Enhancing Drug Discovery and Development through the Integration of Medicinal Chemistry, Chemical Biology, and Academia-Industry Partnerships: Insights from Roche's Endocannabinoid System Projects.

The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples.

Monoacylglycerol lipase blockades the senescence-associated secretory phenotype by interfering with NF-κB activation and promotes docetaxel efficacy in prostate cancer.

Metabolic reprogramming and cellular senescence greatly contribute to cancer relapse and recurrence. In aging and treated prostate, persistent accumulating senescence-associated secretory phenotype (SASP) of cancer cells often limits the overall survival of patients. Novel strategic therapy with monoacylglycerol lipase (MGLL) upregulation that counters the cellular and docetaxel induced SASP might overcome this clinical challenge in prostate cancer (PCa). With primary comparative expression and survival analysis screening of fatty acid (FA) metabolism signature genes in the TCGA PCa dataset and our single center cohort, MGLL was detected to be downregulated in malignancy prostate tissues and its low expression predicted worse progression-free and overall survival. Functionally, overexpression of MGLL mainly suppresses NF-κB-driven SASP (N-SASP) which mostly restricts the cancer cell paracrine and autocrine tumorigenic manners and the corresponding cellular senescence. Further investigating metabolites, we determined that MGLL constitutive expression prevents lipid accumulation, decreases metabolites preferably, and consequently downregulates ATP levels. Overexpressed MGLL inhibited IκBα phosphorylation, NF-κB p65 phosphorylation, and NF-κB nuclear translocation to deactivate NF-κB transcriptional activities, and be responsible for the repressed N-SASP, partially through reducing ATP levels. Preclinically, combinational treatment with MGLL overexpression and docetaxel chemotherapy dramatically delays tumor progression in mouse models. Taken together, our findings identify MGLL as a switch for lipase-related N-SASP suppression and provide a potential drug candidate for promoting docetaxel efficacy in PCa.

Design, synthesis and biological evaluation of naphthyl amide derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors.

Monoacylglycerol lipase (MAGL) is a key enzyme responsible for the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG), and has attracted great interest due to its involvement in various physiological and pathological processes, such as cancer progression. In the past, a number of covalent irreversible inhibitors have been reported for MAGL, however, experimental evidence highlighted some drawbacks associated with the use of these irreversible agents. Therefore, efforts were mainly focused on the development of reversible MAGL inhibitor in recent years. Here, we designed and synthesized a series of naphthyl amide derivatives (12-39) as another type of reversible MAGL inhibitors, exemplified by ± 34, which displayed good MAGL inhibition with a pIC50 of 7.1, and the potency and selectivity against endogenous MAGL were further demonstrated by competitive ABPP. Moreover, the compound showed appreciable antiproliferative activities against several cancer cells, including H460, HT29, CT-26, Huh7 and HCCLM-3. The investigations culminated in the discovery of the naphthyl amide derivative ± 34, and it may represent as a new scaffold for MAGL inhibitor development, particularly for the reversible ones.

Fluorescence-Based Enzyme Activity Assay: Ascertaining the Activity and Inhibition of Endocannabinoid Hydrolytic Enzymes.

The endocannabinoid system, known for its regulatory role in various physiological processes, relies on the activities of several hydrolytic enzymes, such as fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA), monoacylglycerol lipase (MAGL), and α/ß-hydrolase domains 6 (ABHD6) and 12 (ABHD12), to maintain homeostasis. Accurate measurement of these enzymes' activities is crucial for understanding their function and for the development of potential therapeutic agents. Fluorometric assays, which offer high sensitivity, specificity, and real-time monitoring capabilities, have become essential tools in enzymatic studies. This review provides a comprehensive overview of the principles behind these assays, the various substrates and fluorophores used, and advances in assay techniques used not only for the determination of the kinetic mechanisms of enzyme reactions but also for setting up kinetic assays for the high-throughput screening of each critical enzyme involved in endocannabinoid degradation. Through this comprehensive review, we aim to highlight the strengths and limitations of current fluorometric assays and suggest future directions for improving the measurement of enzyme activity in the endocannabinoid system.

Structure-Activity Relationship Studies of Aryl Sulfoxides as Reversible Monoacylglycerol Lipase Inhibitors.

Monoacylglycerol lipase (MAGL) is the key enzyme for the hydrolysis of endocannabinoid 2-arachidonoylglycerol (2-AG). The central role of MAGL in the metabolism of 2-AG makes it an attractive therapeutic target for a variety of disorders, including inflammation-induced tissue injury, pain, multiple sclerosis, and cancer. Previously, we reported LEI-515, an aryl sulfoxide, as a peripherally restricted, covalent reversible MAGL inhibitor that reduced neuropathic pain and inflammation in preclinical models. Here, we describe the structure-activity relationship (SAR) of aryl sulfoxides as MAGL inhibitors that led to the identification of LEI-515. Optimization of the potency of high-throughput screening (HTS) hit 1 yielded compound ±43. However, ±43 was not metabolically stable due to its ester moiety. Replacing the ester group with α-CF2 ketone led to the identification of compound ±73 (LEI-515) as a metabolically stable MAGL inhibitor with subnanomolar potency. LEI-515 is a promising compound to harness the therapeutic potential of MAGL inhibition.

A helping HAND: therapeutic potential of MAGL inhibition against HIV-1-associated neuroinflammation.

Background: Human immunodeficiency virus (HIV) affects nearly 40 million people globally, with roughly 80% of all people living with HIV receiving antiretroviral therapy. Antiretroviral treatment suppresses viral load in peripheral tissues but does not effectively penetrate the blood-brain barrier. Thus, viral reservoirs persist in the central nervous system and continue to produce low levels of inflammatory factors and early viral proteins, including the transactivator of transcription (Tat). HIV Tat is known to contribute to chronic neuroinflammation and synaptodendritic damage, which is associated with the development of cognitive, motor, and/or mood problems, collectively known as HIV-associated neurocognitive disorders (HAND). Cannabinoid anti-inflammatory effects are well documented, but therapeutic utility of cannabis remains limited due to its psychotropic effects, including alterations within brain regions encoding reward processing and motivation, such as the nucleus accumbens. Alternatively, inhibiting monoacylglycerol lipase (MAGL) has demonstrated therapeutic potential through interactions with the endocannabinoid system. Methods: The present study utilized a reward-related operant behavioral task to quantify motivated behavior in female Tat transgenic mice treated with vehicle or MAGL inhibitor MJN110 (1 mg/kg). Brain tissue was collected to assess dendritic injury and neuroinflammatory profiles, including dendritic microtubule-associated protein (MAP2ab) intensity, microglia density, microglia morphology, astrocyte density, astrocytic interleukin-1ß (IL-1ß) colocalization, and various lipid mediators. Results: No significant behavioral differences were observed; however, MJN110 protected against Tat-induced dendritic injury by significantly upregulating MAP2ab intensity in the nucleus accumbens and in the infralimbic cortex of Tat(+) mice. No or only minor effects were noted for Iba-1+ microglia density and/or microglia morphology. Further, Tat increased GFAP+ astrocyte density in the infralimbic cortex and GFAP+ astrocytic IL-1ß colocalization in the nucleus accumbens, with MJN110 significantly reducing these measures in Tat(+) subjects. Lastly, selected HETE-related inflammatory lipid mediators in the striatum were downregulated by chronic MJN110 treatment. Conclusions: These findings demonstrate anti-inflammatory and neuroprotective properties of MJN110 without cannabimimetic behavioral effects and suggest a promising alternative to cannabis for managing neuroinflammation.

Watermelon: setup and validation of an in silico fragment-based approach.

We present a new computational approach, named Watermelon, designed for the development of pharmacophore models based on receptor structures. The methodology involves the sampling of potential hotspots for ligand interactions within a protein target's binding site, utilising molecular fragments as probes. By employing docking and molecular dynamics (MD) simulations, the most significant interactions formed by these probes within distinct regions of the binding site are identified. These interactions are subsequently transformed into pharmacophore features that delineates key anchoring sites for potential ligands. The reliability of the approach was experimentally validated using the monoacylglycerol lipase (MAGL) enzyme. The generated pharmacophore model captured features representing ligand-MAGL interactions observed in various X-ray co-crystal structures and was employed to screen a database of commercially available compounds, in combination with consensus docking and MD simulations. The screening successfully identified two new MAGL inhibitors with micromolar potency, thus confirming the reliability of the Watermelon approach.

Endocannabinoid Hydrolase Inhibitors: Potential Novel Anxiolytic Drugs.

Over the past decade, the idea of targeting the endocannabinoid system to treat anxiety disorders has received increasing attention. Previous studies focused more on developing cannabinoid receptor agonists or supplementing exogenous cannabinoids, which are prone to various adverse effects due to their strong pharmacological activity and poor receptor selectivity, limiting their application in clinical research. Endocannabinoid hydrolase inhibitors are considered to be the most promising development strategies for the treatment of anxiety disorders. More recent efforts have emphasized that inhibition of two major endogenous cannabinoid hydrolases, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), indirectly activates cannabinoid receptors by increasing endogenous cannabinoid levels in the synaptic gap, circumventing receptor desensitization resulting from direct enhancement of endogenous cannabinoid signaling. In this review, we comprehensively summarize the anxiolytic effects of MAGL and FAAH inhibitors and their potential pharmacological mechanisms, highlight reported novel inhibitors or natural products, and provide an outlook on future directions in this field.

Genome-wide identification and mining elite allele variation of the Monoacylglycerol lipase (MAGL) gene family in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Monoacylglycerol lipase (MAGL) genes belong to the alpha/beta hydrolase superfamily, catalyze the terminal step of triglyceride (TAG) hydrolysis, converting monoacylglycerol (MAG) into free fatty acids and glycerol. RESULTS: In this study, 30 MAGL genes in upland cotton have been identified, which have been classified into eight subgroups. The duplication of GhMAGL genes in upland cotton was predominantly influenced by segmental duplication events, as revealed through synteny analysis. Furthermore, all GhMAGL genes were found to contain light-responsive elements. Through comprehensive association and haplotype analyses using resequencing data from 355 cotton accessions, GhMAGL3 and GhMAGL6 were detected as key genes related to lipid hydrolysis processes, suggesting a negative regulatory effect. CONCLUSIONS: In summary, MAGL has never been studied in upland cotton previously. This study provides the genetic mechanism foundation for the discover of new genes involved in lipid metabolism to improve cottonseed oil content, which will provide a strategic avenue for marker-assisted breeding aimed at incorporating desirable traits into cultivated cotton varieties.

Effects of the Dual FAAH/MAGL Inhibitor AKU-005 on Trigeminal Hyperalgesia in Male Rats.

The inhibition of endocannabinoid hydrolysis by enzymatic inhibitors may interfere with mechanisms underlying migraine-related pain. The dual FAAH/MAGL inhibitor AKU-005 shows potent inhibitory activity in vitro. Here, we assessed the effect of AKU-005 in a migraine animal model based on nitroglycerin (NTG) administration. Male rats were treated with AKU-005 (0.5 mg/kg, i.p.) or vehicle 3 h after receiving NTG (10 mg/kg, i.p.) or NTG vehicle. One hour later, rats were subjected to the open field test followed by the orofacial formalin test. At the end of the test, we collected serum samples for assessing calcitonin gene-related peptide (CGRP) levels as well as meninges, trigeminal ganglia, and brain areas to assess mRNA levels of CGRP and pro-inflammatory cytokines, and endocannabinoid and related lipid levels. AKU-005 reduced NTG-induced hyperalgesia during the orofacial formalin test but did not influence NTG-induced changes in the open field test. It significantly reduced serum levels of CGRP, CGRP, and pro-inflammatory cytokine mRNA levels in the meninges, trigeminal ganglia, and central areas. Surprisingly, AKU-005 caused no change in endocannabinoids and related lipids in the regions evaluated. The present findings suggest that AKU-005 may have anti-migraine effects by reducing CGRP synthesis and release and the associated inflammatory events. This effect, however, does not seem mediated via an interference with the endocannabinoid pathway.

P2X7 receptor-dependent increase in endocannabinoid 2-arachidonoyl glycerol production by neuronal cells in culture: Dynamics and mechanism.

BACKGROUND AND PURPOSE: Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2-arachidonoyl glycerol (2-AG). While it is known that extracellular ATP stimulates 2-AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real-time changes in eCB levels with the genetically encoded sensor, GRABeCB2.0, can address this shortfall. EXPERIMENTAL APPROACH: 2-AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC-MS, and GRABeCB2.0 fluorescence changes were detected using live-cell confocal microscopy and a 96-well fluorescence plate reader. KEY RESULTS: 2-AG and AEA increased GRABeCB2.0 fluorescence in N2a cells with EC50 values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB1R) antagonist SR141617 and absent in cells expressing the mutant-GRABeCB2.0. ATP increased only 2-AG levels in N2a cells, as measured by LC-MS, and induced a transient increase in the GRABeCB2.0 signal within minutes primarily via activation of P2X7 receptors (P2X7R). This response was dependent on diacylglycerol lipase ß activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2-AG hydrolysing enzyme, α/ß-hydrolase domain containing 6 (ABHD6). CONCLUSIONS AND IMPLICATIONS: Considering that P2X7R activation increases 2-AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation.

Pharmacological blockade of 2-AG degradation ameliorates clinical, neuroinflammatory and synaptic alterations in experimental autoimmune encephalomyelitis.

The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, researchers have extensively studied the neuroprotective and anti-inflammatory effects of the ECS. Inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) has emerged as a promising strategy to mitigate brain damage in MS. In this study, we investigated the effects of a novel reversible MAGL inhibitor (MAGLi 432) on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We assessed its implications on motor disability, neuroinflammation, and synaptic dysfunction. Systemic in vivo treatment with MAGLi 432 resulted in a less severe EAE disease, accompanied by increased 2-AG levels and decreased levels of arachidonic acid (AA) and prostaglandins (PGs) in the brain. Additionally, MAGLi 432 reduced both astrogliosis and microgliosis, as evidenced by decreased microglia/macrophage density and a less reactive morphology. Flow cytometry analysis further revealed fewer infiltrating CD45+ and CD3+ cells in the brains of MAGLi 432-treated EAE mice. Finally, MAGLi treatment counteracted the striatal synaptic hyperexcitability promoted by EAE neuroinflammation. In conclusion, MAGL inhibition significantly ameliorated EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects. These findings provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and highlight the therapeutic potential of MAGLi-based drugs in mitigating MS-related inflammatory and neurodegenerative brain damage.

Preclinical Evaluation of the Reversible Monoacylglycerol Lipase PET Tracer (R)-[11C]YH132: Application in Drug Development and Neurodegenerative Diseases.

Monoacylglycerol lipase (MAGL) plays a crucial role in the degradation of 2-arachidonoylglycerol (2-AG), one of the major endocannabinoids in the brain. Inhibiting MAGL could lead to increased levels of 2-AG, which showed beneficial effects on pain management, anxiety, inflammation, and neuroprotection. In the current study, we report the characterization of an enantiomerically pure (R)-[11C]YH132 as a novel MAGL PET tracer. It demonstrates an improved pharmacokinetic profile compared to its racemate. High in vitro MAGL specificity of (R)-[11C]YH132 was confirmed by autoradiography studies using mouse and rat brain sections. In vivo, (R)-[11C]YH132 displayed a high brain penetration, and high specificity and selectivity toward MAGL by dynamic PET imaging using MAGL knockout and wild-type mice. Pretreatment with a MAGL drug candidate revealed a dose-dependent reduction of (R)-[11C]YH132 accumulation in WT mouse brains. This result validates its utility as a PET probe to assist drug development. Moreover, its potential application in neurodegenerative diseases was explored by in vitro autoradiography using brain sections from animal models of Alzheimer's disease and Parkinson's disease.

Discovery of a novel class of reversible monoacylglycerol lipase inhibitors for potential treatment of depression.

Biological studies on the endocannabinoid system (ECS) have suggested that monoacylglycerol lipase (MAGL), an essential enzyme responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), is a novel target for developing antidepressants. A decrease of 2-AG levels in the hippocampus of the brain has been observed in depressive-like models induced by chronic stress. Herein, employing a structure-based approach, we designed and synthesized a new class of (piperazine-1-carbonyl) quinolin-2(1H)-one derivatives as potent, reversible and selective MAGL inhibitors. And detailed structure-activity relationships (SAR) studies were discussed. Compound 27 (IC50 = 10.3 nM) exhibited high bioavailability (92.7%) and 2-AG elevation effect in vivo. Additionally, compound 27 exerted rapid antidepressant effects caused by chronic restraint stress (CRS) and didn't show signs of addictive properties in the conditioned place preference (CPP) assays. Our study is the first to report that reversible MAGL inhibitors can treat chronic stress-induced depression effectively, which may provide a new potential therapeutic strategy for the discovery of an original class of safe, rapid antidepressant drugs.

Inhibition of MAGL attenuates Intervertebral Disc Degeneration by Delaying nucleus pulposus senescence through STING.

Intervertebral disc degeneration (IVDD) stands as the primary cause of low back pain (LBP). A significant contributor to IVDD is nucleus pulposus cell (NPC) senescence. However, the precise mechanisms underlying NPC senescence remain unclear. Monoacylglycerol lipase (MAGL) serves as the primary enzyme responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), breaking down monoglycerides into glycerol and fatty acids. It plays a crucial role in various pathological processes, including pain, inflammation, and oxidative stress. In this study, we utilized a lipopolysaccharide (LPS)-induced NPC senescence model and a rat acupuncture-induced IVDD model to investigate the role of MAGL in IVDD both in vitro and in vivo. Initially, our results showed that MAGL expression was increased 2.41-fold and 1.52-fold within NP tissues from IVDD patients and rats induced with acupuncture, respectively. This increase in MAGL expression was accompanied by elevated expression of p16INK4α. Following this, it was noted that the suppression of MAGL resulted in a notable decrease in the quantity of SA-ß-gal-positive cells and hindered the manifestation of p16INK4α and the inflammatory factor IL-1ß in NPCs. MAGL inhibition promotes type II collagen (Col-2) expression and inhibits matrix metalloproteinase 13 (MMP13), thereby restoring the balance of extracellular matrix (ECM) metabolism both in vitro and in vivo. A significant role for STING has also been demonstrated in the regulation of NPC senescence by MAGL. The expression of the STING protein was reduced by 57% upon the inhibition of MAGL. STING activation can replicate the effects of MAGL and substantially increase LPS-induced inflammation while accelerating the senescence of NPCs. These results strongly indicate that the inhibition of MAGL can significantly suppress nucleus pulposus senescence via its interaction with STING, consequently restoring the balance of ECM metabolism. This insight provides new perspectives for potential treatments for IVDD.

Improvement of lower urinary tract dysfunction by a monoacylglycerol lipase inhibitor in mice with spinal cord injury.

AIMS: Activation of the endocannabinoid system by monoacylglycerol lipase (MAGL) blockade may affect the lower urinary tract function. We investigated the effect of an MAGL inhibitor, MJN110, on neurogenic lower urinary tract dysfunction (LUTD) in the mouse model of spinal cord injury (SCI). METHODS: Female C57BL/6 mice that underwent spinal cord transection at T8-10 level were divided into three groups consisting of (1) vehicle-treated SCI mice, (2) 5 mg/kg, or (3) 10 mg/kg of MJN110-treated SCI mice. MJN110 and vehicle were administered intraperitoneally for 7 days from 4 weeks after spinal cord transection. We then conducted awake cystometrograms and compared urodynamic parameters between three groups. The expression of cannabinoid (CB) receptors, TRP receptors, and inflammatory cytokines in L6-S1 dorsal root ganglia (DRG) or the bladder mucosa were evaluated and compared among three groups. Changes in the level of serum 2-arachidonoylglycerol (2-AG) and bladder MAGL were also evaluated. RESULTS: In the cystometrogram, detrusor overactivity (DO) parameters, such as the number of nonvoiding contraction (NVC), a ratio of time to the 1st NVC to intercontraction interval (ICI), and NVC integrals were improved by MJN110 treatment, and some effects were dose dependent. Although MJN110 did not improve voiding efficiency, it decreased bladder capacity, ICI, and residual urine volume compared to vehicle injection. MJN110 treatment groups had lower CB2, TRPV1, TRPA1, and inflammatory cytokines mRNA levels in DRG and bladder mucosa. Serum 2-AG was increased, and bladder MAGL was decreased after MAGL inhibitor treatment. CONCLUSIONS: MAGL inhibition improved LUTD including attenuation of DO after SCI. Thus, MAGL can be a therapeutic target for neurogenic LUTD after SCI.

Identification of (R)-[18F]YH134 for Monoacylglycerol Lipase Neuroimaging and Exploration of Its Use for Central Nervous System and Peripheral Drug Development.

This study aimed to evaluate (R)-[18F]YH134 as a novel PET tracer for imaging monoacylglycerol lipase (MAGL). Considering the ubiquitous expression of MAGL throughout the whole body, the impact of various MAGL inhibitors on (R)-[18F]YH134 brain uptake and its application in brain-periphery crosstalk were explored. Methods: MAGL knockout and wild-type mice were used to evaluate (R)-[18F]YH134 in in vitro autoradiography and PET experiments. To explore the impact of peripheral MAGL occupancy on (R)-[18F]YH134 brain uptake, PET kinetics with an arterial input function were studied in male Wistar rats under baseline and blocking conditions. Results: In in vitro autoradiography, (R)-[18F]YH134 revealed a heterogeneous distribution pattern with high binding to MAGL-rich brain regions in wild-type mouse brain slices, whereas the radioactive signal was negligible in MAGL knockout mouse brain slices. The in vivo brain PET images of (R)-[18F]YH134 in wild-type and MAGL knockout mice demonstrated its high specificity and selectivity in mouse brain. A Logan plot with plasma input function was applied to estimate the distribution volume (V T) of (R)-[18F]YH134. V T was significantly reduced by a brain-penetrant MAGL inhibitor but was unchanged by a peripherally restricted MAGL inhibitor. The MAGL target occupancy in the periphery was estimated using (R)-[18F]YH134 PET imaging data from the brain. Conclusion: (R)-[18F]YH134 is a highly specific and selective PET tracer with favorable kinetic properties for imaging MAGL in rodent brain. Our results showed that blocking of the peripheral target influences brain uptake but not the V T of (R)-[18F]YH134. (R)-[18F]YH134 can be used for estimating the dose of MAGL inhibitor at half-maximal peripheral target occupancy.

Effects of Repeated Treatment with the Monoacylglycerol Lipase Inhibitor MJN110 on Pain-Related Depression of Nesting and Cannabinoid 1 Receptor Function in Male and Female Mice.

MJN110 inhibits the enzyme monoacylglycerol lipase (MAGL) to increase levels of the endocannabinoid 2-arachidonoylglycerol , an endogenous high-efficacy agonist of cannabinoid 1 and 2 receptors (CB1/2R). MAGL inhibitors are under consideration as candidate analgesics, and we reported previously that acute MJN110 produced partial antinociception in an assay of pain-related behavioral depression in mice. Given the need for repeated analgesic administration in many pain patients and the potential for analgesic tolerance during repeated treatment, this study examined antinociceptive effects of repeated MJN110 on pain-related behavioral depression and CB1R-mediated G-protein function. Male and female ICR mice were treated daily for 7 days in a 2 × 2 design with (a) 1.0 mg/kg/d MJN110 or its vehicle followed by (b) intraperitoneal injection of dilute lactic acid (IP acid) or its vehicle as a visceral noxious stimulus to depress nesting behavior. After behavioral testing, G-protein activity was assessed in lumbar spinal cord (LSC) and five brain regions using an assay of CP55,940-stimulated [35S]GTPÉ£S activation. As reported previously, acute MJN110 produced partial but significant relief of IP acid-induced nesting depression on day 1. After 7 days, MJN110 continued to produce significant but partial antinociception in males, while antinociceptive tolerance developed in females. Repeated MJN110 also produced modest decreases in maximum levels of CP55,940-induced [35S]GTPÉ£S binding in spinal cord and most brain regions. These results indicate that repeated treatment with a relatively low antinociceptive MJN110 dose produces only partial and sex-dependent transient antinociception associated with the emergence of CB1R desensitization in this model of IP acid-induced nesting depression. SIGNIFICANCE STATEMENT: The drug MJN110 inhibits monoacylglycerol lipase (MAGL) to increase levels of the endogenous cannabinoid 2-arachidonoylglycerol and produce potentially useful therapeutic effects including analgesia. This study used an assay of pain-related behavioral depression in mice to show that repeated MJN110 treatment produced (1) weak but sustained antinociception in male mice, (2) antinociceptive tolerance in females, and (3) modest cannabinoid-receptor desensitization that varied by region and sex. Antinociceptive tolerance may limit the utility of MJN110 for treatment of pain.

ABHD6 drives endocytosis of AMPA receptors to regulate synaptic plasticity and learning flexibility.

Trafficking of α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs), mediated by AMPAR interacting proteins, enabled neurons to maintain tuning capabilities at rest or active state. α/ß-Hydrolase domain-containing 6 (ABHD6), an endocannabinoid hydrolase, was an AMPAR auxiliary subunit found to negatively regulate the surface delivery of AMPARs. While ABHD6 was found to prevent AMPAR tetramerization in endoplasmic reticulum, ABHD6 was also reported to localize at postsynaptic site. Yet, the role of ABHD6 interacting with AMPAR at postsynaptic site, and the physiological significance of ABHD6 regulating AMPAR trafficking remains elusive. Here, we generated the ABHD6 knockout (ABHD6KO) mice and found that deletion of ABHD6 selectively enhanced AMPAR-mediated basal synaptic responses and the surface expression of postsynaptic AMPARs. Furthermore, we found that loss of ABHD6 impaired hippocampal long-term depression (LTD) and synaptic downscaling in hippocampal synapses. AMPAR internalization assays revealed that ABHD6 was essential for neuronal activity-dependent endocytosis of surface AMPARs, which is independent of ABHD6's hydrolase activity. The defects of AMPAR endocytosis and LTD are expressed as deficits in learning flexibility in ABHD6KO mice. Collectively, we demonstrated that ABHD6 is an endocytic accessory protein promoting AMPAR endocytosis, thereby contributes to the formation of LTD, synaptic downscaling and reversal learning.

Discovery of a non-canonical prototype long-chain monoacylglycerol lipase through a structure-based endogenous reaction intermediate complex.

The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacylglycerol ester reaction intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monoacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzyme's active site. Knowledge about the molecular details of the substrate binding site allowed us to modulate the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.