Allosteric modulators targeting cannabinoid cb1 and cb2 receptors: Implications for drug discovery

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

Development of novel oxazolo[5,4- d ]pyrimidines as competitive CB 2 neutral antagonists based on scaffold hopping

A series of novel oxazolo[5,4-d]pyrimidines was designed via a scaffold hopping strategy and synthesized through a newly developed approach. All these compounds were evaluated for their biological activity toward CB1/CB2 cannabinoid receptors, their metabolic stability in mice liver microsomes and their cytotoxicity against several cell lines. Eight compounds have been identified as CB2 ligands with Ki values less than 1 μM. It is noteworthy that 2-(2-chlorophenyl)-5-methyl-7-(4-methylpiperazin-1-yl) oxazolo[5,4-d]pyrimidine 47 and 2-(2-chlorophenyl)-7-(4-ethylpiperazin-1-yl)- 5-methyloxazolo[5,4-d]pyrimidine 48 showed CB2 binding affinity in the nanomolar range and significant selectivity over CB1 receptors. Interestingly, functionality studies imply that they behave as competitive neutral antagonists. Moreover, all tested compounds are devoid of cytotoxicity toward several cell lines, including Chinese hamster ovary cells (CHO) and human colorectal adenocarcinoma cells HT29

Identification of the first synthetic allosteric modulator of the CB2receptors and evidence of its efficacy for neuropathic pain relief

The direct activation of cannabinoid receptors (CBRs) results in several beneficial effects, therefore numerous CB1R and CB2R ligands have been developed and tested in vitro and in vivo, but none of them reached an advanced stage of clinical development due to several side effects in particular 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 synthetized positive allosteric modulator (PAM) that targets CB2Rs. The evidence for this was obtained using [3H]CP55940 and [35S]GTPγS binding assays. This finding will be useful for the characterization of allosteric binding site(s) on CB2Rs which will be essential for the further development of CB2R allosteric modulators. Moreover, the new CB2R 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

Chemogenomics Knowledgebase and Systems Pharmacology for Hallucinogen Target Identification -Salvinorin A as a Case Study

Drug abuse is a serious problem worldwide. Recently, hallucinogens have been reported as a potential therapy for substance abuse. However, the use of hallucinogens as a drug abuse treatment itself has potential risks. The true mechanisms of hallucinogens are not clear. Thus it is necessary to investigate the mechanism of hallucinogens to make sure they are safe to develop as medicine. So far, no scientific database is available for the mechanism research of hallucinogens. We constructed a hallucinogen-specific chemogenomics database by collecting chemicals, protein targets and pathways closely related to hallucinogens. This information, together with our established computational chemogenomics tools, such as TargetHunter and HTDocking, provided a one-step solution for the mechanism study of hallucinogens. We chose salvinorin A as an example to demonstrate the usability of our platform. Salvinorin A is a potent hallucinogen extracted from the plant Salvia divinorum. It was the first reported non-nitrogenous kappa opioid receptor agonist. Recently, researchers found that oral administration of salvinorin A can affect drug choice in a monkey model, which suggested a potential use of salvinorin A as an abuse-deterrent formulation. However, some complex effects of salvinorin A were reported, including depersonalization or laughing hysterically. Our aim is to identify the potential targets of salvinorin A to further explore the mechanisms of its complex effects. With the help of HTDocking program, we predicted four novel targets for salvinorin A, including muscarinic acetylcholine receptor 2, cannabinoid receptor 1, cannabinoid receptor 2 and dopamine receptor 2. We looked into the interactions between salvinorin A and the predicted targets, and compared their binding modes with the known ligands of these proteins. The similar binding modes, interactions and high docking scores indicate that salvinorin A may interact with these four predicted targets. In the future, we will design experiments or find collaborators to validate our predictions. At the same time, we will continuously enrich our hallucinogen-specific chemogenomics database by adding newest data and building more 3D homology models

Discovery of (E)-3-(4-(Diethylamino) phenyl)-1-phenyl-2-phenylsulfonyl)prop-2-en-1-one as Novel Cannabinoid Receptor 2 Ligands

Cannabinoids (CB) are defined as a class of compounds that can act on Cannabinoid receptors 1 or 2 (CB1 or CB2) and affect human physiology. Both CB1 and CB2 receptors belong to the rhodopsin-like family of G-Protein Coupled Receptors (GPCRs). However, CB1 receptor is mainly expressed in the central nervous system, while CB2 receptor is dominantly located in the peripheral nervous system and immune cells. By now, scientists have discovered many CB ligands that have therapeutic potentials, but the limitation of non-selective ligands is the psychiatric side effect mediated by the activation of CB1 receptor. Although CB1 receptor is crucial in analgesic and anti-inflammatory effects, strategies of designing CB2 selective ligands are made by medicinal chemists to avoid undesirable effects in clinic. In this thesis, we discovered novel CB2 lead compounds with new chemical scaffolds; designed and synthesized four series of analogues for the structure-activity relationship (SAR) studies; tested their binding affinity to both CB2 and CB1 receptors; conducted in-vitro functional studies; and evaluated their potentials for therapeutic treatment. In total, four series of (E)-3-(4-Ethoxy-3-methoxyphenyl)-2-((4-methoxyphenyl) sulfonyl)-1-phenylprop-2-en-1-one have been identified as novel cannabinoid ligands. Physicochemical properties were predicted and docking studies using our CB2 model was conducted. 29 derivatives were then synthesized to conduct SAR studies. The binding affinity and selectivity for cannabinoid receptor CB1 and CB2 were then evaluated. Four compounds showed high CB2 binding affinity (Ki of 10-60 nM) and good selectivity (CB1/CB2 of 20- to 1305 fold). Their off-targets effects were also predicted. Overall, these sulfone derivatives can be used to develop novel therapeutic CB2 ligands

Modeling, Molecular Dynamics Simulation, and Mutation Validation for Structure of Cannabinoid Receptor 2 Based on Known Crystal Structures of GPCRs

The cannabinoid receptor 2 (CB2) plays an important role in the immune system. Although a few of GPCRs crystallographic structures have been reported, it is still challenging to obtain functional transmembrane proteins and high resolution X-ray crystal structures, such as for the CB2 receptor. In the present work, we used 10 reported crystal structures of GPCRs which had high sequence identities with CB2 to construct homology-based comparative CB2 models. We applied these 10 models to perform a prescreen by using a training set consisting of 20 CB2 active compounds and 980 compounds randomly selected from the National Cancer Institute (NCI) database. We then utilized the known 170 cannabinoid receptor 1 (CB1) or CB2 selective compounds for further validation. Based on the docking results, we selected one CB2 model (constructed by β1AR) that was most consistent with the known experimental data, revealing that the defined binding pocket in our CB2 model was well-correlated with the training and testing data studies. Importantly, we identified a potential allosteric binding pocket adjacent to the orthosteric ligand-binding site, which is similar to the reported allosteric pocket for sodium ion Na⁺ in the A_2AAR and the δ-opioid receptor. Our studies in correlation of our data with others suggested that sodium may reduce the binding affinities of endogenous agonists or its analogs to CB2. We performed a series of docking studies to compare the important residues in the binding pockets of CB2 with CB1, including antagonist, agonist, and our CB2 neutral compound (neutral antagonist) XIE35-1001. Then, we carried out 50 ns molecular dynamics (MD) simulations for the CB2 docked with SR144528 and CP55940, respectively. We found that the conformational changes of CB2 upon antagonist/agonist binding were congruent with recent reports of those for other GPCRs. Based on these results, we further examined one known residue, Val113^3.32, and predicted two new residues, Phe183 in ECL2 and Phe281^7.35, that were important for SR144528 and CP55940 binding to CB2. We then performed site-directed mutation experimental study for these residues and validated the predictions by radiometric binding affinity assay

DESIGN, EXPRESSION AND PURIFICATION OF FUNCTIONAL HUMAN CANNABINOID RECEPTOR 2

Cannabinoid receptors (CB1 and CB2) are GPCRs that belong to the rhodopsin-like family. Recent studies have demonstrated a role for CB2 in attenuating bone cancer-induced pain, reducing microglial activation in Alzheimer’s disease, and regulating bone mass in osteoporosis. As such, there is an urgent need for the development of new therapeutic agents targeting CB2. However, structure- or fragment-based in silico drug design is hampered by the absence of 3D structural information on CB2, because of its inherent structural complexity. Recently, it has been shown that specific residues of CB2 (in transmembrane helices III, V, VII and extracellular loop 2) play crucial roles in the binding of the vast majority of cannabinoid ligands. In this report, we performed computer modeling along with site-directed mutagenesis studies and the results suggest that four residues that lie within these regions have a novel importance for receptor recognition of compound XIE95-26, a discovered CB2 inverse agonist in our lab, and known cannabinoid ligands CP-55940 (agonist) and SR144528 (inverse agonist). These mutant CB2 receptors were characterized by western blot, and ligand binding studies. Moreover, functional assays were performed for some mutant CB2 receptor to illustrate the role of altered residues in downstream signaling. Next, we’ve taken further steps to provide a genetically modified functional CB2 for biophysical and biochemical studies with the goal of improving the current purification methodology for the production of chimeric CB2 fusion proteins from the baculovirus system. By introducing a fusion protein and rational modifications to the chimeric receptor, investigators endeavoring to purify other GPCRs have overcome the challenge of obtaining sufficient stable protein for structural studies. Thus, different generations of truncated CB2 receptor were obtained to assess any improvements in receptor expression. Receptor expression and ligand binding capacity were assessed as criteria for pilot scale purification and for detergent selection. The best constructs were subjected to the optimal purification scheme using immobilized metal affinity, anti-M2 affinity, and size exclusion chromatography steps. Collectively, results from this study will provide insights into the structure and functional mechanisms of CB2 and facilitate the design of small molecules that influence CB2 signaling behavior

Contribution du récepteur GPR55 à la synaptogenèse

Les connections synaptiques entre les cellules nerveuses (appelées synapses) sont essentielles à l’établissement de l’architecture du système nerveux. La modification de ces synapses est un des mécanismes par lequel l’apprentissage et la mémoire fonctionnent. On sait depuis plusieurs années déjà que la consommation de cannabis exerce une profonde influence sur l’apprentissage et la mémoire, et que sa consommation chez la femme durant la grossesse ou l’allaitement peut causer des déficits cognitifs chez l’enfant qui perdureront à l’âge adulte. Pour le moment, on ne sait toujours pas si ces effets sont médiés par les récepteurs aux cannabinoïdes classiques (CB1 et CB2) ou par d’autres récepteurs tel le GPR55. Des études récentes du laboratoire du Pr. Bouchard ont démontré un rôle important du système endocannabinoïde dans le développement du système nerveux notamment par la présence du récepteur GPR55 et son implication dans la modulation du guidage et de la croissance des axones durant les périodes foetale et périnatale. Comme certaines molécules et mécanismes cellulaires impliqués dans ces processus peuvent aussi jouer un rôle dans la formation de synapses (synaptogenèse), l’objectif de la présente étude est de déterminer la contribution du GPR55 dans la formation de contacts synaptiques. À partir de cortex d’embryons de souris, nous avons cultivé puis traité des neurones corticaux soit avec un agoniste sélectif de GPR55 (O-1602) ou son antagoniste sélectif (ML-193), soit avec un phytocannabinoïde (cannabidiol) pendant 24 heures au 9e jour in vitro. En immunocytochimie, les neurones traités avec le ML-193 ont démontré une réduction significative du nombre de contacts synaptiques et une augmentation significative avec l’O-1602 et le cannabidiol. Ces changements anatomiques sont corrélés avec des modifications de l’expression des protéines synaptiques GluR1 et synaptophysine au niveau du cortex. En plus de fournir d’importantes informations sur le développement du système nerveux, les résultats de cette étude contribuent à l’amélioration de nos connaissances sur les anomalies du développement induites par la consommation périnatale de cannabis.Functional connections between nerve cells (called synapses) are essential to establish the architecture of the nervous system. The modification of synapses is thought to be one of the mechanisms by which learning and memory occur. It has been known for decades that cannabis consumption has a profound influence on learning and memory, and that maternal marijuana smoking during perinatal period causes cognitive deficits that last in the adulthood of the offspring. For the moment, we do not know if these effects are mediated by the classic CB1 and CB2 cannabinoid receptors or by other receptors such as GPR55. Recent studies by Pr. Bouchard have demonstrated an important role for the endocannabinoid system in the development of the nervous system, including the presence of GPR55 and its involvement in axon growth and target innervation during the fetal and early postnatal periods. As certain molecules and cellular mechanisms involved in these processes may also regulate synapse formation (synaptogenesis), the objective of the present study is to determine the contribution of GPR55 in the formation of new synaptic contacts. Primary cortical neurons isolated from embryonic mice were cultivated and then treated either with a selective agonist of GPR55 (O-1602) or his selective antagonist (ML-193), or with a phytocannabinoid (cannabidiol) for 24h at the ninth day in vitro (DIV9). In immunocytochemistry, neurons treated with ML-193 have shown a decrease in synaptic density, while the treatment with O-1602 or cannabidiol increased it. These anatomical changes were correlated with changes in the expression of synaptic proteins GluR1 and synaptophysin. Results from this study provide important insight on the development of the nervous system and contribute to improving our knowledge on developmental abnormalities induced by perinatal cannabis use

DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL SELECTIVE CANNABINOID RECEPTOR 2 (CB2) LIGANDS WITH THERAPEUTIC POTENTIALS

Cannabinoid receptors 1 and 2 (CB1 and CB2) belong to the rhodopsin-like family of the G-Protein Coupled Receptors (GPCRs). CB1 receptors are highly expressed in the central nervous system, while CB2 receptors are expressed mainly in the immune cells and the periphery. Targeting the CB2 receptors is believed to avoid the psychoactive side effects associated with CB1 receptors. CB2 receptors have been shown to be involved in several physiological functions as well as diseases, such as pain, multiple sclerosis, osteoporosis, and cancer demonstrating the importance of the CB2 receptors. In the present study, we employed chemistry design and discovery to identify novel CB2 ligands, carried out in-vitro functional studies, and evaluated the therapeutic potentials. Several chemical scaffolds were discovered and evaluated. The di-amide scaffold was discovered utilizing pharmacophore drug discovery and molecular docking studies. Several derivatives of the di-amide scaffold demonstrated potent and highly selective CB2 inverse agonists as well as potent anti-osteoclast formation capabilities. The di-amide derivatives suffered from weak anti-multiple myeloma (MM) properties and poor pharmacokinetic properties. A new scaffold was identified utilizing scaffold hopping and molecular docking studies. However, the 2-(sulfonylamino)-2-phenylacetamide scaffold demonstrated weak CB2 binding affinity. Due to the limitation of the two previous scaffolds, virtual high-throughput screening as well as structure-based drug design were utilized for scaffold hopping in order to identify new CB2 scaffolds. A new lead compound was identified and structure activity relationship (SAR) studies were conducted on the scaffold 4-(aminomethyl)-N,N-diethylaniline. Several novel compounds were discovered with high potency and selectivity. Functional experiments showed different functionality (agonist and inverse agonist) of these compounds. Nevertheless, therapeutic studies showed that inverse agonism is essential for the OCL inhibition effects while anti-MM experiments showed that CB2 agonists are more effective than inverse agonists