1-(4-chlorophenyl)-3-(3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl)urea and 3-(2-hydroxy-4-(1-1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol

To determine if diminished orthosteric agonist binding due to mutations in extracellular loops 1 or 2 of the cannabinoid receptor 1 (CB. Binding assays were performed using a range of concentrations of orthosteric compound, in the presence or absence of a set concentration of the allosteric modulator PSNCBAM-1 to determine the EC. Single mutations in extracellular loop 1 or 2 of CB. Based on our findings, we provide evidence of a therapeutic application for allosteric modulators in situations where a mutation in the receptor may hinder its function. By utilizing allosteric modulators, restoration of orthosteric binding may be possible.

Topics: Binding Sites; Cannabinoid Receptor Agonists; Cyclohexanols; HEK293 Cells; Humans; Indoles; Ligands; Mutation; Phenylurea Compounds; Piperidines; Protein Binding; Protein Conformation; Pyridines; Receptor, Cannabinoid, CB1; Rimonabant; Structure-Activity Relationship

While allosteric modulators of the cannabinoid type-1 receptor (CB

Topics: Allosteric Regulation; Animals; Cannabinoid Receptor Modulators; Cerebellum; Cyclohexanols; Discrimination, Psychological; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice, Inbred C57BL; Mice, Inbred ICR; Motor Activity; Phenylurea Compounds; Piperidines; Pregnenolone; Pyrazoles; Pyridines; Receptor, Cannabinoid, CB1; Rimonabant

Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs.

Topics: Affinity Labels; Allosteric Site; Animals; Arrestins; Binding Sites; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Cyclohexanols; Drug Discovery; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Indoles; Ligands; Models, Molecular; Phenylurea Compounds; Piperidines; Pyridines; Radioligand Assay; Rats; Receptor, Cannabinoid, CB1; Structure-Activity Relationship

The cannabinoid receptor type 1 (CB1 ) has an allosteric binding site. The drugs ORG27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} and PSNCBAM-1 {1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non-competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB1 , we examined real-time signalling and trafficking responses of the receptor in the presence of allosteric modulators.. Studies of CB1 signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin-tagged human and rat CB1 . We measured real-time accumulation of cAMP, activation and desensitization of potassium channel-mediated cellular hyperpolarization and CB1 internalization.. ORG27569 and PSNCBAM-1 produce a complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB1 -mediated cellular hyperpolarization and a decrease in agonist-induced receptor internalization.. Contrary to previous studies characterizing allosteric modulators at CB1, this study suggests that the mechanism of action is not non-competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time-dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds.

Topics: Animals; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cell Line, Tumor; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Indoles; Kinetics; Ligands; Membrane Potentials; Mice; Phenylurea Compounds; Piperidines; Potassium Channels, Inwardly Rectifying; Protein Transport; Pyridines; Rats; Receptor, Cannabinoid, CB1; Signal Transduction; Transfection

The human cannabinoid receptor, CB1, a G protein-coupled receptor (GPCR), contains a relatively long (∼110 a.a.) amino terminus, whose function is still not defined. Here we explore a potential role for the CB1 N-terminus in modulating ligand binding to the receptor. Although most of the CB1 N-terminus is not necessary for ligand binding, previous studies have found that mutations introduced into its conserved membrane proximal region (MPR) do impair the receptors ability to bind ligand. Moreover, within the highly conserved MPR (∼ residues 90-110) lie two cysteine residues that are invariant in all CB1 receptors. We find these two cysteines (C98 and C107) form a disulfide in heterologously expressed human CB1, and this C98-C107 disulfide is much more accessible to reducing agents than the previously known disulfide in extracellular loop 2 (EL2). Interestingly, the presence of the C98-C107 disulfide modulates ligand binding to the receptor in a way that can be quantitatively analyzed by an allosteric model. The C98-C107 disulfide also alters the effects of allosteric ligands for CB1, Org 27569 and PSNCBAM-1. Together, these results provide new insights into how the N-terminal MPR and EL2 act together to influence the high-affinity orthosteric ligand binding site in CB1 and suggest that the CB1 N-terminal MPR may be an area through which allosteric modulators can act.

Topics: Allosteric Regulation; Amino Acid Sequence; Animals; Binding Sites; Chlorocebus aethiops; COS Cells; Cyclohexanols; Cysteine; Disulfides; Dithiothreitol; Humans; Indoles; Ligands; Phenylurea Compounds; Piperidines; Pyridines; Rats; Receptor, Cannabinoid, CB1

Rimonabant (Acomplia, SR141716A), a cannabinoid CB1 receptor inverse agonist, has recently been approved for the treatment of obesity. There are, however, concerns regarding its side effect profile. Developing a CB1 antagonist with a different pharmacological mechanism may lead to a safer alternative. To this end we have screened a proprietary small molecule library and have discovered a novel class of allosteric antagonist at CB1 receptors. Herein, we have characterized an optimized prototypical molecule, PSNCBAM-1, and its hypophagic effects in vivo.. A CB1 yeast reporter assay was used as a primary screen. PSNCBAM-1 was additionally characterized in [35S]-GTPgammaS, cAMP and radioligand binding assays. An acute rat feeding model was used to evaluate its effects on food intake and body weight in vivo.. In CB1 receptor yeast reporter assays, PSNCBAM-1 blocked the effects induced by agonists such as CP55,940, WIN55212-2, anandamide (AEA) or 2-arachidonoyl glycerol (2-AG). The antagonist characteristics of PSNCBAM-1 were confirmed in [35S]-GTPgammaS binding and cAMP assays and was shown to be non-competitive by Schild analyses. PSNCBAM-1 did not affect CB2 receptors. In radioligand binding assays, PSNCBAM-1 increased the binding of [3H]CP55,940 despite its antagonist effects. In an acute rat feeding model, PSNCBAM-1 decreased food intake and body weight.. PSNCBAM-1 exerted its effects through selective allosteric modulation of the CB1 receptor. The acute effects on food intake and body weight induced in rats provide a first report of in vivo activity for an allosteric CB1 receptor antagonist.

Topics: Allosteric Regulation; Animals; Appetite Depressants; Cell Line; Cell Membrane; Cerebral Cortex; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; Eating; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Molecular Structure; Phenylurea Compounds; Piperidines; Pyrazoles; Pyridines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Saccharomyces cerevisiae; Silicone Elastomers; Sulfur Radioisotopes; Weight Gain