Phospholipase A₂ (PLA₂) enzymes are considered the primary source of arachidonic acid for cyclooxygenase (COX)-mediated biosynthesis of prostaglandins. Here, we show that a distinct pathway exists in ...brain, where monoacylglycerol Upase (MAGL) hydrolyzes the endocannabinoid 2-arachidonoylglycerol to generate a major arachidonate precursor pool for neuroinflammatory prostaglandins. MAGL-disrupted animals show neuroprotection in a parkinsonian mouse model. These animals are spared the hemorrhaging caused by COX inhibitors in the gut, where prostaglandins are instead regulated by cytosolic PLA₂ . These findings identify MAGL as a distinct metabolic node that couples endocannabinoid to prostaglandin signaling networks in the nervous system and suggest that inhibition of this enzyme may be a new and potentially safer way to suppress the proinflammatory cascades that underlie neurodegenerative disorders.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The serine hydrolase α/β hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and ...physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6’s role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.
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•ABHD6 inhibition reduces high-fat-diet-induced obesity/insulin resistance•ABHD6 inhibition protects against high-fat-diet-induced hepatic steatosis•ABHD6 is a critical regulator of hepatic de novo lipogenesis•ABHD6 is both a monoacylglycerol lipase and a lysophospholipase
Metabolic syndrome has become a leading health concern. Now, Brown and colleagues show that that serine hydrolase ABHD6 is a key driver of the metabolic syndrome and that inhibition of ABHD6 protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic and in vitro enzymology approaches, the authors show that ABHD6 is a promiscuous lipase that hydrolyzes monoacylglycerols and lysophospholipids and argue that ABHD6 inhibitors may hold therapeutic promise.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hydrolytic enzymes constitute one of the largest and most diverse protein classes in Nature and play key roles in nearly all physiological and pathological processes. The mammalian serine hydrolase ...superfamily contains a remarkable number of uncharacterized members, with at least 40−50% of these enzymes lacking experimentally verified endogenous substrates and products. Assignment of metabolic and cellular functions to these enzymes requires the development of pharmacological tools to selectively perturb their activity. We describe herein a functional proteomic strategy to systematically develop potent and selective inhibitors for uncharacterized serine hydrolases and its application to the brain-enriched enzyme α/β-hydrolase-6. We anticipate that the methods described herein will facilitate the development of selective chemical probes to annotate the metabolic and (patho)physiological functions of many of the uncharacterized serine hydrolases that currently populate eukaryotic and prokaryotic proteomes.
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IJS, KILJ, NUK, PNG, UL, UM
The nicotinamide adenine dinucleotide hydrolase (NADase) sterile alpha toll/interleukin receptor motif containing-1 (SARM1) acts as a central executioner of programmed axon death and is a possible ...therapeutic target for neurodegenerative disorders. While orthosteric inhibitors of SARM1 have been described, this multidomain enzyme is also subject to intricate forms of autoregulation, suggesting the potential for allosteric modes of inhibition. Previous studies have identified multiple cysteine residues that support SARM1 activation and catalysis, but which of these cysteines, if any, might be selectively targetable by electrophilic small molecules remains unknown. Here, we describe the chemical proteomic discovery of a series of tryptoline acrylamides that site-specifically and stereoselectively modify cysteine-311 (C311) in the noncatalytic, autoregulatory armadillo repeat (ARM) domain of SARM1. These covalent compounds inhibit the NADase activity of WT-SARM1, but not C311A or C311S SARM1 mutants, show a high degree of proteome-wide selectivity for SARM1_C311 and stereoselectively block vincristine- and vacor-induced neurite degeneration in primary rodent dorsal root ganglion neurons. Our findings describe selective, covalent inhibitors of SARM1 targeting an allosteric cysteine, pointing to a potentially attractive therapeutic strategy for axon degeneration-dependent forms of neurological disease.
Functional proteomic profiling reveals several brain hydrolase targets for endocannabinoid biosynthesis inhibitors.
The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key ...retrograde mediator in the nervous system based on pharmacological studies using inhibitors of the 2-AG biosynthetic enzymes diacyglycerol lipase α and β (DAGL-α/β). Here, we show by competitive activity-based protein profiling that the DAGL-α/β inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was observed for THL and RHC80267, suggesting that pharmacological effects observed with both agents may be viewed as good initial evidence for DAGL-dependent events.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The endogenous cannabinoid (endocannabinoid) anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH). Pharmacological blockade of FAAH has emerged as a ...potentially attractive strategy for augmenting endocannabinoid signaling and retaining the beneficial effects of cannabinoid receptor activation, while avoiding the undesirable side effects, such as weight gain and impairments in cognition and motor control, observed with direct cannabinoid receptor 1 agonists. Here, we report the detailed mechanistic and pharmacological characterization of N-pyridazin-3-yl-4-(3-{5-(trifluoromethyl)pyridin-2-yloxy}benzylidene)piperidine-1-carboxamide (PF-04457845), a highly efficacious and selective FAAH inhibitor. Mechanistic studies confirm that PF-04457845 is a time-dependent, covalent FAAH inhibitor that carbamylates FAAH's catalytic serine nucleophile. PF-04457845 inhibits human FAAH with high potency (k(inact)/K(i) = 40,300 M(-1)s(-1); IC(50) = 7.2 nM) and is exquisitely selective in vivo as determined by activity-based protein profiling. Oral administration of PF-04457845 produced potent antinociceptive effects in both inflammatory complete Freund's adjuvant (CFA) and noninflammatory (monosodium iodoacetate) pain models in rats, with a minimum effective dose of 0.1 mg/kg (CFA model). PF-04457845 displayed a long duration of action as a single oral administration at 1 mg/kg showed in vivo efficacy for 24 h with a concomitant near-complete inhibition of FAAH activity and maximal sustained elevation of anandamide in brain. Significantly, PF-04457845-treated mice at 10 mg/kg elicited no effect in motility, catalepsy, and body temperature. Based on its exceptional selectivity and in vivo efficacy, combined with long duration of action and optimal pharmacokinetic properties, PF-04457845 is a clinical candidate for the treatment of pain and other nervous system disorders.
Non‐Technical Summary 2‐Arachidonoylglycerol (2‐AG) is an endogenous marijuana‐like chemical that regulates synaptic transmission via the stimulation of the type I cannabinoid receptor (CB1). It is ...inactivated by an enzyme called monoacylglycerol lipase (MAGL). 2‐AG inactivation is impaired in MAGL knockout mice. We show that 2‐AG accumulation in the brain of MAGL knockout mice alters several forms of 2‐AG‐mediated synaptic depression in the cerebellum via tonic activation and desensitization of CB1 receptors.
The endocannabinoid (eCB) 2‐arachidonoylglycerol (2‐AG) is hydrolysed primarily by monoacylglycerol lipase (MAGL). Here, we investigated whether eCB‐mediated retrograde synaptic depression in cerebellar slices was altered in MAGL knockout (MAGL−/−) mice. Depolarization‐induced suppression of excitation (DSE) and metabotropic glutamate receptor (mGluR1)‐mediated synaptic depression are mediated by 2‐AG‐induced activation of CB1 receptors. We show that genetic deletion of MAGL prolonged DSE at parallel fibre (PF) or climbing fibre (CF) to Purkinje cell (PC) synapses. Likewise, mGluR1‐mediated synaptic depression, induced either by high‐frequency stimulation of PF or mGluR1 agonist DHPG, was prolonged in MAGL−/− mice. About 15% of 2‐AG in the brain is hydrolysed by serine hydrolase α‐β‐hydrolase domain 6 and 12 (ABHD6 and ABHD12). However, the selective ABHD6 inhibitor WWL123 had no significant effect on cerebellar DSE in MAGL+/+ and −/− mice. The CB1 receptor antagonist SR141716 significantly increased the amplitude of basal excitatory postsynaptic currents (EPSCs) in MAGL−/− mice but not in MAGL+/+ mice. Conversely, the CB1 agonist WIN55212 induced less depression of basal EPSCs in MAGL−/− mice than in MAGL+/+ mice. These results provide genetic evidence that inactivation of 2‐AG by MAGL determines the time course of eCB‐mediated retrograde synaptic depression and that genetic deletion of MAGL causes tonic activation and consequential desensitization of CB1 receptors.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Delta(9)-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana, has useful medicinal properties but also undesirable side effects. The brain receptor for THC, CB(1), is also activated ...by the endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG). Augmentation of endocannabinoid signaling by blockade of their metabolism may offer a more selective pharmacological approach compared with CB(1) agonists. Consistent with this premise, inhibitors of the anandamide-degrading enzyme fatty acid amide hydrolase (FAAH) produce analgesic and anxiolytic effects without cognitive defects. In contrast, we show that dual blockade of the endocannabinoid-degrading enzymes monoacylglycerol lipase (MAGL) and FAAH by selected organophosphorus agents leads to greater than ten-fold elevations in brain levels of both 2-AG and anandamide and to robust CB(1)-dependent behavioral effects that mirror those observed with CB(1) agonists. Arachidonic acid levels are decreased by the organophosphorus agents in amounts equivalent to elevations in 2-AG, which indicates that endocannabinoid and eicosanoid signaling pathways may be coordinately regulated in the brain.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
BACKGROUND AND PURPOSE Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non‐noxious stimuli are perceived as painful. One potential target ...to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB1 and CB2 cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with Δ9‐tetrahydrocannabinol (THC).
EXPERIMENTAL APPROACH Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS‐induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses.
KEY RESULTS FAAH (−/−) mice or wild‐type mice treated with FAAH inhibitors (URB597, OL‐135 and PF‐3845) displayed an anti‐allodynic phenotype. Furthermore, i.p. PF‐3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF‐3845 produced a partial reduction in allodynia. However, the anti‐allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti‐allodynic effects of FAAH‐compromised mice required activation of both CB1 and CB2 receptors, but other potential targets of FAA substrates (i.e. µ‐opioid, TRPV1 and PPARα receptors) had no apparent role.
CONCLUSIONS AND IMPLICATIONS AEA is the primary FAAH substrate reducing LPS‐induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain.
LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue‐8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue‐7
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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