Chemoproteomic identification of a DPP4 homolog in Bacteroides thetaiotaomicron.

Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

Attenuating ABHD17 enhances S- palmitoylation, membrane localization and signal transduction of NOD2 and Crohn's disease-associated variants.

NOD2 is an intracellular innate immune receptor that senses bacterial peptidoglycans. Although soluble in the cytosol, a portion of the protein is associated with the plasma membrane and endosomal compartments for microbial surveillance. Palmitoylation of NOD2 by zDHHC5 promotes its membrane recruitment to drive proinflammatory and antimicrobial responses to pathogenic invasion. A depalmitoylation step by an unknown protein, thioesterase, releases NOD2 from membranes into the cytosol, where the protein can then enter a new cycle of palmitoylation-depalmitoylation. Here, we identify α/ß -hydrolase domain-containing protein 17 isoforms (ABHD17A, 17B, 17C) as the thioesterases responsible for depalmitoylation of NOD2. Inhibiting ABHD17 increased the plasmalemmal localization of both wild-type NOD2 and a subset of hypo-palmitoylated Crohn's disease-associated variants, resulting in increased NF-κB activation and production of pro-inflammatory cytokines in epithelial cells. These results suggest that targeted inhibition of ABHD17 may rescue some Crohn's disease-associated NOD2 variants.

Inhibitory Neurotransmission Is Sex-Dependently Affected by Tat Expression in Transgenic Mice and Suppressed by the Fatty Acid Amide Hydrolase Enzyme Inhibitor PF3845 via Cannabinoid Type-1 Receptor Mechanisms.

(1) Background. The endocannabinoid (eCB) system, which regulates physiological and cognitive processes, presents a promising therapeutic target for treating HIV-associated neurocognitive disorders (HAND). Here we examine whether upregulating eCB tone has potential protective effects against HIV-1 Tat (a key HIV transactivator of transcription) protein-induced alterations in synaptic activity. (2) Methods. Whole-cell patch-clamp recordings were performed to assess inhibitory GABAergic neurotransmission in prefrontal cortex slices of Tat transgenic male and female mice, in the presence and absence of the fatty acid amide hydrolase (FAAH) enzyme inhibitor PF3845. Western blot and mass spectrometry analyses assessed alterations of cannabinoid receptor and enzyme protein expression as well as endogenous ligands, respectively, to determine the impact of Tat exposure on the eCB system. (3) Results. GABAergic activity was significantly altered upon Tat exposure based on sex, whereas the effectiveness of PF3845 to suppress GABAergic activity in Tat transgenic mice was not altered by Tat or sex and involved CB1R-related mechanisms that depended on calcium signaling. Additionally, our data indicated sex-dependent changes for AEA and related non-eCB lipids based on Tat induction. (4) Conclusion. Results highlight sex- and/or Tat-dependent alterations of GABAergic activity and eCB signaling in the prefrontal cortex of Tat transgenic mice and further increase our understanding about the role of FAAH inhibition in neuroHIV.

Monoacylglycerol Lipase Inhibitor MJN110 Reduces Neuronal Hyperexcitability, Restores Dendritic Arborization Complexity, and Regulates Reward-Related Behavior in Presence of HIV-1 Tat.

While current therapeutic strategies for people living with human immunodeficiency virus type 1 (HIV-1) suppress virus replication peripherally, viral proteins such as transactivator of transcription (Tat) enter the central nervous system early upon infection and contribute to chronic inflammatory conditions even alongside antiretroviral treatment. As demand grows for supplemental strategies to combat virus-associated pathology presenting frequently as HIV-associated neurocognitive disorders (HAND), the present study aimed to characterize the potential utility of inhibiting monoacylglycerol lipase (MAGL) activity to increase inhibitory activity at cannabinoid receptor-type 1 receptors through upregulation of 2-arachidonoylglycerol (2-AG) and downregulation of its degradation into proinflammatory metabolite arachidonic acid (AA). The MAGL inhibitor MJN110 significantly reduced intracellular calcium and increased dendritic branching complexity in Tat-treated primary frontal cortex neuron cultures. Chronic MJN110 administration in vivo increased 2-AG levels in the prefrontal cortex (PFC) and striatum across Tat(+) and Tat(-) groups and restored PFC N-arachidonoylethanolamine (AEA) levels in Tat(+) subjects. While Tat expression significantly increased rate of reward-related behavioral task acquisition in a novel discriminative stimulus learning and cognitive flexibility assay, MJN110 altered reversal acquisition specifically in Tat(+) mice to rates indistinguishable from Tat(-) controls. Collectively, our results suggest a neuroprotective role of MAGL inhibition in reducing neuronal hyperexcitability, restoring dendritic arborization complexity, and mitigating neurocognitive alterations driven by viral proteins associated with latent HIV-1 infection.

Toxoplasma gondii serine hydrolases regulate parasite lipid mobilization during growth and replication within the host.

The intracellular protozoan parasite Toxoplasma gondii must scavenge cholesterol and other lipids from the host to facilitate intracellular growth and replication. Enzymes responsible for neutral lipid synthesis have been identified but there is no evidence for enzymes that catalyze lipolysis of cholesterol esters and esterified lipids. Here, we characterize several T. gondii serine hydrolases with esterase and thioesterase activities that were previously thought to be depalmitoylating enzymes. We find they do not cleave palmitoyl thiol esters but rather hydrolyze short-chain lipid esters. Deletion of one of the hydrolases results in alterations in levels of multiple lipids species. We also identify small-molecule inhibitors of these hydrolases and show that treatment of parasites results in phenotypic defects reminiscent of parasites exposed to excess cholesterol or oleic acid. Together, these data characterize enzymes necessary for processing lipids critical for infection and highlight the potential for targeting parasite hydrolases for therapeutic applications.

ABHD17 regulation of plasma membrane palmitoylation and N-Ras-dependent cancer growth.

Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.

Single-Cell Profiling and SCOPE-Seq Reveal Lineage Dynamics of Adult Ventricular-Subventricular Zone Neurogenesis and NOTUM as a Key Regulator.

In the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum, which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny.

AIG1 and ADTRP are endogenous hydrolases of fatty acid esters of hydroxy fatty acids (FAHFAs) in mice.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS-based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9th carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.

Inhibitory Control Deficits Associated with Upregulation of CB1R in the HIV-1 Tat Transgenic Mouse Model of Hand.

In the era of combined antiretroviral therapy, HIV-1 infected individuals are living longer lives; however, longevity is met with an increasing number of HIV-1 associated neurocognitive disorders (HAND) diagnoses. The transactivator of transcription (Tat) is known to mediate the neurotoxic effects in HAND by acting directly on neurons and also indirectly via its actions on glia. The Go/No-Go (GNG) task was used to examine HAND in the Tat transgenic mouse model. The GNG task involves subjects discriminating between two stimuli sets in order to determine whether or not to inhibit a previously trained response. Data reveal inhibitory control deficits in female Tat(+) mice (p = .048) and an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group (p < .05). A significant negative correlation was noted between inhibitory control and IL CB1R expression (r = -.543, p = .045), with CB1R expression predicting 30% of the variance of inhibitory control (R2 = .295, p = .045). Furthermore, there was a significant increase in spontaneous excitatory postsynaptic current (sEPSC) frequencies in Tat(+) compared to Tat(-) mice (p = .008, across sexes). The increase in sEPSC frequency was significantly attenuated by bath application of PF3845, a fatty acid amide hydrolase (FAAH) enzyme inhibitor (p < .001). Overall, the GNG task is a viable measure to assess inhibitory control deficits in Tat transgenic mice and results suggest a potential therapeutic treatment for the observed deficits with drugs which modulate endocannabinoid enzyme activity. Graphical Abstract Results of the Go/No-Go operant conditioning task reveal inhibitory control deficits in female transgenic Tat(+) mice without significantly affecting males. The demonstrated inhibitory control deficits appear to be associated with an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group.

Quantification of In Vivo Target Engagement Using Microfluidic Activity-Based Protein Profiling.

Accurate measurement of drug-target interactions in vivo is critical for both preclinical development and translation to clinical studies, yet many assays rely on indirect measures such as biomarkers associated with target activity. Activity-based protein profiling (ABPP) is a direct method of quantifying enzyme activity using active site-targeted small-molecule covalent probes that selectively label active but not inhibitor-bound enzymes. Probe-labeled enzymes in complex proteomes are separated by polyacrylamide gel electrophoresis and quantified by fluorescence imaging. To accelerate workflows and avoid imaging artifacts that make conventional gels challenging to quantify, we adapted protocols for a commercial LabChip GXII microfluidic instrument to permit electrophoretic separation of probe-labeled proteins in tissue lysates and plasma, and quantification of fluorescence (probe/protein labeling ratio of 1:1). Electrophoretic separation on chips occurred in 40 s per sample, and instrument software automatically identified and quantified peaks, resulting in an overall time savings of 3-5 h per 96-well sample plate. Calculated percent inhibition was not significantly different between the two formats. Chip performance was consistent between chips and sample replicates. Conventional gel imaging was more sensitive but required five times higher sample volume than microfluidic chips. Microfluidic chips produced results comparable to those of gels but with much lower sample consumption, facilitating assay miniaturization for scarce biological samples. The time savings afforded by microfluidic electrophoresis and automatic quantification has allowed us to incorporate microfluidic ABPP early in the drug discovery workflow, enabling routine assessments of tissue distribution and engagement of targets and off-targets in vivo.

Monoacylglycerol Lipase Inhibition in Human and Rodent Systems Supports Clinical Evaluation of Endocannabinoid Modulators.

Monoacylglycerol lipase (MGLL) is the primary degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). The first MGLL inhibitors have recently entered clinical development for the treatment of neurologic disorders. To support this clinical path, we report the pharmacological characterization of the highly potent and selective MGLL inhibitor ABD-1970 [1,1,1,3,3,3-hexafluoropropan-2-yl 4-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-4-chlorobenzyl)piperazine-1-carboxylate]. We used ABD-1970 to confirm the role of MGLL in human systems and to define the relationship between MGLL target engagement, brain 2-AG concentrations, and efficacy. Because MGLL contributes to arachidonic acid metabolism in a subset of rodent tissues, we further used ABD-1970 to evaluate whether selective MGLL inhibition would affect prostanoid production in several human assays known to be sensitive to cyclooxygenase inhibitors. ABD-1970 robustly elevated brain 2-AG content and displayed antinociceptive and antipruritic activity in a battery of rodent models (ED50 values of 1-2 mg/kg). The antinociceptive effects of ABD-1970 were potentiated when combined with analgesic standards of care and occurred without overt cannabimimetic effects. ABD-1970 also blocked 2-AG hydrolysis in human brain tissue and elevated 2-AG content in human blood without affecting stimulated prostanoid production. These findings support the clinical development of MGLL inhibitors as a differentiated mechanism to treat pain and other neurologic disorders.

Neuroprotective effects of fatty acid amide hydrolase catabolic enzyme inhibition in a HIV-1 Tat model of neuroAIDS.

The HIV-1 transactivator of transcription (Tat) is a neurotoxin involved in the pathogenesis of HIV-1 associated neurocognitive disorders (HAND). The neurotoxic effects of Tat are mediated directly via AMPA/NMDA receptor activity and indirectly through neuroinflammatory signaling in glia. Emerging strategies in the development of neuroprotective agents involve the modulation of the endocannabinoid system. A major endocannabinoid, anandamide (N-arachidonoylethanolamine, AEA), is metabolized by fatty acid amide hydrolase (FAAH). Here we demonstrate using a murine prefrontal cortex primary culture model that the inhibition of FAAH, using PF3845, attenuates Tat-mediated increases in intracellular calcium, neuronal death, and dendritic degeneration via cannabinoid receptors (CB1R and CB2R). Live cell imaging was used to assess Tat-mediated increases in [Ca2+]i, which was significantly reduced by PF3845. A time-lapse assay revealed that Tat potentiates cell death while PF3845 blocks this effect. Additionally PF3845 blocked the Tat-mediated increase in activated caspase-3 (apoptotic marker) positive neurons. Dendritic degeneration was characterized by analyzing stained dendritic processes using Imaris and Tat was found to significantly decrease the size of processes while PF3845 inhibited this effect. Incubation with CB1R and CB2R antagonists (SR141716A and AM630) revealed that PF3845-mediated calcium effects were dependent on CB1R, while reduced neuronal death and degeneration was CB2R-mediated. PF3845 application led to increased levels of AEA, suggesting the observed effects are likely a result of increased endocannabinoid signaling at CB1R/CB2R. Our findings suggest that modulation of the endogenous cannabinoid system through inhibition of FAAH may be beneficial in treatment of HAND.

Monoacylglycerol Lipase Inhibitors Reverse Paclitaxel-Induced Nociceptive Behavior and Proinflammatory Markers in a Mouse Model of Chemotherapy-Induced Neuropathy.

Although paclitaxel effectively treats various cancers, its debilitating peripheral neuropathic pain side effects often persist long after treatment has ended. Therefore, a compelling need exists for the identification of novel pharmacologic strategies to mitigate this condition. As inhibitors of monoacylglycerol lipase (MAGL), the primary hydrolytic enzyme of the endogenous cannabinoid, 2-arachidonyolglycerol, produces antinociceptive effects in numerous rodent models of pain, we investigated whether inhibitors of this enzyme (i.e., JZL184 and MJN110) would reverse paclitaxel-induced mechanical allodynia in mice. These drugs dose dependently reversed allodynia with respective ED50 values (95% confidence limit) of 8.4 (5.2-13.6) and 1.8 (1.0-3.3) mg/kg. Complementary genetic and pharmacologic approaches revealed that the antiallodynic effects of each drug require both cannabinoid receptors, CB1 and CB2 MJN110 reduced paclitaxel-mediated increased expression of monocyte chemoattractant protein-1 (MCP-1, CCL2) and phospho-p38 MAPK in dorsal root ganglia as well as MCP-1 in spinal dorsal horn. Whereas the antinociceptive effects of high dose JZL184 (40 mg/kg) underwent tolerance following 6 days of repeated dosing, repeated administration of a threshold dose (i.e., 4 mg/kg) completely reversed paclitaxel-induced allodynia. In addition, we found that the administration of MJN110 to control mice lacked intrinsic rewarding effects in the conditioned place preference (CPP) paradigm. However, it produced a CPP in paclitaxel-treated animals, suggesting a reduced paclitaxel-induced aversive state. Importantly, JZL184 did not alter the antiproliferative and apoptotic effects of paclitaxel in A549 and H460 non-small cell lung cancer cells. Taken together, these data indicate that MAGL inhibitors reverse paclitaxel-induced neuropathic pain without interfering with chemotherapeutic efficacy.

Inhibition of the endocannabinoid-regulating enzyme monoacylglycerol lipase elicits a CB1 receptor-mediated discriminative stimulus in mice.

Substantial challenges exist for investigating the cannabinoid receptor type 1 (CB1)-mediated discriminative stimulus effects of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA), compared with exogenous CB1 receptor agonists, such as Δ9-tetrahydrocannabinol (THC) and the synthetic cannabinoid CP55,940. Specifically, each endocannabinoid is rapidly degraded by the respective hydrolytic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Whereas MAGL inhibitors partially substitute for THC and fully substitute for CP55,940, FAAH inhibitors do not substitute for either drug. Interestingly, combined FAAH-MAGL inhibition results in full THC substitution, and the dual FAAH-MAGL inhibitor SA-57 serves as its own discriminative training stimulus. Because MAGL inhibitors fully substitute for SA-57, we tested whether the selective MAGL inhibitor MJN110 would serve as a training stimulus. Twelve of 13 C57BL/6J mice learned to discriminate MJN110 from vehicle, and the CB1 receptor antagonist rimonabant dose-dependently blocked its discriminative stimulus. CP55,940, SA-57, and another MAGL inhibitor JZL184, fully substituted for MJN110. In contrast, the FAAH inhibitor PF-3845 failed to substitute for the MJN110 discriminative stimulus, but produced a 1.6 (1.1-2.2; 95% confidence interval) leftward shift in the MJN110 dose-response curve. Inhibitors of other relevant enzymes (i.e., ABHD6, COX-2) and nicotine did not engender substitution. Diazepam partially substituted for MJN110, but rimonabant failed to block this partial effect. These findings suggest that MAGL normally throttles 2-AG stimulation of CB1 receptors to a magnitude insufficient to produce cannabimimetic subjective effects. Accordingly, inhibitors of this enzyme may release this endogenous brake producing effects akin to those produced by exogenously administered cannabinoids.

The endocannabinoid hydrolysis inhibitor SA-57: Intrinsic antinociceptive effects, augmented morphine-induced antinociception, and attenuated heroin seeking behavior in mice.

Although opioids are highly efficacious analgesics, their abuse potential and other untoward side effects diminish their therapeutic utility. The addition of non-opioid analgesics offers a promising strategy to reduce required antinociceptive opioid doses that concomitantly reduce opioid-related side effects. Inhibitors of the primary endocannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) show opioid-sparing effects in preclinical models of pain. As simultaneous inhibition of these enzymes elicits enhanced antinociceptive effects compared with single enzyme inhibition, the present study tested whether the dual FAAH-MAGL inhibitor SA-57 [4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester] produces morphine-sparing antinociceptive effects, without major side effects associated with either drug class. SA-57 dose-dependently reversed mechanical allodynia in the constriction injury (CCI) of the sciatic nerve model of neuropathic pain and carrageenan inflammatory pain model. As previously reported, SA-57 was considerably more potent in elevating anandamide (AEA) than 2-arachidonyl glycerol (2-AG) in brain. Its anti-allodynic effects required cannabinoid (CB)1 and CB2 receptors; however, only CB2 receptors were necessary for the anti-edematous effects in the carrageenan assay. Although high doses of SA-57 alone were required to produce antinociception, low doses of this compound, which elevated AEA and did not affect 2-AG brain levels, augmented the antinociceptive effects of morphine, but lacked cannabimimetic side effects. Because of the high abuse liability of opioids and implication of the endocannabinoid system in the reinforcing effects of opioids, the final experiment tested whether SA-57 would alter heroin seeking behavior. Strikingly, SA-57 reduced heroin-reinforced nose poke behavior and the progressive ratio break point for heroin. In conclusion, the results of the present study suggest that inhibition of endocannabinoid degradative enzymes represents a promising therapeutic approach to decrease effective doses of opioids needed for clinical pain control, and may also possess therapeutic potential to reduce opioid abuse.

Robust anti-nociceptive effects of monoacylglycerol lipase inhibition in a model of osteoarthritis pain.

BACKGROUND AND PURPOSE: Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain relief. We evaluated the effects of a potent and selective monoacylglycerol (MAG) lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat. EXPERIMENTAL APPROACH: Intra-articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA-induced weight-bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined. KEY RESULTS: Acute MJN110 (5 mg·kg-1 ) significantly reversed MIA-induced weight-bearing asymmetry (MIA/vehicle: 68 ± 6 g; MIA/MJN110: 35 ± 4 g) and lowered ipsilateral PWTs (MIA/vehicle: 7 ± 0.8 g; MIA/MJN110: 11 ± 0.6 g), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5 mg·kg-1 ) resulted in anti-nociceptive tolerance. A lower dose of MJN110 (1 mg·kg-1 ) acutely inhibited pain behaviour, which was maintained for 1 week of repeated administration but had no effect on joint histology. MJN110 significantly inhibited expression of membrane-associated PGE synthase-1 in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared with vehicle-treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2-arachidonoylglycerol. CONCLUSIONS AND IMPLICATIONS: Our data support further assessment of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain.

Discriminative Stimulus Properties of the Endocannabinoid Catabolic Enzyme Inhibitor SA-57 in Mice.

Whereas the inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the respective major hydrolytic enzymes of N-arachidonoyl ethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), elicits no or partial substitution for Δ(9)-tetrahydrocannabinol (THC) in drug-discrimination procedures, combined inhibition of both enzymes fully substitutes for THC, as well as produces a constellation of cannabimimetic effects. The present study tested whether C57BL/6J mice would learn to discriminate the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) from vehicle in the drug-discrimination paradigm. In initial experiments, 10 mg/kg SA-57 fully substituted for CP55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a high-efficacy CB1 receptor agonist in C57BL/6J mice and for AEA in FAAH (-/-) mice. Most (i.e., 23 of 24) subjects achieved criteria for discriminating SA-57 (10 mg/kg) from vehicle within 40 sessions, with full generalization occurring 1 to 2 hours postinjection. CP55,940, the dual FAAH-MAGL inhibitor JZL195 (4-​nitrophenyl 4-​(3-​phenoxybenzyl)piperazine-​1-​carboxylate), and the MAGL inhibitors MJN110 (2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate) and JZL184 (4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) fully substituted for SA-57. Although the FAAH inhibitors PF-3845 ((N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) and URB597 (cyclohexylcarbamic acid 3'-(aminocarbonyl)-[1,1'-biphenyl]-3-yl ester) did not substitute for SA-57, PF-3845 produced a 2-fold leftward shift in the MJN110 substitution dose-response curve. In addition, the CB1 receptor antagonist rimonabant blocked the generalization of SA-57, as well as substitution of CP55,940, JZL195, MJN110, and JZL184. These findings suggest that MAGL inhibition plays a major role in the CB1 receptor-mediated SA-57 training dose, which is further augmented by FAAH inhibition.

Elevation of 2-AG by monoacylglycerol lipase inhibition in the visceral insular cortex interferes with anticipatory nausea in a rat model.

Anticipatory nausea (AN) is a conditioned nausea reaction experienced by chemotherapy patients upon returning to the clinic. Currently, there are no specific treatments for this phenomenon, with the classic antiemetic treatments (e.g., ondansetron) providing no relief. The rat model of AN, contextually elicited conditioned gaping reactions in rats, provides a tool for assessing potential treatments for this difficult to treat disorder. Systemically administered drugs which elevate the endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), by interfering with their respective degrading enzymes, fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) interfere with AN in the rat model. We have shown that MAGL inhibition within the visceral insular cortex (VIC) interferes with acute nausea in the gaping model (Sticht et al., 2015). Here we report that bilateral infusion of the MAGL inhibitor, MJN110 (but neither the FAAH inhibitor, PF3845, nor ondansetron) into the VIC suppressed contextually elicited conditioned gaping, and this effect was reversed by coadministration of the CB1 antagonist, AM251. These findings suggest that 2-AG within the VIC plays a critical role in the regulation of both acute nausea and AN. Because there are currently no specific therapeutics for chemotherapy patients that develop anticipatory nausea, MAGL inhibition by MJN110 may be a candidate treatment. (PsycINFO Database Record

The Selective Monoacylglycerol Lipase Inhibitor MJN110 Produces Opioid-Sparing Effects in a Mouse Neuropathic Pain Model.

Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Δ(9)-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid 2-arachidonoylglycerol through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor 2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were 2.4 (1.9-3.0) mg/kg and 0.43 (0.23-0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required µ-opioid, CB1, and CB2 receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure.

Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.