Correction: Eid et al. Interference with TGFß1-Mediated Inflammation and Fibrosis Underlies Reno-Protective Effects of the CB1 Receptor Neutral Antagonists AM6545 and AM4113 in a Rat Model of Metabolic Syndrome. Molecules 2021, 26, 866.

The authors wish to make the following changes to the paper [...].

The neutral CB1 antagonist AM6527 reduces ethanol seeking, binge-like consumption, reinforcing, and withdrawal effects in male and female mice.

RATIONALE: Alcohol use disorder (AUD) is a debilitating physiological and psychiatric disorder which affects individuals globally. The current pharmacological interventions to treat AUD are limited, and hence there is an urgent need for a novel pharmacological therapy which can be effective and safe across the population. OBJECTIVE: We aimed to investigate a novel neutral cannabinoid receptor-1 (CB1R) antagonist, AM6527, in several preclinical models of ethanol consumption using male and female C57BL6/J mice. METHODS: Independent groups of male and female mice were subjected to repeated cycles of drinking in the dark (DID), or intermittent access to alcohol (IAA) procedures. Twenty minutes prior to ethanol access in each procedure, animals were treated with intraperitoneal injections of either 1, 3, and 10 mg/kg of AM6527 or its respective vehicle. Acamprosate (100, 200, 300, and 400 mg/kg) or its respective vehicle was used as a positive control. Separate groups of male mice were subjected to a chain schedule of ethanol reinforcement to gain access to ethanol wherein completion of a fixed interval (FI; 5 min) schedule (link 1: "Seeking") was reinforced with continuous access to ethanol (fixed ratio; FR1) for up to 1.8 g/kg (link 2: "consumption"). All the animals were treated with 1, 3, and 10 mg/kg of AM6527 or its respective vehicle 20 mins prior to the start of the FI chain of the procedure. Separately, AM6527 was also evaluated in male and female mice undergoing acute ethanol withdrawal following 8 weeks of intermittent or continuous access to 20% ethanol drinking. RESULTS: In both DID and IAA procedures, AM6527 reduced ethanol consumption in a dose-related manner in both male and female mice. AM6527 produced no tolerance in the DID procedure; mice treated with 3 mg/kg of AM6527 for 3 weeks continuously drank significantly smaller amounts of ethanol as compared to vehicle-treated mice over a period of three DID cycles. Moreover, in the IAA procedure, AM6527 caused an increase in water intake over the 24-h period. Acamprosate transiently reduced ethanol intake in male mice in both the DID and the IAA procedures but failed to produce any significant effect in female mice. AM6527 also produced a decrease in the FI responding ("ethanol seeking") in animals trained to self-administer ethanol. Lastly, AM6527 mitigated neurological withdrawal signs, i.e., handling induced convulsions (HIC) in mice undergoing acute ethanol withdrawal. CONCLUSIONS: Current findings support previous studies with CB1R neutral antagonist in reducing voluntary ethanol intake and seeking behavior. Based on results shown in this work, AM6527 can be developed as a first in class CB1R neutral antagonist to treat AUD in both males and females.

Effects of the CB1 receptor antagonists AM6545 and AM4113 on metabolic syndrome-induced prostatic hyperplasia in rats.

Metabolic syndrome (MetS) is a combination of metabolic disorders that can predispose individuals to benign prostatic hyperplasia (BPH). The inhibition of the cannabinoid 1 (CB1) receptor has been used to treat metabolic disorders in animal models. This study reports the use of a peripherally restricted CB1 antagonist (AM6545) and a neutral CB1 antagonist (AM4113) to improve MetS-related BPH in rats. Animals were divided into three control groups to receive either a normal rodent diet, AM6545, or AM4113. MetS was induced in the fourth, fifth, and sixth groups using a concentrated fructose solution and high-salt diet delivered as food pellets for eight weeks. The fifth and sixth groups were further given AM6545 or AM4113 for additional four weeks. Body and prostate weights were measured and prostate sections were stained with hematoxylin eosin. Cyclin D1, markers of oxidative stress and inflammation, and levels of the endocannabinoids were recorded. BPH in rats with MetS was confirmed through increased prostate weight and index, as well as histopathology. Treatment with either AM6545 or AM4113 significantly decreased prostate weight, improved prostate histology, and reduced cyclin D1 expression compared with the MetS group. Groups treated with CB1 antagonists experienced reduced lipid peroxidation, recovered glutathione depletion, restored catalase activity, and had lower inflammatory markers interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). MetS rats treated with either AM6545 or AM4113 showed reduced concentrations of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the prostate compared with the MetS group. In conclusion, the CB1 antagonists AM6545 and AM4113 protect against MetS-induced BPH through their anti-proliferative, antioxidant, and anti-inflammatory effects.

Role of CB1 receptors in the acute regulation of small intestinal permeability: effects of high-fat diet.

The endocannabinoid system of the gastrointestinal tract is involved in the control of intestinal barrier function. Whether the cannabinoid 1 (CB1) receptor is expressed on the intestinal epithelium and acutely regulates barrier function has not been determined. Here, we tested the hypothesis that ligands of the CB1 receptor acutely modulate small intestinal permeability and that this is associated with altered distribution of tight junction proteins. We examined the acute effects of CB1 receptor ligands on small intestinal permeability both in chow-fed and 2-wk high-fat diet (HFD)-fed mice using Ussing chambers. We assessed the distribution of CB1 receptor and tight junction proteins using immunofluorescence and the expression of CB1 receptor using PCR. A low level of CB1 expression was found on the intestinal epithelium. CB1 receptor was highly expressed on enteric nerves in the lamina propria. Neither the CB1/CB2 agonist CP55,940 nor the CB1 neutral antagonist AM6545 altered the flux of 4kDa FITC dextran (FD4) across the jejunum or ileum of chow-fed mice. Remarkably, both CP55,940 and AM6545 reduced FD4 flux across the jejunum and ileum in HFD-fed mice that have elevated baseline intestinal permeability. These effects were absent in CB1 knockout mice. CP55,940 reduced the expression of claudin-2, whereas AM6545 had little effect on claudin-2 expression. Neither ligand altered the expression of ZO-1. Our data suggest that CB1 receptor on the intestinal epithelium regulates tight junction protein expression and restores barrier function when it is increased following exposure to a HFD for 2 wk.NEW & NOTEWORTHY The endocannabinoid system of the gastrointestinal tract regulates homeostasis by acting as brake on motility and secretion. Here we show that when exposed to a high fat diet, intestinal permeability is increased and activation of the CB1 receptor on the intestinal epithelium restores barrier function. This work further highlights the role of the endocannabinoid system in regulating intestinal homeostasis when it is perturbed.

Blockade of CB1 or Activation of CB2 Cannabinoid Receptors Is Differentially Efficacious in the Treatment of the Early Pathological Events in Streptozotocin-Induced Diabetic Rats.

Oxidative stress, neurodegeneration, neuroinflammation, and vascular leakage are believed to play a key role in the early stage of diabetic retinopathy (ESDR). The aim of this study was to investigate the blockade of cannabinoid receptor 1 (CB1R) and activation of cannabinoid receptor 2 (CB2R) as putative therapeutics for the treatment of the early toxic events in DR. Diabetic rats [streptozotocin (STZ)-induced] were treated topically (20 µL, 10 mg/mL), once daily for fourteen days (early stage DR model), with SR141716 (CB1R antagonist), AM1710 (CB2R agonist), and the dual treatment SR141716/AM1710. Immunohistochemical-histological, ELISA, and Evans-Blue analyses were performed to assess the neuroprotective and vasculoprotective properties of the pharmacological treatments on diabetes-induced retinal toxicity. Activation of CB2R or blockade of CB1R, as well as the dual treatment, attenuated the nitrative stress induced by diabetes. Both single treatments protected neural elements (e.g., RGC axons) and reduced vascular leakage. AM1710 alone reversed all toxic insults. These findings provide new knowledge regarding the differential efficacies of the cannabinoids, when administered topically, in the treatment of ESDR. Cannabinoid neuroprotection of the diabetic retina in ESDR may prove therapeutic in delaying the development of the advanced stage of the disease.

Interference with TGFß1-Mediated Inflammation and Fibrosis Underlies Reno-Protective Effects of the CB1 Receptor Neutral Antagonists AM6545 and AM4113 in a Rat Model of Metabolic Syndrome.

The role of cannabinoid receptors in nephropathy is gaining much attention. This study investigated the effects of two neutral CB1 receptor antagonists, AM6545 and AM4113, on nephropathy associated with metabolic syndrome (MetS). MetS was induced in rats by high-fructose high-salt feeding for 12 weeks. AM6545, the peripheral silent antagonist and AM4113, the central neutral antagonist were administered in the last 4 weeks. At the end of study, blood and urine samples were collected for biochemical analyses while the kidneys were excised for histopathological investigation and transforming growth factor beta 1 (TGFß1) measurement. MetS was associated with deteriorated kidney function as indicated by the elevated proteinuria and albumin excretion rate. Both compounds equally inhibited the elevated proteinuria and albumin excretion rate while having no effect on creatinine clearance and blood pressure. In addition, AM6545 and AM4113 alleviated the observed swelling and inflammatory cells infiltration in different kidney structures. Moreover, AM6545 and AM4113 alleviated the observed histopathological alterations in kidney structure of MetS rats. MetS was associated with a ten-fold increase in urine uric acid while both compounds blocked this increase. Furthermore, AM6545 and AM4113 completely prevented the collagen deposition and the elevated expression of the TGFß1 seen in MetS animals. In conclusion, AM6545 and AM4113, possess reno-protective effects by interfering with TGFß1-mediated renal inflammation and fibrosis, via peripheral action.

Effects of the CB1 Receptor Antagonists AM6545 and AM4113 on Insulin Resistance in a High-Fructose High-Salt Rat Model of Metabolic Syndrome.

Background and Objectives: Insulin resistance (IR) is a serious condition leading to development of diabetes and cardiovascular complications. Hyper-activation of cannabinoid receptors-1 (CB1) has been linked to the development of metabolic disorders such as IR. Therefore, the effect of blocking CB1 on the development of IR was investigated in the present study. Materials and Methods: A 12-week high-fructose/high-salt feeding model of metabolic syndrome was used to induce IR in male Wistar rats. For this purpose, two different CB1-antagonists were synthesized and administered to the rats during the final four weeks of the study, AM6545, the peripheral neutral antagonist and AM4113, the central neutral antagonist. Results: High-fructose/salt feeding for 12 weeks led to development of IR while both AM6545 and AM4113, administered in the last 4 weeks, significantly inhibited IR. This was correlated with increased animal body weight wherein both AM6545 and AM4113 decreased body weight in IR animals but with loss of IR/body weight correlation. While IR animals showed significant elevations in serum cholesterol and triglycerides with no direct correlation with IR, both AM6545 and AM4113 inhibited these elevations, with direct IR/cholesterol correlation in case of AM6545. IR animals had elevated serum uric acid, which was reduced by both AM6545 and AM4113. In addition, IR animals had decreased adiponectin levels and elevated liver TNFα content with strong IR/adiponectin and IR/TNFα correlations. AM6545 inhibited the decreased adiponectin and the increased TNFα levels and retained the strong IR/adiponectin correlation. However, AM4113 inhibited the decreased adiponectin and the increased TNFα levels, but with loss of IR/adiponectin and IR/TNFα correlations. Conclusions: Both CB1 neutral antagonists alleviated IR peripherally, and exerted similar effects on rats with metabolic syndrome. They also displayed anti-dyslipidemic, anti-hyperurecemic and anti-inflammatory effects. Overall, these results should assist in the development of CB1 neutral antagonists with improved safety profiles for managing metabolic disorders.

Conformational gating, dynamics and allostery in human monoacylglycerol lipase.

Inhibition of human Monoacylglycerol Lipase (hMGL) offers a novel approach for treating neurological diseases. The design of inhibitors, targeting active-inactive conformational transitions of the enzyme, can be aided by understanding the interplay between structure and dynamics. Here, we report the effects of mutations within the catalytic triad on structure, conformational gating and dynamics of hMGL by combining kinetics, NMR, and HDX-MS data with metadynamics simulations. We found that point mutations alter delicate conformational equilibria between active and inactive states. HDX-MS reveals regions of the hMGL that become substantially more dynamic upon substitution of catalytic acid Asp-239 by alanine. These regions, located far from the catalytic triad, include not only loops but also rigid α-helixes and ß-strands, suggesting their involvement in allosteric regulation as channels for long-range signal transmission. The results identify the existence of a preorganized global communication network comprising of tertiary (residue-residue contacts) and quaternary (rigid-body contacts) networks that mediate robust, rapid intraprotein signal transmission. Catalytic Asp-239 controls hMGL allosteric communications and may be considered as an essential residue for the integration and transmission of information to enzymes' remote regions, in addition to its well-known role to facilitate Ser-122 activation. Our findings may assist in the identification of new druggable sites in hMGL.

Reversal of diet-induced hepatic steatosis by peripheral CB1 receptor blockade in mice is p53/miRNA-22/SIRT1/PPARα dependent.

OBJECTIVE: The endocannabinoid (eCB) system is increasingly recognized as being crucially important in obesity-related hepatic steatosis. By activating the hepatic cannabinoid-1 receptor (CB1R), eCBs modulate lipogenesis and fatty acid oxidation. However, the underlying molecular mechanisms are largely unknown. METHODS: We combined unbiased bioinformatics techniques, mouse genetic manipulations, multiple pharmacological, molecular, and cellular biology approaches, and genomic sequencing to systematically decipher the role of the hepatic CB1R in modulating fat utilization in the liver and explored the downstream molecular mechanisms. RESULTS: Using an unbiased normalized phylogenetic profiling analysis, we found that the CB1R evolutionarily coevolves with peroxisome proliferator-activated receptor-alpha (PPARα), a key regulator of hepatic lipid metabolism. In diet-induced obese (DIO) mice, peripheral CB1R blockade (using AM6545) induced the reversal of hepatic steatosis and improved liver injury in WT, but not in PPARα-/- mice. The antisteatotic effect mediated by AM6545 in WT DIO mice was accompanied by increased hepatic expression and activity of PPARα as well as elevated hepatic levels of the PPARα-activating eCB-like molecules oleoylethanolamide and palmitoylethanolamide. Moreover, AM6545 was unable to rescue hepatic steatosis in DIO mice lacking liver sirtuin 1 (SIRT1), an upstream regulator of PPARα. Both of these signaling molecules were modulated by the CB1R as measured in hepatocytes exposed to lipotoxic conditions or treated with CB1R agonists in the absence/presence of AM6545. Furthermore, using microRNA transcriptomic profiling, we found that the CB1R regulated the hepatic expression, acetylation, and transcriptional activity of p53, resulting in the enhanced expression of miR-22, which was found to specifically target SIRT1 and PPARα. CONCLUSIONS: We provide strong evidence for a functional role of the p53/miR-22/SIRT1/PPARα signaling pathway in potentially mediating the antisteatotic effect of peripherally restricted CB1R blockade.

Cannabinoid Antagonist Drug Discrimination in Nonhuman Primates.

Despite a growing acceptance that withdrawal symptoms can emerge following discontinuation of cannabis products, especially in high-intake chronic users, there are no Food and Drug Administration (FDA)-approved treatment options. Drug development has been hampered by difficulties studying cannabis withdrawal in laboratory animals. One preclinical approach that has been effective in studying withdrawal from drugs in several pharmacological classes is antagonist drug discrimination. The present studies were designed to examine this paradigm in squirrel monkeys treated daily with the long-acting CB1 agonist AM2389 (0.01 mg/kg) and trained to discriminate the CB1 inverse agonist/antagonist rimonabant (0.3 mg/kg) from saline. The discriminative-stimulus effects of rimonabant were both dose and time dependent and, importantly, could be reproduced by discontinuation of agonist treatment. Antagonist substitution tests with the CB1 neutral antagonists AM4113 (0.03-0.3 mg/kg), AM6527 (0.03-1.0 mg/kg), and AM6545 (0.03-1.0 mg/kg) confirmed that the rimonabant discriminative stimulus also could be reproduced by CB1 antagonists lacking inverse agonist action. Agonist substitution tests with the phytocannabinoid ∆9-tetrahydrocannabinol (0.1-1.0 mg/kg), synthetic CB1 agonists nabilone (0.01-0.1 mg/kg), AM4054 (0.01-0.03 mg/kg), K2/Spice compound JWH-018 (0.03-0.3 mg/kg), FAAH-selective inhibitors AM3506 (0.3-5.6 mg/kg), URB597 (3.0-5.6 mg/kg), and nonselective FAAH/MGL inhibitor AM4302 (3.0-10.0 mg/kg) revealed that only agonists with CB1 affinity were able to reduce the rimonabant-like discriminative stimulus effects of withholding daily agonist treatment. Although the present studies did not document physiologic disturbances associated with withdrawal, the results are consistent with the view that the cannabinoid antagonist drug discrimination paradigm provides a useful screening procedure for examining the ability of candidate medications to attenuate the interoceptive stimuli provoked by cannabis discontinuation. SIGNIFICANCE STATEMENT: Despite a growing acceptance that withdrawal symptoms can emerge following the discontinuation of cannabis products, especially in high-intake chronic users, there are no FDA-approved pharmacotherapies to assist those seeking treatment. The present studies systematically examined cannabinoid antagonist drug discrimination, a preclinical animal model that is designed to appraise the ability of candidate medications to attenuate the interoceptive effects that accompany abrupt cannabis abstinence.

Probing the CB1 Cannabinoid Receptor Binding Pocket with AM6538, a High-Affinity Irreversible Antagonist.

Cannabinoid receptor 1 (CB1) is a potential therapeutic target for the treatment of pain, obesity and obesity-related metabolic disorders, and addiction. The crystal structure of human CB1 has been determined in complex with the stabilizing antagonist AM6538. In the present study, we characterize AM6538 as a tight-binding/irreversible antagonist of CB1, as well as two derivatives of AM6538 (AM4112 and AM6542) as slowly dissociating CB1 antagonists across binding simulations and cellular signaling assays. The long-lasting nature of AM6538 was explored in vivo wherein AM6538 continues to block CP55,940-mediated behaviors in mice up to 5 days after a single injection. In contrast, the effects of SR141716A abate in mice 2 days after injection. These studies demonstrate the functional outcome of CB1 antagonist modification and open the path for development of long-lasting CB1 antagonists.

In vitro determination of the efficacy of illicit synthetic cannabinoids at CB1 receptors.

BACKGROUND AND PURPOSE: The morbidity and mortality associated with recreational use of synthetic cannabinoid receptor agonists (SCRAs) may reflect strong activation of CB1 receptors and is a major health concern. The properties of SCRA at CB1 receptors are not well defined. Here we have developed an assay to determine acute CB1 receptor efficacy using receptor depletion with the irreversible CB1 receptor antagonist AM6544, with application of the Black and Leff operational model to calculate efficacy. EXPERIMENTAL APPROACH: Receptor depletion in mouse AtT-20 pituitary adenoma cells stably expressing human CB1 receptors was achieved by pretreatment of cells with AM6544 (10 µM, 60 min). The CB1 receptor-mediated hyperpolarisation of AtT-20 cells was measured using fluorescence-based membrane potential dye. From data fit to the operational model, the efficacy (τ) and affinity (KA ) parameters were obtained for each drug. KEY RESULTS: AM6544 did not affect the potency or maximal effect of native somatostatin receptor-induced hyperpolarization. The τ value of ∆9 -THC was 80-fold less than the reference CB receptor agonist CP55940 and 260-fold less than the highest efficacy SCRA, 5F-MDMB-PICA. The operational efficacy of SCRAs ranged from 233 (5F-MDMB-PICA) to 28 (AB-PINACA), with CP55940 in the middle of the efficacy rank order. There was no correlation between the τ and KA values. CONCLUSIONS AND IMPLICATIONS: All SCRAs tested showed substantially higher efficacy at CB1 receptors than ∆9 -THC, which may contribute to the adverse effects seen with these drugs but not ∆9 -THC.

Crystal Structure of the Human Cannabinoid Receptor CB2.

The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.

Cannabinoid CB1 receptor neutral antagonist AM4113 inhibits heroin self-administration without depressive side effects in rats.

Cannabinoid CB1 receptors (CB1Rs) have been shown to be a promising target in medication development for the treatment of addiction. However, clinical trials with SR141716A (rimonabant, a selective CB1R antagonist/inverse agonist) for the treatment of obesity and smoking cessation failed due to unwanted side effects, such as depression, anxiety, and suicidal tendencies. Recent preclinical studies suggest that the neutral CB1R antagonist AM4113 may retain the therapeutic anti-addictive effects of SR141716A in nicotine self-administration models and possibly has fewer unwanted side effects. However, little is known about whether AM4113 is also effective for other drugs of abuse, such as opioids and psychostimulants, and whether it produces depressive side effects similar to SR141716A in experimental animals. In this study, we demonstrated that systemic administration of AM4113 (3 and 10 mg/kg) dose-dependently inhibited the self-administration of intravenous heroin but not cocaine or methamphetamine, whereas SR141716A (3 and 10 mg/kg) dose-dependently inhibited the self-administration of heroin and methamphetamine but not cocaine. In the electrical brain-stimulation reward (BSR) paradigm, SR141716A (3 and 10 mg/kg) dose-dependently increased the BSR stimulation threshold (i.e., decreased the stimulation reward), but AM4113 had no effect on BSR at the same doses, suggesting that SR141716A may produce aversive effects while AM4113 may not. Together, these findings show that neutral CB1R antagonists such as AM4113 deserve further research as a new class of CB1R-based medications for the treatment of opioid addiction without SR141716A-like aversive effects.

Long-Lasting In Vivo Effects of the Cannabinoid CB1 Antagonist AM6538.

AM6538 is a cannabinoid antagonist that binds CB1 receptors expressed in HEK-293 cells in a wash-resistant manner. The effects of AM6538 in live animals has not previously been established. We characterized the antagonist effects of AM6538 in male mice, using a warm-water tail-withdrawal assay, and in male squirrel monkeys trained to discriminate the CB1 agonist AM4054 from vehicle. The cannabinoid agonists WIN 55,212, Δ9-tetrahydrocannabinol (THC), and AM4054 all produced 100% maximum possible antinociceptive effects in mice following vehicle pretreatment. One-hour pretreatment with increasing doses of AM6538 (0.1-10 mg/kg) produced first rightward, then downward shifts of the agonist dose-effect functions. Rimonabant, 1-10 mg/kg, produced parallel rightward shifts of the AM4054 dose-effect functions, and baseline effects of AM4054 were nearly recovered within 24 hours following 10 mg/kg of rimonabant. In contrast, in mice treated with 10 mg/kg of AM6538, antagonism of THC or AM4054 lasted up to 7 days. AM6538 also antagonized the discriminative stimulus effects of AM4054 in squirrel monkeys in a dose-related manner, and the effects of 3.2 mg/kg of AM6538 endured for more than 7 days. The effective reduction in CB1 receptor reserve was used to calculate the relative efficacy (tau values) of WIN 55,212, THC, and AM4054 in mice and of AM4054 monkeys, with results indicating that THC has a lower efficacy than WIN 55,212 or AM4054 in mice. These results demonstrate that AM6538 is a long-acting CB antagonist in vivo, and further suggest that differences in CB efficacy can be revealed in behavioral assays following AM6538 treatment.

Effects of Distal Mutations on the Structure, Dynamics and Catalysis of Human Monoacylglycerol Lipase.

An understanding of how conformational dynamics modulates function and catalysis of human monoacylglycerol lipase (hMGL), an important pharmaceutical target, can facilitate the development of novel ligands with potential therapeutic value. Here, we report the discovery and characterization of an allosteric, regulatory hMGL site comprised of residues Trp-289 and Leu-232 that reside over 18 Å away from the catalytic triad. These residues were identified as critical mediators of long-range communication and as important contributors to the integrity of the hMGL structure. Nonconservative replacements of Trp-289 or Leu-232 triggered concerted motions of structurally distinct regions with a significant conformational shift toward inactive states and dramatic loss in catalytic efficiency of the enzyme. Using a multimethod approach, we show that the dynamically relevant Trp-289 and Leu-232 residues serve as communication hubs within an allosteric protein network that controls signal propagation to the active site, and thus, regulates active-inactive interconversion of hMGL. Our findings provide new insights into the mechanism of allosteric regulation of lipase activity, in general, and may provide alternative drug design possibilities.

Cannabinoid-1 receptor neutral antagonist reduces binge-like alcohol consumption and alcohol-induced accumbal dopaminergic signaling.

Binge alcohol (ethanol) drinking is associated with profound adverse effects on our health and society. Rimonabant (SR141716A), a CB1 receptor inverse agonist, was previously shown to be effective for nicotine cessation and obesity. However, studies using rimonabant were discontinued as it was associated with an increased risk of depression and anxiety. In the present study, we examined the pharmacokinetics and effects of AM4113, a novel CB1 receptor neutral antagonist on binge-like ethanol drinking in C57BL/6J mice using a two-bottle choice drinking-in-dark (DID) paradigm. The results indicated a slower elimination of AM4113 in the brain than in plasma. AM4113 suppressed ethanol consumption and preference without having significant effects on body weight, ambulatory activity, preference for tastants (saccharin and quinine) and ethanol metabolism. AM4113 pretreatment reduced ethanol-induced increase in dopamine release in nucleus accumbens. Collectively, these data suggest an important role of CB1 receptor-mediated regulation of binge-like ethanol consumption and mesolimbic dopaminergic signaling, and further points to the potential utility of CB1 neutral antagonists for the treatment of binge ethanol drinking.

CB1 receptor antagonism in the bed nucleus of the stria terminalis interferes with affective opioid withdrawal in rats.

The bed nucleus of the stria terminalis (BNST) is a region of the extended amygdala that is implicated in addiction, anxiety, and stress related behaviors. This region has been identified in mediating the aversive state of naloxone-precipitated morphine withdrawal (MWD) and cannabinoid Type I (CB1) receptors have been found to modulate neurotransmission within this region. Previous findings suggest that the CB1 antagonist/inverse agonist, AM251, administered systemically or by infusion into the central nucleus of the amygdala (CeA) prevented the aversive affective properties of MWD as measured by conditioned place aversion learning. As well, when administered systemically or by infusion into the basolateral nucleus of the amygdala (BLA) or the interoceptive insular cortex, the monoaclyglycerol lipase (MAGL) inhibitor, MJN110 (which elevates 2-arachidonlyglycerol), also prevented a naloxone-precipitated MWD induced place aversion. Given the connectivity of these regions and the BNST, the present study sought to determine whether cannabinoid modulation of the BNST would also prevent the affective properties of naloxone precipitated MWD-induced place aversion learning. Prior to conditioning trials, rats received intra-BNST infusions of AM251, in Experiment 1, or MJN110 in Experiment 2. AM251, but not MJN110, prevented the establishment of the MWD-induced place aversion. The current findings emphasize an important role for the BNST in opioid withdrawal and suggest that the ameliorative effects of systemically administered CB1 antagonists are mediated, in part, by their actions within this region. (PsycINFO Database Record

Crystal structures of agonist-bound human cannabinoid receptor CB1.

The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC). Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1-agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a 'twin toggle switch' of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros-Weinstein numbering) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties.

Binding Site Characterization of AM1336, a Novel Covalent Inverse Agonist at Human Cannabinoid 2 Receptor, Using Mass Spectrometric Analysis.

Cannabinoid 2 receptor (CB2R), a Class-A G-protein coupled receptor (GPCR), is a promising drug target under a wide array of pathological conditions. Rational drug design has been hindered due to our poor understanding of the structural features involved in ligand binding. Binding of a high-affinity biarylpyrazole inverse agonist AM1336 to a library of the human CB2 receptor (hCB2R) cysteine-substituted mutants provided indirect evidence that two cysteines in transmembrane helix-7 (H7) were critical for the covalent attachment. We used proteomics analysis of the hCB2R with bound AM1336 to directly identify peptides with covalently attached ligand and applied in silico modeling for visualization of the ligand-receptor interactions. The hCB2R, with affinity tags (FlaghCB2His6), was produced in a baculovirus-insect cell expression system and purified as a functional receptor using immunoaffinity chromatography. Using mass spectrometry-based bottom-up proteomic analysis of the hCB2R-AM1336, we identified a peptide with AM1336 attached to the cysteine C284(7.38) in H7. The hCB2R homology model in lipid bilayer accommodated covalent attachment of AM1336 to C284(7.38), supporting both biochemical and mass spectrometric data. This work consolidates proteomics data and in silico modeling and integrates with our ligand-assisted protein structure (LAPS) experimental paradigm to assist in structure-based design of cannabinoid antagonist/inverse agonists.