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G protein-coupled receptors (GPCRs) exist within a landscape of interconvertible conformational states and in dynamic equilibrium between monomers and higher-order oligomers, both influenced by ligand binding. Here, we show that a homobivalent ligand formed by equal chromenopyrazole moieties as pharmacophores, connected by 14 methylene units, can modulate the dynamics of the cannabinoid CB 2 receptor (CB 2 R) homodimerization by simultaneously binding both protomers of the CB 2 R-CB 2 R homodimer. Computational and pharmacological experiments showed that one of the ligand pharmacophores binds to the orthosteric site of one protomer, and the other pharmacophore to a membrane-oriented pocket between transmembranes 1 and 7 of the partner protomer. This results in unique pharmacological properties, including increased potency in G i -mediated signaling and enhanced recruitment of β-arrestin. Thus, by modulating dimerization dynamics, it may be possible to fine-tune CB 2 R activity, potentially leading to improved therapeutic outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial or other interests that could have influenced the published work in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The bioinformatic analysis of cannabinoid receptors (CBRs) CB 1 and CB 2 reveals a detailed picture of their structure, evolution, and physiological significance within the endocannabinoid system (ECS). The study highlights the evolutionary conservation of these receptors evidenced by sequence alignments across diverse species including humans, amphibians, and fish. Both CBRs share a structural hallmark of seven transmembrane (TM) helices, characteristic of class A G-protein-coupled receptors (GPCRs), which are critical for their signalling functions. The study reports a similarity of 44.58 % between both CBR sequences, which suggests that while their evolutionary paths and physiological roles may differ, there is considerable conservation in their structures. Pathway databases like KEGG, Reactome, and WikiPathways were employed to determine the involvement of the receptors in various signalling pathways. The pathway analyses integrated within this study offer a detailed view of the CBRs interactions within a complex network of cannabinoid-related signalling pathways. High-resolution crystal structures (PDB ID: 5U09 for CB 1 and 5ZTY for CB 2 ) provided accurate structural information, showing the binding pocket volume and surface area of the receptors, essential for ligand interaction. The comparison between these receptors' natural sequences and their engineered pseudo-CBRs (p-CBRs) showed a high degree of sequence identity, confirming the validity of using p-CBRs in receptor-ligand interaction studies. This comprehensive analysis enhances the understanding of the structural and functional dynamics of cannabinoid receptors, highlighting their physiological roles and their potential as therapeutic targets within the ECS., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The cannabinoid type 2 receptor (CB 2 R), a G protein-coupled receptor, is an important regulator of immune cell function and a promising target to treat chronic inflammation and fibrosis. While CB 2 R is typically targeted by small molecules, including endo-, phyto-, and synthetic cannabinoids, peptides-owing to their size-may offer a different interaction space to facilitate differential interactions with the receptor. Here, we explore plant-derived cyclic cystine-knot peptides as ligands of the CB 2 R. Cyclotides are known for their exceptional biochemical stability. Recently, they gained attention as G protein-coupled receptor modulators and as templates for designing peptide ligands with improved pharmacokinetic properties over linear peptides. Cyclotide-based ligands for CB 2 R were profiled based on a peptide-enriched extract library comprising nine plants. Employing pharmacology-guided fractionation and peptidomics, we identified the cyclotide vodo-C1 from sweet violet (Viola odorata) as a full agonist of CB 2 R with an affinity (K i ) of 1 μM and a potency (EC 50 ) of 8 μM. Leveraging deep learning networks, we verified the structural topology of vodo-C1 and modeled its molecular volume in comparison to the CB 2 R ligand binding pocket. In a fragment-based approach, we designed and characterized vodo-C1-based bicyclic peptides (vBCL1-4), aiming to reduce size and improve potency. Opposite to vodo-C1, the vBCL peptides lacked the ability to activate the receptor but acted as negative allosteric modulators or neutral antagonists of CB 2 R. This study introduces a macrocyclic peptide phytocannabinoid, which served as a template for the development of synthetic CB 2 R peptide modulators. These findings offer opportunities for future peptide-based probe and drug development at cannabinoid receptors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

3-3'-Diindolylmethane (DIM) is a biologically active dimer derived from the endogenous conversion of indole-3-carbinol (I3C), a naturally occurring glucosinolate found in many cruciferous vegetables (i.e., Brassicaceae ). DIM was the first pure androgen receptor antagonist isolated from the Brassicaceae family and has been recently investigated for its potential pharmacological use in prostate cancer prevention and treatment. Interestingly, there is evidence that DIM can also interact with cannabinoid receptors. In this context, by considering the well-known involvement of the endocannabinoid system in prostate cancer, we have pharmacologically characterized the properties of DIM on both CB 1 and CB 2 cannabinoid receptors in two human prostate cancer cell lines: PC3 (androgen-independent/androgen receptor negative) and LNCaP (androgen-dependent). In the PC3 cell line, DIM was able to activate CB 2 receptors and potentially associated apoptotic pathways. On the other hand, although DIM was also able to activate CB 2 receptors in the LNCaP cell line, no apoptotic effects were observed. Our evidence confirms that DIM is a CB 2 receptor ligand and, moreover, it has a potential anti-proliferative effect on androgen-independent/androgen receptor-negative prostate cancer cells.

A series of novel cannabinoid-type derivatives were synthesized by the coupling of (1S,4R)-(+) and (1R,4S)-(-)-fenchones with various resorcinols/phenols. The fenchone-resorcinol derivatives were fluorinated using Selectfluor and demethylated using sodium ethanethiolate in dimethylformamide (DMF). The absolute configurations of four compounds were determined by X-ray single crystal diffraction. The fenchone-resorcinol analogs possessed high affinity and selectivity for the CB2 cannabinoid receptor. One of the analogues synthesized, 2-(2',6'-dimethoxy-4'-(2″-methyloctan-2″-yl)phenyl)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol ( 1d ), had a high affinity (K i = 3.51 nM) and selectivity for the human CB2 receptor (hCB2). In the [ 35 S]GTPγS binding assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC 50 = 2.59 nM, E (max) = 89.6%). Two of the fenchone derivatives were found to possess anti-inflammatory and analgesic properties. Molecular-modeling studies elucidated the binding interactions of 1d within the CB2 binding site.

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

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 pK i of 5.15-6.66, among which five compounds showed antagonistic activities with pIC 50 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 pK i of 6.66 and pIC 50 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.

Evidence supports a role of host genetic diversity in the clinical course of coronavirus disease 2019 (COVID-19). Variation in the cannabinoid CB2 receptor gene (CNR2) could affect the regulatory action of endocannabinoids on the immune system, resulting in an increased risk of various inflammatory diseases. The present study investigated the relationship between the CNR2-Q63R variant and COVID-19 severity. A total of 200 Iranian COVID-19 patients were enrolled in the study and genotyped using a TaqMan assay. The co-dominant, dominant, recessive, over-dominant, and additive inheritance models were analyzed using SNPStats software. In silico molecular docking was also performed to simulate the effects of the Q63R variation on CB2 binding with a ligand and with the G-protein. A significant difference in the Q63R allele and genotype distribution was found between expired and discharged COVID-19 patients in co-dominant, recessive, and additive inheritance models. The molecular docking results showed that the predicted structure of mutant CB2 (63R type) could not bind to the G-protein in the correct position. The data indicated that the Q63R variation in the CNR2 gene may affect the severity of COVID-19. Identification of genes related to susceptibility and severity of COVID-19 may lead to specific targets for drug repurposing or development., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The activation of the human cannabinoid receptor type II (CB2R) is known to mediate analgesic and anti-inflammatory processes without the central adverse effects related to cannabinoid receptor type I (CB1R). In this work we describe the synthesis and evaluation of a novel series of N-aryl-2-pyridone-3-carboxamide derivatives tested as human cannabinoid receptor type II (CB2R) agonists. Different cycloalkanes linked to the N-aryl pyridone by an amide group displayed CB2R agonist activity as determined by intracellular [cAMP] levels. The most promising compound 8d exhibited a non-toxic profile and similar potency (EC50 = 112 nM) to endogenous agonists Anandamide (AEA) and 2-Arachidonoylglycerol (2-AG) providing new information for the development of small molecules activating CB2R. Molecular docking studies showed a binding pose consistent with two structurally different agonists WIN-55212-2 and AM12033 and suggested structural requirements on the pyridone substituents that can satisfy the orthosteric pocket and induce an agonist response. Our results provide additional evidence to support the 2-pyridone ring as a suitable scaffold for the design of CB2R agonists and represent a starting point for further optimization and development of novel compounds for the treatment of pain and inflammation.

The Sterling Research Group identified pravadoline as an aminoalkylindole (AAI) non-steroidal anti-inflammatory pain reliever. As drug design progressed, the ability of AAI analogs to block prostaglandin synthesis diminished, and antinociceptive activity was found to result from action at the CB 1 cannabinoid receptor, a G-protein-coupled receptor (GPCR) abundant in the brain. Several laboratories applied computational chemistry methods to ultimately conclude that AAI and cannabinoid ligands could overlap within a common binding pocket but that WIN55212-2 primarily utilized steric interactions via aromatic stacking, whereas cannabinoid ligands required some electrostatic interactions, particularly involving the CB 1 helix-3 lysine. The Huffman laboratory identified strategies to establish CB 2 receptor selectivity among cannabimimetic indoles to avoid their CB 1 -related adverse effects, thereby stimulating preclinical studies to explore their use as anti-hyperalgesic and anti-allodynic pharmacotherapies. Some AAI analogs activate novel GPCRs referred to as "Alkyl Indole" receptors, and some AAI analogs act at the colchicine-binding site on microtubules. The AAI compounds having the greatest potency to interact with the CB 1 receptor have found their way into the market as "Spice" or "K2". The sale of these alleged "herbal products" evades FDA consumer protections for proper labeling and safety as a medicine, as well as DEA scheduling as compounds having no currently accepted medical use and a high potential for abuse. The distribution to the public of potent alkyl indole synthetic cannabimimetic chemicals without regard for consumer safety contrasts with the adherence to regulatory requirements for demonstration of safety that are routinely observed by ethical pharmaceutical companies that market medicines.

Azobenzene-embedded photoswitchable ligands are the widely used chemical tools in photopharmacological studies. Current approaches to azobenzene introduction rely mainly on the isosteric replacement of typical azologable groups. However, atypical scaffolds may offer more opportunities for photoswitch remodeling, which are chemically in an overwhelming majority. Herein, we investigate the rational remodeling of atypical scaffolds for azobenzene introduction, as exemplified in the development of photoswitchable ligands for the cannabinoid receptor 2 (CB2). Based on the analysis of residue-type clusters surrounding the binding pocket, we conclude that among the three representative atypical arms of the CB2 antagonist, AM10257, the adamantyl arm is the most appropriate for azobenzene remodeling. The optimizing spacer length and attachment position revealed AzoLig 9 with excellent thermal bistability, decent photopharmacological switchability between its two configurations, and high subtype selectivity. This structure-guided approach gave new impetus in the extension of new chemical spaces for tool customization for increasingly diversified photo-pharmacological studies and beyond.

β-arrestins are partners of the G protein-coupled receptors (GPCRs), regulating their intracellular trafficking and signaling. Development of biased GPCR agonists, selectively targeting either G protein or β-arrestin pathways, are in the focus of interest due to their therapeutic potential in different pathological conditions. The CB 2 cannabinoid receptor (CB 2 R) is a GPCR involved in various functions in the periphery and the central nervous system. Two common occurring variants of CB 2 R, harboring Q63R or L133I missense mutations, have been implicated in the development of a diverse set of disorders. To evaluate the effect of these mutations, we characterized the binding profile of these mutant CB 2 receptors to G proteins and β-arrestin2. Although their ability to inhibit cAMP signaling was similar, the Q63R mutant had increased, whereas the L133I mutant receptor had decreased β-arrestin2 binding. In line with these observations, the variants also had altered intracellular trafficking. Our results show that two common variants of the CB 2 receptor have biased signaling properties, which may contribute to the pathogenesis of the associated disorders and may offer CB 2 R as a target for further development of biased receptor activation strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Turu, Soltész-Katona, Tóth, Juhász, Cserző, Misák, Balla, Caron and Hunyady.)

Cannabinoid receptors type 2 (CB2R) represent an attractive therapeutic target for neurodegenerative diseases and cancer. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor receptor density and/or occupancy during a CB2R-tailored therapy, we herein describe the radiosynthesis of cis -[ 18 F]1-(4-fluorobutyl- N -((1s,4s)-4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide ([ 18 F] LU14 ) starting from the corresponding mesylate precursor. The first biological evaluation revealed that [ 18 F] LU14 is a highly affine CB2R radioligand with >80% intact tracer in the brain at 30 min p.i. Its further evaluation by PET in a well-established rat model of CB2R overexpression demonstrated its ability to selectively image the CB2R in the brain and its potential as a tracer to further investigate disease-related changes in CB2R expression.

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)., Competing Interests: The authors declare no competing interests., (© 2021.)

Cannabis sativa L. is a source of over 150 active compounds known as phytocannabinoids that are receiving renewed interest due to their diverse pharmacologic activities. Indeed, phytocannabinoids mimic the endogenous bioactive endocannabinoids effects through activation of CB1 and CB2 receptors widely described in the central nervous system and peripheral tissues. All phytocannabinoids have been studied for their protective actions towards different biological mechanisms, including inflammation, immune response, oxidative stress that, altogether, result in an inhibitory activity against the carcinogenesis. The role of the endocannabinoid system is not yet completely clear in cancer, but several studies indicate that cannabinoid receptors and endogenous ligands are overexpressed in different tumor tissues. Recently, in vitro and in vivo evidence support the effectiveness of phytocannabinoids against various cancer types, in terms of proliferation, metastasis, and angiogenesis, actions partially due to their ability to regulate signaling pathways critical for cell growth and survival. The aim of this review was to report the current knowledge about the action of phytocannabinoids from Cannabis sativa L. against cancer initiation and progression with a specific regard to brain, breast, colorectal, and lung cancer as well as their possible use in the therapies. We will also report the known molecular mechanisms responsible for such positive effects. Finally, we will describe the actual therapeutic options for Cannabis sativa L. and the ongoing clinical trials.

Cannabinoids comprise different classes of compounds, which aroused interest in recent years because of their several pharmacological properties. Such properties include analgesic activity, bodyweight reduction, the antiemetic effect, the reduction of intraocular pressure and many others, which appear correlated to the affinity of cannabinoids towards CB 1 and/or CB 2 receptors. Within the search aiming to identify novel chemical scaffolds for cannabinoid receptor interaction, the CB 1 antagonist/inverse agonist pyrazole-based derivative rimonabant has been modified, giving rise to several tricyclic pyrazole-based compounds, most of which endowed of high affinity and selectivity for CB 1 or CB 2 receptors. The aim of this review is to present the synthesis and summarize the SAR study of such tricyclic pyrazole-based compounds, evidencing, for some derivatives, their potential in the treatment of neuropathic pain, obesity or in the management of glaucoma.

Single chemical entities with potential to simultaneously interact with two binding sites are emerging strategies in medicinal chemistry. We have designed, synthesized and functionally characterized the first bitopic ligands for the CB 2 receptor. These compounds selectively target CB 2 versus CB 1 receptors. Their binding mode was studied by molecular dynamic simulations and site-directed mutagenesis., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

The selective targeting of the cannabinoid receptor 2 (CB2) is crucial for the development of peripheral system-acting cannabinoid analgesics. This work aimed at computer-assisted identification of prospective CB2-selective compounds among the constituents of Cannabis Sativa . The molecular structures and corresponding binding affinities to CB1 and CB2 receptors were collected from ChEMBL. The molecular structures of Cannabis Sativa constituents were collected from a phytochemical database. The collected records were curated and applied for the development of quantitative structure-activity relationship (QSAR) models with a machine learning approach. The validated models predicted the affinities of Cannabis Sativa constituents. Four structures of CB2 were acquired from the Protein Data Bank (PDB) and the discriminatory ability of CB2-selective ligands and two sets of decoys were tested. We succeeded in developing the QSAR model by achieving Q 2 5-CV > 0.62. The QSAR models helped to identify three prospective CB2-selective molecules that are dissimilar to already tested compounds. In a complementary structure-based virtual screening study that used available PDB structures of CB2, the agonist-bound, Cryogenic Electron Microscopy structure of CB2 showed the best statistical performance in discriminating between CB2-active and non-active ligands. The same structure also performed best in discriminating between CB2-selective ligands from non-selective ligands.

Citral ((2 E )-3,7-dimethylocta-2,6-dienal), a bioactive component of lemongrass, inhibits oxidant activity, nuclear factor kappa B (NF-κB) activation, and cyclooxygenase-2 (COX-2) expression, even as it activates peroxisome proliferator-activated receptor (PPAR)-α and γ. Additionally, citral produces long-lasting inhibition of transient receptor potential (TRP) channels that are found in sensory neurons, such as TRPV1-3 and TRPM8, while it transiently blocks TRPV4 and TRPA1. Here, the effect of citral in experimental models of acute inflammation and hyperalgesia in mice, and the underlying citral mechanisms of action were investigated. ADMET properties and molecular targets were predicted using the online server. The immunomodulatory and antihyperalgesic effects of citral were evaluated, using mechanical and thermal stimuli, at different time-points on carrageenan, lipopolysaccharides (LPS), and zymosan-induced paw edema and hyperalgesia in mice. ADMET analysis ensures that the citral has not violated Lipinski's rule of five, indicating its safety consumption, and molecular target prediction software identified that citral is a potential fatty acid amide hydrolase (FAAH) inhibitor. Oral treatment with citral (50-300 mg/kg) significantly inhibited carrageenan-induced paw edema and thermal allodynia. Furthermore, citral modulated the inflammation induced by LPS and zymosan, toll-like receptor (TLR) 4, and TLR2/dectin-1 ligands, respectively. Moreover, pretreatment with cannabinoid receptor type 2 (CB2R) antagonists and ATP-sensitive K + channel inhibitor, but not with a cannabinoid receptor type 1 (CB1R) antagonist, significantly reversed the anti-inflammatory effect of citral. Intriguingly, citral did not cause any relevant action in the central nervous system, and it was safe when assessed in a 14 day toxicity assay in male mice. Therefore, citral constitutes a promising, innovative, and safe molecule for the management of immunoinflammatory conditions and pain states.

Cannabinoids are a group of chemical compounds that have been used for thousands of years due to their psychoactive function and systemic physiological effects. There are at least two types of cannabinoid receptors, CB1 and CB2, which belong to the G protein-coupled receptor superfamily and can trigger different signaling pathways to exert their physiological functions. In this study, several representative agonists and antagonists of both CB1 and CB2 were systematically studied to predict their binding affinities and selectivity against both cannabinoid receptors using a set of hierarchical molecular modeling and simulation techniques, including homology modeling, molecular docking, molecular dynamics (MD) simulations and end point binding free energy calculations using the molecular mechanics/Poisson-Boltzmann surface area-WSAS (MM-PBSA-WSAS) method, and molecular mechanics/generalized Born surface area (MM-GBSA) free energy decomposition. Encouragingly, the calculated binding free energies correlated very well with the experimental values and the correlation coefficient square ( R 2 ), 0.60, was much higher than that of an efficient but less accurate docking scoring function ( R 2 = 0.37). The hotspot residues for CB1 and CB2 in both active and inactive conformations were identified via MM-GBSA free energy decomposition analysis. The comparisons of binding free energies, ligand-receptor interaction patterns, and hotspot residues among the four systems, namely, agonist-bound CB1, agonist-bound CB2, antagonist-bound CB1, and antagonist-bound CB2, enabled us to investigate and identify distinct binding features of these four systems, with which one can rationally design potent, selective, and function-specific modulators for the cannabinoid receptors.

Background and Purpose: Primary dysmenorrhea is the most common gynaecologic problem in menstruating women and is characterized by spasmodic uterine contraction and pain symptoms associated with inflammatory disturbances. Paeonol is an active phytochemical component that has shown anti-inflammatory and analgesic effects in several animal models. The aim of this study was to explore whether paeonol is effective against dysmenorrhea and to investigate the potential mechanism of cannabinoid receptor signalling., Experimental Approach: Dysmenorrhea was established by injecting oestradiol benzoate into female mice. The effects of paeonol on writhing time and latency, uterine pathology and inflammatory mediators were explored. Isolated uterine smooth muscle was used to evaluate the direct effect of paeonol on uterine contraction., Key Results: The oral administration of paeonol reduced dysmenorrhea pain and PGE2 and TNF-α expression in the uterine tissues of mice, and paeonol was found to be distributed in lesions of the uterus. Paeonol almost completely inhibited oxytocin-, high potassium- and Ca 2+ -induced contractions in isolated uteri. Antagonists of CB2R (AM630) and the MAPK pathway (U0126), but not of CB1R (AM251), reversed the inhibitory effect of paeonol on uterine contraction. Paeonol significantly blocked L-type Ca 2+ channels and calcium influx in uterine smooth muscle cells via CB2R. Molecular docking results showed that paeonol fits well with the binding site of CB2R., Conclusions and Implications: Paeonol partially acts through CB2R to restrain calcium influx and uterine contraction to alleviate dysmenorrhea in mice. These results suggest that paeonol has therapeutic potential for the treatment of dysmenorrhea., Competing Interests: Declaration of Competing Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2019. Published by Elsevier GmbH.)

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with G i , as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Drugs selectively targeting CB2 hold promise for treating neurodegenerative disorders, inflammation, and pain while avoiding psychotropic side effects mediated by CB1. The mechanisms underlying CB2 activation and signaling are poorly understood but critical for drug design. Here we report the cryo-EM structure of the human CB2-G i signaling complex bound to the agonist WIN 55,212-2. The 3D structure reveals the binding mode of WIN 55,212-2 and structural determinants for distinguishing CB2 agonists from antagonists, which are supported by a pair of rationally designed agonist and antagonist. Further structural analyses with computational docking results uncover the differences between CB2 and CB1 in receptor activation, ligand recognition, and G i coupling. These findings are expected to facilitate rational structure-based discovery of drugs targeting the cannabinoid system., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The endocannabinoid (eCB) system is implied in various human diseases ranging from central nervous system to autoimmune disorders. Cannabinoid receptor 2 (CB 2 R) is an integral component of the eCB system. Yet, the downstream effects elicited by this G protein-coupled receptor upon binding of endogenous or synthetic ligands are insufficiently understood-likely due to the limited arsenal of reliable biological and chemical tools. Herein, we report the design and synthesis of CB 2 R-selective cannabinoids along with their in vitro pharmacological characterization (binding and functional studies). They combine structural features of HU-308 and AM841 to give chimeric ligands that emerge as potent CB 2 R agonists with high selectivity over the closely related cannabinoid receptor 1 (CB 1 R). The synthesis work includes convenient preparation of substituted resorcinols often found in cannabinoids. The utility of the synthetic cannabinoids in this study is showcased by preparation of the most selective high-affinity fluorescent probe for CB 2 R to date., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The butyl homologues of Δ 9 -tetrahydrocannabinol, Δ 9 -tetrahydrocannabutol (Δ 9 -THCB), and cannabidiol, cannabidibutol (CBDB), were isolated from a medicinal Cannabis sativa variety (FM2) inflorescence. Appropriate spectroscopic and spectrometric characterization, including NMR, UV, IR, ECD, and HRMS, was carried out on both cannabinoids. The chemical structures and absolute configurations of the isolated cannabinoids were confirmed by comparison with the spectroscopic data of the respective compounds obtained by stereoselective synthesis. The butyl homologue of Δ 9 -THC, Δ 9 -THCB, showed an affinity for the human CB1 ( K i = 15 nM) and CB2 receptors ( K i = 51 nM) comparable to that of (-)- trans -Δ 9 -THC. Docking studies suggested the key bonds responsible for THC-like binding affinity for the CB1 receptor. The formalin test in vivo was performed on Δ 9 -THCB in order to reveal possible analgesic and anti-inflammatory properties. The tetrad test in mice showed a partial agonistic activity of Δ 9 -THCB toward the CB1 receptor.

G protein-coupled receptors (GPCRs) comprise a large class of integral membrane proteins involved in the regulation of a broad spectrum of physiological processes and are a major target for pharmaceutical drug development. Structural studies can help advance the rational design of novel specific pharmaceuticals that target GPCRs, but such studies require expression of significant quantities of these proteins in pure, homogenous, and sufficiently stable form. An essential precursor for these structural studies is an assessment of protein stability under experimental conditions. Here we report that solubilization of a GPCR, type II cannabinoid receptor CB 2 , in a Façade detergent enables radioligand thermostability assessments of this receptor with low background from nonspecific interactions with lipophilic cannabinoid ligand. Furthermore, this detergent is compatible with a [ 35 S]GTPγS radionucleotide exchange assay measuring guanine exchange factor activity that can be applied after heat treatment to further assess receptor thermostability. We demonstrate that both assays can be utilized to determine differences in CB 2 thermostability caused by mutations, detergent composition, and the presence of stabilizing ligands. We report that a constitutively active CB 2 variant has higher thermostability than the WT receptor, a result that differs from a previous thermostability assessment of the analogous CB 1 mutation. We conclude that both ligand-binding and activity-based assays under optimized detergent conditions can support selection of thermostable variants of experimentally demanding GPCRs.

Selective activation of the cannabinoid receptor subtype 2 (CB2) shows promise for treating pain, inflammation, multiple sclerosis, cancer, ischemic/reperfusion injury and osteoporosis. Target selectivity and off-target side effects are two major limiting factors for orthosteric ligands, and therefore, the search for allosteric modulators (AMs) is a widely used drug discovery approach. To date, only a limited number of negative CB2 AMs have been identified, possessing only micromolar activity at best, and the CB2 receptor's allosteric site(s) are not well characterized. Herein, we used computational approaches including receptor modeling, site mapping, docking, molecular dynamics (MD) simulations and binding free energy calculations to predict, characterize and validate allosteric sites within the complex of the CB2 receptor with bound orthosteric agonist CP55,940. After docking of known negative CB2 allosteric modulators (NAMs), dihydro-gambogic acid (DHGA) and trans -β-caryophyllene (TBC) (note that TBC also shows agonist activity), at the predicted allosteric sites, the best total complex with CB2, CP55,940 and NAM was embedded into a hydrated lipid bilayer and subjected to a 200 ns MD simulation. The presence of an AM affected the CB2-CP55,940 complex, altering the relative positioning of the toggle switch residues and promoting a strong π-π interaction between Phe117 3.36 and Trp258 6.48 . Binding of either TBC or DHGA to a putative allosteric pocket directly adjacent to the orthosteric ligand reduced the binding free energy of CP55,940, which is consistent with the expected effect of a negative AM. The identified allosteric sites present immense scope for the discovery of novel classes of CB2 AMs.

The cannabinoid type 2 (CB2) receptor has emerged as a valuable target for therapy and imaging of immune-mediated pathologies. With the aim to find a suitable radiofluorinated analogue of the previously reported CB2 positron emission tomography (PET) radioligand [ 11 C]RSR-056, 38 fluorinated derivatives were synthesized and tested by in vitro binding assays. With a K i (hCB2) of 6 nM and a selectivity factor of nearly 700 over cannabinoid type 1 receptors, target compound 3 exhibited optimal in vitro properties and was selected for evaluation as a PET radioligand. [ 18 F] 3 was obtained in an average radiochemical yield of 11 ± 4% and molar activities between 33 and 114 GBq/μmol. Specific binding of [ 18 F] 3 to CB2 was demonstrated by in vitro autoradiography and in vivo PET experiments using the CB2 ligand GW-405 833. Metabolite analysis revealed only intact [ 18 F] 3 in the rat brain. [ 18 F] 3 detected CB2 upregulation in human amyotrophic lateral sclerosis spinal cord tissue and may thus become a candidate for diagnostic use in humans.

Activation of the CB 2 receptor is an attractive therapeutic strategy for the treatment of a wide range of inflammatory diseases. However, receptor subtype selectivity is necessary in order to circumvent the psychoactive effects associated with activation of the CB 1 receptor. We aimed to use potent, non-selective synthetic cannabinoids designer drugs to develop selective CB 2 receptor agonists. Simple structural modifications such as moving the amide substituent of 3-amidoalkylindole synthetic cannabinoids to the 2-position and bioisosteric replacement of the indole core to the 7-azaindole scaffold are shown to be effective and general strategies to impart receptor subtype selectivity. 2-Amidoalkylindole 16 (EC 50 CB 1  > 10 μM, EC 50 CB 2  = 189 nM) and 3-amidoalkyl-7-azaindole 21 (EC 50 CB 1  > 10 μM, EC 50  = 49 nM) were found to be potent and selective agonists with favourable physicochemical properties. Docking studies were used to elucidate the molecular basis for the observed receptor subtype selectivity for these compounds., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

In earlier work, we reported a novel class of CB2 selective ligands namely cannabilactones. These compounds carry a dimethylheptyl substituent at C3, which is typical for synthetic cannabinoids. In the current study with the focus on the pharmacophoric side chain at C3 we explored the effect of replacing the C1'- gem -dimethyl group with the bulkier cyclopentyl ring, and, we also probed the chain's length and terminal carbon substitution with bromo or cyano groups. One of the analogs synthesized namely 6-[1-(1,9-dihydroxy-6-oxo-6 H -benzo[ c ]chromen-3-yl) cyclopentyl] hexanenitrile (AM4346) has very high affinity ( K i = 4.9 nM) for the mouse CB2 receptor (mCB2) and 131-fold selectivity for that target over the rat CB1 (rCB1). The species difference in the affinities of AM4346 between the mouse (m) and the human (h) CB2 receptors is reduced when compared to our first-generation cannabilactones. In the cyclase assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC 50 = 3.7 ± 1.5 nM, E (max) = 89%). We have also extended our structure-activity relationship (SAR) studies to include biphenyl synthetic intermediates that mimic the structure of the phytocannabinoid cannabinodiol.

(1) Background: The cannabinoid 2 receptor (CB 2 R) is a promising anti-inflammatory drug target and development of selective CB 2 R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB 2 R ligands: CB 2 R agonists, RO6871304, and RO6871085, as well as a CB 2 R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB 2 R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB 2 R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB 2 R -/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB 2 R -/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB 2 R -/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB 2 R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB 2 R and selectivity for CB 2 R > CB 1 R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC 50 s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB 2 R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB 2 R -/- , and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB 2 R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB 2 R agonists support selective targeting of CB 2 R for treating ocular inflammatory diseases.

Allosteric modulators of cannabinoid receptors hold great therapeutic potential, as they do not possess intrinsic efficacy, but instead enhance or diminish the receptor's response of orthosteric ligands allowing for the tempering of cannabinoid receptor signaling without the desensitization, tolerance and dependence. Allosteric modulators of cannabinoid receptors have numerous advantages over the orthosteric ligands such as higher receptor type selectivity, probe dependence and biased signaling, so they have a great potential to separate the therapeutic benefits from side effects own of orthosteric ligands. This review aims to give an overview of the CB1 and CB2 receptor allosteric modulators highlighting the structure-activity relationship and pharmacological profile of each classes, and their future promise.

Cannabinoid receptor type II, or CB 2 , is an integral membrane protein that belongs to a large class of G-protein-coupled receptors (GPCR)s. CB 2 is a part of the endocannabinoid system, which plays an important role in the regulation of immune response, inflammation, and pain. Information about the structure and function of CB 2 is essential for the development of specific ligands targeting this receptor. We present here a methodology for recombinant expression of CB 2 and its stable isotope labeling, purification, and reconstitution into liposomes, in preparation for its characterization by nuclear magnetic resonance (NMR). Correctly folded, functional CB 2 labeled with [ 13 C, 15 N]tryptophan or uniformly labeled with 13 C and 15 N is expressed in a medium of defined composition, under controlled aeration, pH, and temperature conditions. The receptor is purified by affinity chromatography and reconstituted into lipid bilayers in the form of proteoliposomes suitable for analysis by NMR spectroscopy. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Although human epidermal growth factor receptor 2 (HER2)-targeted therapies have dramatically improved the clinical outcome of HER2-positive breast cancer patients, innate and acquired resistance remains an important clinical challenge. New therapeutic approaches and diagnostic tools for identification, stratification, and treatment of patients at higher risk of resistance and recurrence are therefore warranted. Here, we unveil a mechanism controlling the oncogenic activity of HER2: heteromerization with the cannabinoid receptor CB 2 R. We show that HER2 physically interacts with CB 2 R in breast cancer cells, and that the expression of these heteromers correlates with poor patient prognosis. The cannabinoid Δ 9 -tetrahydrocannabinol (THC) disrupts HER2-CB 2 R complexes by selectively binding to CB 2 R, which leads to ( i ) the inactivation of HER2 through disruption of HER2-HER2 homodimers, and ( ii ) the subsequent degradation of HER2 by the proteasome via the E3 ligase c-CBL. This in turn triggers antitumor responses in vitro and in vivo. Selective targeting of CB 2 R transmembrane region 5 mimicked THC effects. Together, these findings define HER2-CB 2 R heteromers as new potential targets for antitumor therapies and biomarkers with prognostic value in HER2-positive breast cancer., Competing Interests: Conflict of interest statement: M.G. and C.S. are members of the Zelda Therapeutics Medical Advisory Board.

CB2 receptor belongs to the family of G-protein coupled receptors (GPCRs), which extensively controls a range of pointer transduction. CB2 plays an essential role in the immune system. It also associates in the pathology of different ailment conditions. In this scenario, the synthetic drugs are inducing side effects to the human beings after the drug use. Therefore, this study is seeking novel alternate drug molecules with least side effects than conventional drugs. The alternative drug molecules were chosen from the natural sources. These molecules were selected from cyanobacteria with the help of earlier research findings. The target and ligand molecules were obtained from recognized databases. The bioactive molecules are selected from various cyanobacterial species, which are selected by their biological and pharmacological properties, after, which we incorporated to the crucial findings such as homology modelling, molecular docking, MD simulations along with absorption, distribution, metabolism, and excretion (ADME) analysis. Initially, the homology modelling was performed to frame the target from unknown sequences of CB2, which revealed 44% of similarities and 66% of identities with the A2A receptor. Subsequently, the CB2 protein molecule has docked with already known and prepared bioactive molecules, agonists and antagonist complex. In the present study, the agonists (5) and antagonist (1) were also taken for comparing the results with natural molecules. At the end of the docking analysis, the cyanobacterial molecules and an antagonist TNC-201 are revealed better docking scores with well binding contacts than the agonists. Especially, the usneoidone shows better results than other cyanobacterial molecules, and it is very close docking scores with that of TCN-201. Therefore, the usneoidone has incorporated to MD simulation with Cannabinoid receptors 2 (CB2). In MD simulations, the complex (CB2 and usneoidone) reveals better stability in 30 ns. Based on the computational outcome, we concluded that usneoidone is an effectual and appropriate drug candidate for activating CB2 receptors and it will be serving as a better component for the complications of CB2. Moreover, these computational approaches can be motivated to discover novel drug candidates in the pharmacological and healthcare sectors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The direct activation of cannabinoid receptors (CBRs) results in several beneficial effects; therefore several CBRs ligands have been synthesized and tested in vitro and in vivo. However, none of them reached an advanced phase of clinical development due mainly to side effects on the CNS. Medicinal chemistry approaches are now engaged to develop allosteric modulators that might offer a novel therapeutic approach to achieve potential therapeutic benefits avoiding inherent side effects of orthosteric ligands. Here we identify the first ever synthesized positive allosteric modulator (PAM) that targets CB 2 Rs. The evidence for this was obtained using [ 3 H]CP55940 and [ 35 S]GTPγS binding assays. This finding will be useful for the characterization of allosteric binding site(s) on CB 2 Rs which will be essential for the further development of CB 2 R allosteric modulators. Moreover, the new CB 2 R PAM displayed antinociceptive activity in vivo in an experimental mouse model of neuropathic pain, raising the possibility that it might be a good candidate for clinical development.

Herein we describe the design and synthesis of a new series of heteroarylpyrimidine/heteroaryltriazine derivatives on the basis of quinazoline-2,4(1H,3H)-diones as CB 2 R-selective ligands using a bioisosterism strategy. An acetamide group was explored to displace the enamine linker of the lead compound for the purpose of stereoisomerism elimination and hydrophilicity increase. As a result, some of the synthesized compounds showed high bioactivity and selectivity for CB 2 R in calcium mobilization assays, and four displayed CB 2 R agonist activity, with EC 50 values below 30 nm. The compound exhibiting the highest agonist activity toward CB 2 R (EC 50 =7.53±3.15 nm) had a selectivity over CB 1 R of more than 1328-fold. Moreover, structure-activity relationship (SAR) studies indicated that the substituents on the nucleus play key roles in the functionality of a ligand, with one such example demonstrating CB 2 R antagonist activity. Additionally, molecular docking simulations were conducted with the aim of better understanding of these new derivatives in relation to the structural requirements for agonists/antagonists binding to CB 2 R., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Activation of high affinity receptor for IgE (FcεRI) by IgE/antigen complexes in mast cells (MCs) leads to the release of preformed pro-inflammatory mediators stored in granules by a Ca 2+ -dependent process known as anaphylactic degranulation. Degranulation inhibition has been proposed as a strategy to control allergies and chronic inflammation conditions. Cannabinoids are important inhibitors of inflammatory reactions but their effects on IgE/Ag-mediated MCs responses are not well described. In this study, we analyzed the effect of the endocannabinoid anandamide (AEA), the selective CB 2 receptor agonist HU308, and the GPR55 receptor agonist lysophosphatidylinositol (LPI) on FcεRI-induced activation in murine bone marrow-derived mast cells (BMMCs). Our results show that AEA, HU380 and LPI inhibited FcεRI-induced degranulation in a concentration-dependent manner. This effect was mediated by CB 2 and GPR55 receptor activation through a mechanism insensitive to pertussis toxin. Degranulation inhibition was prevented by CB 2 and GPR55 antagonism, but not by CB 1 receptor blockage. AEA also inhibited calcium-dependent cytokine mRNA synthesis induced by FcεRI crosslinking, without affecting early phosphorylation events. In addition, AEA, HU308 and LPI inhibited intracellular Ca 2+ rise in response to IgE/Ag. CB 2 and GPR55 receptor antagonism could not prevent the inhibition produced by AEA and HU308, but partially blocked the one caused by LPI. These results indicate that AEA inhibits IgE/Ag-induced degranulation through a mechanism that includes the participation of CB 2 and GPR55 receptors acting in close crosstalk, and show that CB 2 -GPR55 heteromers are important negative regulators of FcεRI-induced responses in MCs., (Copyright © 2018. Published by Elsevier B.V.)

During these last years, the CB 2 cannabinoid receptor has emerged as a potential anti-inflammatory target in diseases such as multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, ischemic stroke, autoimmune diseases, osteoporosis, and cancer. However, the development of clinically useful CB 2 agonists reveals to be very challenging. Allosterism and biased-signaling mechanisms at CB 2 receptor may offer new avenues for the development of improved CB 2 receptor-targeted therapies. Although there has been some exploration of CB 1 receptor activation by new CB 1 allosteric or biased-signaling ligands, the CB 2 receptor is still at initial stages in this domain. In an effort to understand the molecular basis behind these pharmacological approaches, we have analyzed and summarized the structural data reported so far at CB 2 receptor., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Two 3D quantitative structure⁻activity relationships (3D-QSAR) models for predicting Cannabinoid receptor 1 and 2 (CB₁ and CB₂) ligands have been produced by way of creating a practical tool for the drug-design and optimization of CB₁ and CB₂ ligands. A set of 312 molecules have been used to build the model for the CB₁ receptor, and a set of 187 molecules for the CB₂ receptor. All of the molecules were recovered from the literature among those possessing measured K i values, and Forge was used as software. The present model shows high and robust predictive potential, confirmed by the quality of the statistical analysis, and an adequate descriptive capability. A visual understanding of the hydrophobic, electrostatic, and shaping features highlighting the principal interactions for the CB₁ and CB₂ ligands was achieved with the construction of 3D maps. The predictive capabilities of the model were then used for a scaffold-hopping study of two selected compounds, with the generation of a library of new compounds with high affinity for the two receptors. Herein, we report two new 3D-QSAR models that comprehend a large number of chemically different CB₁ and CB₂ ligands and well account for the individual ligand affinities. These features will facilitate the recognition of new potent and selective molecules for CB₁ and CB₂ receptors.

Antagonists of peripheral type 1 cannabinoid receptors (CB1) may have utility in the treatment of obesity, liver disease, metabolic syndrome and dyslipidemias. We have targeted analogues of the purine inverse agonist otenabant (1) for this purpose. The non-tissue selective CB1 antagonist rimonabant (2) was approved as a weight-loss agent in Europe but produced centrally mediated adverse effects in some patients including dysphoria and suicidal ideation leading to its withdrawal. Efforts are now underway to produce compounds with limited brain exposure. While many structure-activity relationship (SAR) studies of 2 have been reported, along with peripheralized compounds, 1 remains relatively less studied. In this report, we pursued analogues of 1 in which the 4-aminopiperidine group was switched to piperazine group to enable a better understanding of SAR to eventually produce compounds with limited brain penetration. To access a binding pocket and modulate physical properties, the piperazine was functionalized with alkyl, heteroalkyl, aryl and heteroaryl groups using a variety of connectors, including amides, sulfonamides, carbamates and ureas. These studies resulted in compounds that are potent antagonists of hCB1 with high selectivity for hCB1 over hCB2. The SAR obtained led to the discovery of 65 (Ki = 4 nM, >1,000-fold selective for hCB1 over hCB2), an orally bioavailable aryl urea with reduced brain penetration, and provides direction for discovering peripherally restricted compounds with good in vitro and in vivo properties., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In recent years, cannabinoid type 2 receptors (CB 2 R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB 2 R are devoid of the psychoactive effects typically observed for CB 1 R ligands. Based on our recent studies on a class of pyridazinone 4-carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure-activity relationships for this promising scaffold with the aim to develop potent CB 2 R ligands. In binding assays, two of the new synthesized compounds [6-(3,4-dichlorophenyl)-2-(4-fluorobenzyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (2) and 6-(4-chloro-3-methylphenyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxamide (22)] showed high CB 2 R affinity, with K i values of 2.1 and 1.6 nm, respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB 2 R inverse agonists. Compound 22 displayed the highest CB 2 R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle-switch residue, W6.48., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ferrocene analogs of known fatty acid amide hydrolase inhibitors and CB 2 ligands have been synthesized and characterized spectroscopically and crystallographically. The resulting bio-organometallic isoxazoles were assayed for their effects on CB 1 and CB 2 receptors as well as on fatty acid amide hydrolase. None had any fatty acid amide hydrolase activity but compound 3, 5-(2-(pentyloxy)phenyl)-N-ferrocenylisoxazole-3-carboxamide, was found to be a potent CB 2 ligand (K i  = 32.5 nM).

Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), which is an increasing problem in the clinic and has been associated with elevated rates of mortality. Therapies to treat AKI are currently not available, so identification of new targets that can be modulated to ameliorate renal damage upon diagnosis of AKI is essential. In this study, a novel cannabinoid receptor 2 (CB2) agonist, SMM-295 [3'-methyl-4-(2-(thiophen-2-yl)propan-2-yl)biphenyl-2,6-diol], was designed, synthesized, and tested in vitro and in silico. Molecular docking of SMM-295 into a CB2 active-state homology model showed that SMM-295 interacts well with key amino acids to stabilize the active state. In human embryonic kidney 293 cells, SMM-295 was capable of reducing cAMP production with 66-fold selectivity for CB2 versus cannabinoid receptor 1 and dose-dependently increased mitogen-activated protein kinase and Akt phosphorylation. In vivo testing of the CB2 agonist was performed using a mouse model of bilateral IRI, which is a common model to mimic human AKI, where SMM-295 was immediately administered upon reperfusion of the kidneys after the ischemia episode. Histologic damage assessment 48 hours after reperfusion demonstrated reduced tubular damage in the presence of SMM-295. This was consistent with reduced plasma markers of renal dysfunction (i.e., creatinine and neutrophil gelatinase-associated lipocalin) in SMM-295-treated mice. Mechanistically, kidneys treated with SMM-295 were shown to have elevated activation of Akt with reduced terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL)-positive cells compared with vehicle-treated kidneys after IRI. These data suggest that selective CB2 receptor activation could be a potential therapeutic target in the treatment of AKI., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Novel 1,4-dihydropyrazolo[3,4-a]pyrrolizine-, 4,5-dihydro-1H-pyrazolo[4,3-g]indolizine- and 1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]azepine-3-carboxamide-based compounds were designed and synthesized for cannabinoid CB 1 and CB 2 receptor interactions. Any of the new synthesized compounds showed high affinity for CB 2 receptor with K i values superior to 314 nm, whereas some of them showed moderate affinity for CB 1 receptor with K i values inferior to 400 nm. 7-Chloro-1-(2,4-dichlorophenyl)-N-(homopiperidin-1-yl)-4,5-dihydro-1H-pyrazolo[4,3-g]indolizine-3-carboxamide (2j) exhibited good affinity for CB 1 receptor (K i CB 1  = 81 nm) and the highest CB 2 /CB 1 selectively ratio (>12). Docking studies carried out on such compounds were performed using the hCB 1 X-ray in complex with the close pyrazole analogue AM6538 and disclosed specific pattern of interactions related to the tricyclic pyrrolopyrazole scaffolds as CB 1 ligands., (© 2017 John Wiley & Sons A/S.)

Pepcan-12 (RVD-hemopressin; RVDPVNFKLLSH) is the major peptide of a family of endogenous peptide endocannabinoids (pepcans) shown to act as negative allosteric modulators (NAM) of cannabinoid CB1 receptors. Noradrenergic neurons have been identified to be a specific site of pepcan production. However, it remains unknown whether pepcans occur in the periphery and interact with peripheral CB2 cannabinoid receptors. Here, it is shown that pepcan-12 acts as a potent (K i value ~50 nM) hCB2 receptor positive allosteric modulator (PAM). It significantly potentiated the effects of CB2 receptor agonists, including the endocannabinoid 2-arachidonoyl glycerol (2-AG), for [35S]GTPγS binding and cAMP inhibition (5-10 fold). In mice, the putative precursor pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH) was identified with pepcan-12 in brain, liver and kidney. Pepcan-12 was increased upon endotoxemia and ischemia reperfusion damage where CB2 receptors play a protective role. The adrenals are a major endocrine site of production/secretion of constitutive pepcan-12, as shown by its marked loss after adrenalectomy. However, upon I/R damage pepcan-12 was strongly increased in the liver (from ~100 pmol/g to ~500 pmol/g) independent of adrenals. The wide occurrence of this endogenous hormone-like CB2 receptor PAM, with unforeseen opposite allosteric effects on cannabinoid receptors, suggests its potential role in peripheral pathophysiological processes.

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.

Cannabinoid receptor 2 (CB2R)-dependent signaling is implicated in neuronal physiology and immune surveillance by brain microglia. Selective CB2R agonists hold therapeutic promise for inflammatory and other neurological disorders. Information on human CB2R (hCB2R) ligand-binding and functional domains is needed to inform the rational design and optimization of candidate druglike hCB2R agonists. Prior demonstration that hCB2R transmembrane helix 2 (TMH2) cysteine C2.59(89) reacts with small-molecule methanethiosulfonates showed that this cysteine residue is accessible to sulfhydryl derivatization reagents. We now report the design and application of two novel, pharmacologically active, high-affinity molecular probes, AM4073 and AM4099, as chemical reporters to interrogate directly the interaction of classical cannabinoid agonists with hCB2R cysteine residues. AM4073 has one electrophilic isothiocyanate (NCS) functionality at the C9 position of its cyclohexenyl C-ring, whereas AM4099 has NCS groups at that position and at the terminus of its aromatic A-ring C3 side chain. Pretreatment of wild-type hCB2R with either probe reduced subsequent [ 3 H]CP55,940 specific binding by ∼60%. Conservative serine substitution of any hCB2R TMH cysteine residue except C2.59(89) did not affect the reduction of [ 3 H]CP55,940 specific binding by either probe, suggesting that AM4073 and AM4099 interact irreversibly with this TMH2 cysteine. In contrast, AM841, an exceptionally potent hCB2R megagonist and direct AM4073/4099 congener bearing a single electrophilic NCS group at the terminus of its C3 side chain, had been demonstrated to bind covalently to TMH6 cysteine C6.47(257) and not C2.59(89). Molecular modeling indicates that the AM4073-hCB2R* interaction at C2.59(89) orients this classical cannabinoid away from TMH6 and toward the TMH2-TMH3 interface in the receptor's hydrophobic binding pocket, whereas the AM841-hCB2R* interaction at C6.47(257) favors agonist orientation toward TMH6/7. These data constitute initial evidence that TMH2 cysteine C2.59(89) is a component of the hCB2R binding pocket for classical cannabinoids. The results further demonstrate how interactions between classical cannabinoids and specific amino acids within the hCB2R* ligand-binding domain act as determinants of agonist pharmacological properties and the architecture of the agonist-hCB2R* conformational ensemble, allowing the receptor to adopt distinct activity states, such that interaction of classical cannabinoids with TMH6 cysteine C6.47(257) favors a binding pose more advantageous for agonist potency than does their interaction with TMH2 cysteine C2.59(89).

The preceding years have brought an exponential increase in our understanding of the endocannabinoid system (ECS), including the knowledge of CB1 and CB2 cannabinoid receptors, endocannabinoids, and the enzymes that synthesize and degrade endocannabinoids. Among these ECS components CB2 receptors have been the subject of considerable attention, primarily due to their promising therapeutic potential to treat numerous pathologies while avoiding the adverse psychotropic effects that can accompany CB1 receptor-based therapies. Recently, our research group has reported a new series of non-cytotoxic benzo[d]imidazoles and benzo[b]thiophenes displaying high CB2/CB1 selectivity index. In order to investigate the structural requirements for CB2 ligands and to derive a predictive model that can be used for the design of novel selective CB2 ligands, a three-dimensional quantitative structure-activity relationship (3D-QSAR) study was performed on the above mentioned chemical series employing comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) techniques. The CoMFA and CoMSIA models displayed high external predictability (r pred 2 0.919 and 0.908) and good statistical robustness. Valuable information regarding the steric, electrostatic and hydrophobic properties of the molecules was obtained, and several modifications around both heterocycles were evaluated with the aim to generate new promising series of benzo[d]imidazoles and benzo[b]thiophenes derivatives displaying high CB2 selectivity and low toxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)