerol lipase was sensitive to URB602, but not URB754. The lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis was also observed for intact RBL2H3 basophilic leukaemia cells. C6 glioma expressed mRNA for monoacylglycerol lipase, and hydrolyzed 2-oleoylglycerol in a manner sensitive to inhibition by methyl arachidonoyl fluorophosphonate but not URB754 or URB597. MC3T3-E1 mouse osteoblastic cells, which did not express mRNA for monoacylglycerol lipase, hydrolyzed 2-oleoylglycerol in the presence of URB597, but the hydrolysis was less sensitive to methyl arachidonoyl fluorophosphonate than for C6 cells. CONCLUSIONS AND IMPLICATIONS: The data demonstrate that the compounds URB602 and URB754 do not behave as selective and/or potent inhibitors of monoacylglycerol lipase.