Virtual Screening of a Chemically Diverse "Superscaffold" Library Enables Ligand Discovery for a Key GPCR Target.

The advent of ultra-large libraries of drug-like compounds has significantly broadened the possibilities in structure-based virtual screening, accelerating the discovery and optimization of high-quality lead chemotypes for diverse clinical targets. Compared to traditional high-throughput screening, which is constrained to libraries of approximately one million compounds, the ultra-large virtual screening approach offers substantial advantages in both cost and time efficiency. By expanding the chemical space with compounds synthesized from easily accessible and reproducible reactions and utilizing a large, diverse set of building blocks, we can enhance both the diversity and quality of the discovered lead chemotypes. In this study, we explore new chemical spaces using reactions of sulfur(VI) fluorides to create a combinatorial library consisting of several hundred million compounds. We screened this virtual library for cannabinoid type II receptor (CB2) antagonists using the high-resolution structure in conjunction with a rationally designed antagonist, AM10257. The top-predicted compounds were then synthesized and tested in vitro for CB2 binding and functional antagonism, achieving an experimentally validated hit rate of 55%. Our findings demonstrate the effectiveness of reliable reactions, such as sulfur fluoride exchange, in diversifying ultra-large chemical spaces and facilitate the discovery of new lead compounds for important biological targets.

Cannabinoid Receptor 2 Blockade Prevents Anti-Depressive-like Effect of Cannabidiol Acid Methyl Ester in Female WKY Rats.

The pathophysiology of major depressive disorder (MDD) is diverse and multi-factorial, yet treatment strategies remain limited. While women are twice as likely to develop the disorder as men, many animal model studies of antidepressant response rely solely on male subjects. The endocannabinoid system has been linked to depression in clinical and pre-clinical studies. Cannabidiolic Acid-Methyl Ester (CBDA-ME, EPM-301) demonstrated anti-depressive-like effects in male rats. Here, we explored acute effects of CBDA-ME and some possible mediating mechanisms, using a depressive-like genetic animal model, the Wistar-Kyoto (WKY) rat. In Experiment 1, Female WKY rats underwent the Forced swim test (FST) following acute CBDA-ME oral ingestion (1/5/10 mg/kg). In Experiment 2, Male and female WKY rats underwent the FST after injection of CB1 (AM-251) and CB2 (AM-630) receptor antagonists 30 min before acute CBDA-ME ingestion (1 mg/kg, males; 5 mg/kg, females). Serum levels of Brain-Derived Neurotrophic Factor (BDNF), numerous endocannabinoids and hippocampal Fatty Acid Amide Hydrolase (FAAH) levels were assessed. Results indicate that females required higher doses of CBDA-ME (5 and 10 mg/kg) to induce an anti-depressive-like effect in the FST. AM-630 blocked the antidepressant-like effect in females, but not in males. The effect of CBDA-ME in females was accompanied by elevated serum BDNF and some endocannabinoids and low hippocampal expression of FAAH. This study shows a sexually diverse behavioral anti-depressive response to CBDA-ME and possible underlying mechanisms in females, supporting its potential use for treating MDD and related disorders.

Identification of a novel fatty acid binding protein-5-CB2 receptor-dependent mechanism regulating anxiety behaviors in the prefrontal cortex.

The endocannabinoid (eCB) system represents a promising neurobiological target for novel anxiolytic pharmacotherapies. Previous clinical and preclinical evidence has revealed that genetic and/or pharmacological manipulations altering eCB signaling modulate fear and anxiety behaviors. Water-insoluble eCB lipid anandamide requires chaperone proteins for its intracellular transport to degradation, a process that requires fatty acid-binding proteins (FABPs). Here, we investigated the effects of a novel FABP-5 inhibitor, SBFI-103, on fear and anxiety-related behaviors using rats. Acute intra-prelimbic cortex administration of SBFI-103 induced a dose-dependent anxiolytic response and reduced contextual fear expression. Surprisingly, both effects were reversed when a cannabinoid-2 receptor (CB2R) antagonist, AM630, was co-infused with SBFI-103. Co-infusion of the cannabinoid-1 receptor antagonist Rimonabant with SBFI-103 reversed the contextual fear response yet showed no reversal effect on anxiety. Furthermore, in vivo neuronal recordings revealed that intra-prelimbic region SBFI-103 infusion altered the activity of putative pyramidal neurons in the basolateral amygdala and ventral hippocampus, as well as oscillatory patterns within these regions in a CB2R-dependent fashion. Our findings identify a promising role for FABP5 inhibition as a potential target for anxiolytic pharmacotherapy. Furthermore, we identify a novel, CB2R-dependent FABP-5 signaling pathway in the PFC capable of strongly modulating anxiety-related behaviors and anxiety-related neuronal transmission patterns.

Targeting cannabinoid receptor 2 (CB2) limits collagen production-An in vitro study in a primary culture of human fibroblasts.

Previous studies showed that cannabinoid 2 (CB2) receptor is involved in skin inflammation, fibrogenesis and re-epithelialization in mice, indicating that this receptor may be implicated in wound healing. Thus, topical use of cannabinoids may have a role in local fibrotic and wound healing diseases such as scars or keloids. We investigate the effect of the CB2 selective receptor agonist (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (JWH133) and the CB2 selective receptor antagonist (6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl)(4-methoxyphenyl)-methanone (AM630), on primary cultures of human fibroblasts. Primary cultures of adult human fibroblasts were obtained from abdominal human skin samples. Fibroblasts pretreated with JWH133 and/or AM630 were stimulated with TGF-ß (10 ng/ml). Fibroblast activation into myofibroblasts was quantified by the expression of alpha-smooth muscle actin (α-SMA) using Immunocytochemistry and Western Blot assays. Collagen content was quantified with the Sirius red staining assay. Upon human fibroblasts stimulation with TGF-ß, a significant increase on α-SMA and CB2 receptor expression was observed. In these cells, JWH133 decreased α-SMA expression and collagen content. However, this effect was not observed in resting human fibroblasts. AM630 decreased α-SMA expression and collagen content in both resting and activated fibroblasts. This effect was time- and concentration-dependent with an IC50 value of 11 µM. The CB2 receptor appears to be involved in fibroblast repair during skin wound healing in humans, as TGF-ß increases CB2 receptor expression and JWH133 produces an anti-fibrotic effect in human fibroblasts. AM630 also showed an anti-fibrotic effect hypothesizing that other cannabinoid receptors, such as TRPV, may be involved in this response.

Identification of Novel Antagonists Targeting Cannabinoid Receptor 2 Using a Multi-Step Virtual Screening Strategy.

The endocannabinoid system plays an essential role in the regulation of analgesia and human immunity, and Cannabinoid Receptor 2 (CB2) has been proved to be an ideal target for the treatment of liver diseases and some cancers. In this study, we identified CB2 antagonists using a three-step "deep learning-pharmacophore-molecular docking" virtual screening approach. From the ChemDiv database (1,178,506 compounds), 15 hits were selected and tested by radioligand binding assays and cAMP functional assays. A total of 7 out of the 15 hits were found to exhibit binding affinities in the radioligand binding assays against CB2 receptor, with a pKi of 5.15-6.66, among which five compounds showed antagonistic activities with pIC50 of 5.25-6.93 in the cAMP functional assays. Among these hits, Compound 8 with the 4H-pyrido[1,2-a]pyrimidin-4-one scaffold showed the best binding affinity and antagonistic activity with a pKi of 6.66 and pIC50 of 6.93, respectively. The new scaffold could serve as a lead for further development of CB2 drugs. Additionally, we hope that the model in this study could be further utilized to identify more novel CB2 receptor antagonists, and the developed approach could also be used to design potent ligands for other therapeutic targets.

Virtual screening using docking and molecular dynamics of cannabinoid analogs against CB1 and CB2 receptors.

BACKGROUND: Cannabis sativa has been attributed to different pharmacological properties. A number of secondary metabolites such as tetrahydrocannabinol (THC), cannabinol (CBD), and different analogs, with highly promising biological activity on CB1 and CB2 receptors, have been identified. METHODS: Thus, this study aimed was to evaluate the activity of THC, CBD, and their analogs using molecular docking and molecular dynamics simulations (MD) methods. Initially, the molecules (ligands) were selected by bioinformatics searches in databases. Subsequently, CB1 and CB2 receptors were retrieved from the protein data bank database. Afterward, each receptor and its ligands were optimized to perform molecular docking. Then, MD Simulation was performed with the most stable ligand-receptor complexes. Finally, the Molecular Mechanics-Generalized Born Surface Area (MM-PBSA) method was applied to analyze the binding free energy between ligands and cannabinoid receptors. RESULTS: The results obtained showed that ligand LS-61176 presented the best affinity in the molecular docking analysis. Also, this analog could be a CB1 negative allosteric modulator like CBD and probably an agonist in CB2 like THC and CBD according to their dynamic behavior in silico. The possibility of having a THC and a CBD analog (LS-61176) as a promising molecule for experimental evaluation since it could have no central side-effects on CB1 and have effects of CB2 useful in pain, inflammation, and some immunological disorders. Docking results were validate using ROC curve for both cannabinoids receptor where AUC for CB1 receptor was 0.894±0.024, and for CB2 receptor AUC was 0.832±0032, indicating good affinity prediction.

Activation of CB2 receptor inhibits pyroptosis and subsequently ameliorates cecal ligation and puncture-induced sepsis.

BACKGROUND: Cannabinoid receptor 2 (CB2), whose activities are upregulated during sepsis, may be related to the regulation of inflammatory programmed cell death called pyroptosis. The aim of this study is to investigate the role of CB2 activation in attenuation of inflammation through inhibiting pyroptosis in cecal ligation puncture (CLP)-induced sepsis andlipopolysaccharide (LPS) + ATP-stimulated macrophages. METHODS: C57BL/6 mice were subjected to CLP procedure and treated with CB2 agonist HU308 and CB2 antagonist AM630. Lung tissues were collected for analyses of lung W/D ratio, inflammatory factors levels, and pyroptosis-related protein expression. Murine bone-marrow-derived macrophages (BMDM) were treated with LPS and ATP to construct a septic model in vitro in the presence of HU308 and AM630 for assessment of cell injury, cytokine levels and pyroptosis-related protein expression accordingly. To verify the relationship between CB2 receptors and pyroptosis in the process of inflammatory response, BMDM were transduced with CB2 receptors knockdown lentiviral vectors in the presence of HU308 and AM630 for assessment of pyroptosis-related protein expression. RESULTS: CB2 activation ameliorated the release of inflammatory mediators. The results showed that CLP-induced pyroptosis was elevated, and CB2 agonist HU308 treatment inhibited the pyroptosis activity through a decrease of the protein levels of NLRP3 as well as caspase-1 and GSDMD activation. Similar results were obtained in BMDM after LPS and ATP treatment. Treatment with CB2 knockdown lentiviral particles prevented the HU308-induced decreases in cell pyroptosis, demonstrating that endogenous CB2 receptors are required for the cannabinoid-induced cell protection. CONCLUSIONS: CB2 receptors activation plays a protective role in sepsis through inhibition of pyroptosis. The effect of CB2 receptors against pyroptosis depends on the existence of endogenous CB2 receptors.

Zerumbone Ameliorates Neuropathic Pain Symptoms via Cannabinoid and PPAR Receptors Using In Vivo and In Silico Models.

Neuropathic pain is a chronic pain condition persisting past the presence of any noxious stimulus or inflammation. Zerumbone, of the Zingiber zerumbet ginger plant, has exhibited anti-allodynic and antihyperalgesic effects in a neuropathic pain animal model, amongst other pharmacological properties. This study was conducted to further elucidate the mechanisms underlying zerumbone's antineuropathic actions. Research on therapeutic agents involving cannabinoid (CB) and peroxisome proliferator-activated receptors (PPARs) is rising. These receptor systems have shown importance in causing a synergistic effect in suppressing nociceptive processing. Behavioural responses were assessed using the von Frey filament test (mechanical allodynia) and Hargreaves plantar test (thermal hyperalgesia), in chronic constriction injury (CCI) neuropathic pain mice. Antagonists SR141716 (CB1 receptor), SR144528 (CB2 receptor), GW6471 (PPARα receptor) and GW9662 (PPARγ receptor) were pre-administered before the zerumbone treatment. Our findings indicated the involvement of CB1, PPARα and PPARγ in zerumbone's action against mechanical allodynia, whereas only CB1 and PPARα were involved against thermal hyperalgesia. Molecular docking studies also suggest that zerumbone has a comparable and favourable binding affinity against the respective agonist on the CB and PPAR receptors studied. This finding will contribute to advance our knowledge on zerumbone and its significance in treating neuropathic pain.

Protocol for crystal structure determination of the antagonist-bound human cannabinoid receptor CB2.

Human cannabinoid receptor CB2 plays an important role in the immune system and is an attractive therapeutic target for pain and for inflammatory and neurodegenerative diseases. However, the structural basis of CB2 agonist selectivity is still elusive. Here, we describe a detailed protocol for the determination of the crystal structure of antagonist AM10257-bound CB2. This methodology could be applied to the structural studies of CB2 with diverse antagonists and agonists or to other class A G-protein-coupled receptors. For complete details on the use and execution of this protocol, please refer to Li et al. (2019).

Synthesis, Molecular Pharmacology, and Structure-Activity Relationships of 3-(Indanoyl)indoles as Selective Cannabinoid Type 2 Receptor Antagonists.

Synthetic indole cannabinoids characterized by a 2',2'-dimethylindan-5'-oyl group at the indole C3 position constitute a new class of ligands possessing high affinity for human CB2 receptors at a nanomolar concentration and a good selectivity index. Starting from the neutral antagonist 4, the effects of indole core modification on the pharmacodynamic profile of the ligands were investigated. Several N1 side chains afforded potent and CB2-selective neutral antagonists, notably derivatives 26 (R1 = n-propyl, R2 = H) and 35 (R1 = 4-pentynyl, R2 = H). Addition of a methyl group at C2 improved the selectivity for the CB2 receptor. Moreover, C2 indole substitution may control the CB2 activity as shown by the functionality switch in 35 (antagonist) and 49 (R1 = 4-pentynyl, R2 = CH3, partial agonist).

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2.

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB2 by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB2 in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB2.

Targeting 2-arachidonoylglycerol signalling in the neurobiology and treatment of depression.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is an atypical neurotransmitter synthesized on demand in response to a wide range of stimuli, including exposure to stress. Through the activation of cannabinoid receptors, 2-AG can interfere with excitatory and inhibitory neurotransmission in different brain regions and modulate behavioural, endocrine and emotional components of the stress response. Exposure to chronic or intense unpredictable stress predisposes to maladaptive behaviour and is one of the main risk factors involved in developing mood disorders, such as major depressive disorder (MDD). In this review, we describe the molecular mechanisms involved in 2-AG signalling in the brain of healthy and stressed animals and discuss how such mechanisms could modulate stress adaptation and susceptibility to depression. Furthermore, we review preclinical evidence indicating that the pharmacological modulation of 2-AG signalling stands as a potential new therapeutic target in treating MDD. Particular emphasis is given to the pharmacological augmentation of 2-AG levels by monoacylglycerol lipase (MAGL) inhibitors and the modulation of CB2 receptors.

CB2 receptors modulate seizure-induced expression of pro-inflammatory cytokines in the hippocampus but not neocortex.

We compared neuroinflammatory responses induced by nonconvulsive and convulsive seizures and analyzed the role that may be played by cannabinoid CB2 receptors in the neuroinflammatory response induced by generalized tonic-clonic seizures (GTCS). Using quantitative PCR, we analyzed expression of interleukin-1b, CCL2, interleukin-6, tumor necrosis factor (TNF), transforming growth factor beta 1 (TGFb1), fractalkine, and cannabinoid receptor type 2 in the neocortex, dorsal and ventral hippocampus, cortical leptomeninges, dura mater, and spleen in 3 and 6 h after induction of GTCS by a high dose of pentylenetetrazole (PTZ, 70 mg/kg) and absence-like activity by a low dose of PTZ (30 mg/kg). The low dose of PTZ had no effect on the gene expression 3 and 6 h after PTZ injection. In 3 and 6 h after high PTZ dose, the expression of CCL2 and TNF increased in the neocortex. Both ventral and dorsal parts of the hippocampus responded to seizures by elevation of CCL2 expression 3 h after PTZ. Cortical leptomeninges but not dura mater also had elevated CCL2 level and decreased TGFb1 expression 3 h after GTCS. Activation of CB2 receptors by HU308 suppressed an inflammatory response only in the dorsal hippocampus but not neocortex. Suppression of CB2 receptors by AM630 potentiated expression of inflammatory cytokines also in the hippocampus but not in the neocortex. Thus, we showed that GTCS, but not the absence-like activity, provoke inflammatory response in the neocortex, dorsal and ventral hippocampus, and cortical leptomeninges. Modulation of CB2 receptors changes seizure-induced neuroinflammation only in the hippocampus but not neocortex.

Cannabinoid Receptor Modulation of Neurogenesis: ST14A Striatal Neural Progenitor Cells as a Simplified In Vitro Model.

The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches. We report that ST14A cells express various ECS components, supporting the presence of an active ECS. While CB1 and CB2 receptor blockade did not affect ST14A cell number, exogenous administration of the endocannabinoid 2-AG and the synthetic CB2 agonist JWH133 increased ST14A cell proliferation. Phospholipase C (PLC), but not PI3K pharmacological blockade negatively modulated CB2-induced ST14A cell proliferation, suggesting that a PLC pathway is involved in the steps downstream to CB2 activation. On the basis of our results, we propose ST14A neural progenitor cells as a useful in vitro model for studying ECS modulation of neurogenesis, also in prospective in vivo pharmacological studies.

Analogues of cannabinoids as multitarget drugs in the treatment of Alzheimer's disease.

Given that neuronal degeneration in Alzheimer's disease (AD) is caused by the combination of multiple neurotoxic insults, current directions in the research of novel therapies to treat this disease attempts to design multitarget strategies that could be more effective than the simply use of acetylcholinesterase inhibitors; currently, the most used therapy for AD. One option, explored recently, is the synthesis of new analogues of cannabinoids that could competitively inhibit the acetylcholinesterase (AChE) enzyme and showing the classic neuroprotective profile of cannabinoid compounds. In this work, molecular docking has been used to design some cannabinoid analogues with such multitarget properties, based on the similarities of donepezil and Δ9-tetrahydrocannabinol. The analogues synthesized, compounds 1 and 2, demonstrated to have two interesting characteristics in different in vitro assays: competitive inhibition of AChE and competitive antagonism at the CB1/CB2 receptors. They are highly lipophilic, highlighting that they could easily reach the CNS, and apparently presented a low toxicity. These results open the door to the synthesis of new compounds for a more effective treatment of AD.

Modern approaches to the development of synthetic cannabinoid receptor probes.

The endocannabinoid system, which spans the central and peripheral nervous systems and regulates many biologic processes, is an important target for probe discovery and medications development. Whereas the earliest endocannabinoid receptor probes were derivatives of the non-selective phytocannabinoids isolated from Cannabis species, modern drug discovery techniques have expanded the definitions of what constitutes a CB1R or CB2R cannabinoid receptor ligand. This review highlights recent advances in synthetic cannabinoid receptor chemistry and pharmacology. We provide examples of new CB1R- and CB2R-selective probes, and discuss rational approaches to the design of peripherally-restricted agents. We also describe structural classes of positive- and negative allosteric modulators (PAMs and NAMs) of CB1R and CB2R. Finally, we introduce new opportunities for cannabinoid receptor probe development that have emerged in recent years, including biased agonists that may lead to medications lacking adverse effects.

Endocannabinoid System as a Promising Therapeutic Target in Inflammatory Bowel Disease - A Systematic Review.

Inflammatory bowel disease (IBD) is a general term used to describe a group of chronic inflammatory conditions of the gastrointestinal tract of unknown etiology, including two primary forms: Crohn's disease (CD) and ulcerative colitis (UC). The endocannabinoid system (ECS) plays an important role in modulating many physiological processes including intestinal homeostasis, modulation of gastrointestinal motility, visceral sensation, or immunomodulation of inflammation in IBD. It consists of cannabinoid receptors (CB1 and CB2), transporters for cellular uptake of endocannabinoid ligands, endogenous bioactive lipids (Anandamide and 2-arachidonoylglycerol), and the enzymes responsible for their synthesis and degradation (fatty acid amide hydrolase and monoacylglycerol lipase), the manipulation of which through agonists and antagonists of the system, shows a potential therapeutic role for ECS in inflammatory bowel disease. This review summarizes the role of ECS components on intestinal inflammation, suggesting the advantages of cannabinoid-based therapies in inflammatory bowel disease.

CB2R induces a protective response against epileptic seizures through ERK and p38 signaling pathways.

BACKGROUND AND PURPOSE: Epilepsy is a pivotal neurological disorder characterized by the synchronous discharging of neurons to induce momentary brain dysfunction. Temporal lobe epilepsy is the most common type of epilepsy, with seizures originating from the mesial temporal lobe. The hippocampus forms part of the mesial temporal lobe and plays a significant role in epileptogenesis; it also has a vital influence on the mental development of children. In this study, we aimed to explore the effects of CB2 receptor (CB2R) activation on ERK and p38 signaling in nerve cells of a rat epilepsy model. MATERIALS AND METHODS: We treated Sprague-Dawley rats with pilocarpine to induce an epilepsy model and treated such animals with a CB2R agonist (JWH133) alone or with a CB2R antagonist (AM630). Nissl's stain showed the neuron conditon in different groups. Western blot analyzed the level of p-ERK and p-p38. RESULTS: JWH133 can increase the latent period of first seizure attack and decrease the Grades IV-V magnitude ratio after the termination of SE. Nissl's stain showed JWH133 protected neurons in the hippocampus while AM630 inhibited the functioning of CB2R in neurons. Western blot analysis showed that JWH133 decreased levels of p-ERK and p-p38, which is found at increased levels in the hippocampus of our epilepsy model. In contrast, AM630 inhibited the protective function of JWH133 and also enhanced levels of p-ERK and p-p38. CONCLUSIONS: CB2R activation can induce neurons proliferation and survival through activation of ERK and p38 signaling pathways.

URB597 abrogates anxiogenic and depressive behaviors in the methamphetamine-withdrawal mice: Role of the cannabinoid receptor type 1, cannabinoid receptor type 2, and transient receptor potential vanilloid 1 channels.

BACKGROUND: Methamphetamine is an addictive stimulant that possesses toxicity in the brain when taken repeatedly or at higher doses. Methamphetamine neurotoxicity is associated with numerous forms of mental impairment, including depression and anxiety. Evidence has also demonstrated that the endocannabinoid system is involved in the regulation of anxiety and depression. AIMS: This study was designed to determine the involvement of the endocannabinoid system in anxiety- and depression-related behaviors in methamphetamine-withdrawal male NMRI mice. METHODS: The elevated plus maze and forced swim test were used to assess the level of anxiety and depression. RESULTS: We found that methamphetamine (30 mg/kg, intraperitoneal) evoked depressive- and anxiogenic-like effects at 3 days post-administration. Injection of URB597 (5-10 ng/mouse, intracerebroventricular), 10 min before the test, prevented the emotional deficits induced by methamphetamine withdrawal. Moreover, the cannabinoid receptor type 1 antagonist AM251 (1 µg/mouse) or cannabinoid receptor type 2 antagonist AM630 (5 and 10 µg/mouse) suppressed the antidepressant activity in the methamphetamine-withdrawal mice treated with URB597. The transient receptor potential vanilloid 1 antagonist capsazepine (25 µg/mouse) prevented while capsazepine (100 µg/mouse) potentiated the antidepressant efficacy in the methamphetamine-withdrawal mice treated with URB597. The higher dose of AM630 and two higher doses of capsazepine had antidepressant efficacy, by themselves. Furthermore, capsazepine (50 µg/mouse) increased locomotion in the methamphetamine-withdrawal mice treated with URB597. CONCLUSIONS: The results suggest that URB597 has a potential for preventing methamphetamine withdrawal-evoked anxiety and depression. Cannabinoid type 1 receptors, cannabinoid type 2 receptors and transient receptor potential vanilloid 1 differently affect depression-related behaviors in methamphetamine-withdrawal mice treated with URB597.

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands.

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure-activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 µM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.