Drugs that interfere with cannabinoid CB1 transmission suppress various food-motivated behaviors, and it has been suggested that such drugs could be useful as appetite suppressants. Biochemical studies indicate that most of these drugs assessed thus far have been CB1 inverse agonists, and although they have been shown to suppress food intake, they also appear to induce nausea and malaise. The present studies were undertaken to characterize the behavioral effects of AM4113, which is a CB1 neutral antagonist, and to examine whether this drug can reduce food-reinforced behaviors and feeding on diets with varying macronutrient compositions. Biochemical data demonstrated that AM4113 binds to CB1 receptors, but does not show inverse agonist properties (ie no effects on cyclic-AMP production). In tests of spontaneous locomotion and analgesia, AM4113 reversed the effects of the CB1 agonist AM411. AM4113 suppressed food-reinforced operant responding with rats responding on fixed ratio (FR) 1 and 5 schedules of reinforcement in a dosedependent manner, and also suppressed feeding on high-fat, high-carbohydrate, and lab chow diets. However, in the same dose range that suppressed feeding, AM4113 did not induce conditioned gaping, which is a sign of nausea and food-related malaise in rats. These results suggest that AM4113 may decrease appetite by blocking endogenous cannabinoid tone, and that this drug may be less associated with nausea than CB1 inverse agonists. Neuropsychopharmacology (2008) 33, 946-955;

The CB1 cannabinoid receptor has been shown to play important physiological roles in the central nervous system, as well as peripherally, and is a target for development of therapeutic medications. To gain insight on the ligand binding site(s) and structural features of activation, we designed and synthesized (Ϫ)-7Ј-isothiocyanato-11-hydroxy-1Ј,1Ј-dimethylheptylhexahydrocannabinol (AM841), a classical cannabinoid affinity label that incorporates an isothiocyanate substituent as an electrophilic reactive group capable of interacting irreversibly with a suitably located and properly oriented nucleophilic amino acid residue at or near the binding site. To obtain evidence for the site of covalent attachment of AM841, C6.47, identified in part by interactive ligand docking, was mutated to serine, alanine, and leucine to reduce or eliminate the nucleophilic character. Wild-type (WT) and mutant CB1 receptors were evaluated for their abilities to recognize a series of cannabinergic ligands. Each bound comparably to WT, excluding C6.47L, which displayed a reduced affinity for It is noteworthy that AM841 was shown to bind irreversibly to WT CB1 but exhibited no covalent attachment with the mutants and behaved as an agonist suggesting irreversible attachment to C6.47 maintains CB1 in its active state. The evidence presented identifies C6.47 as the site of covalent bond formation with AM841 and combined with the binding data fully supports the molecular modeling. These studies present the first report of tandem applications of affinity labeling, site-directed mutagenesis, and interactive ligand docking for CB1.The CB1 and CB2 cannabinoid receptors are relatively new members in the G-protein-coupled receptor (GPCR) superfamily. They have been shown to play important physiological roles and represent targets for development of therapeutic medications. From a pharmacological standpoint, agonist activation of both receptors results in the release of G␣ iproteins, causing a concomitant reduction in intracellular This work has been supported by National Institute on Drug Abuse grants DA05955 (to R.P.P.) DA00355 (to A.D.K.), DA09158, DA03801, DA07215, DA07312 (to A.M.), DA03934, DA00489 (to P.H.R.), DA05274, and DA09978 (to M.E.A.).

SUMMARY The extensive physiological influence of transmission through the CB2 cannabinoid receptor makes this G protein-coupled receptor (GPCR) a promising therapeutic target for treating neuropathic pain, inflammation, and immune disorders. However, there is little direct structural information pertaining to either GPCR or CB2-receptor ligand recognition and activation. The present work helps characterize experimentally the ligand-binding interactions of the human CB2 (hCB2) receptor. This study illustrates how our overall experimental approach, “ligand-assisted protein structure” (LAPS), affords direct determination of the requirements for ligand binding to the hCB2 receptor and discrimination among the binding motifs for ligands that activate therapeutically relevant GPCRs.

The cannabinoid 1 receptor (CB1R) is one of the most widely expressed metabotropic G protein-coupled receptors in brain, and its participation in various (patho)physiological processes has made CB1R activation a viable therapeutic modality. Adverse psychotropic effects limit the clinical utility of CB1R orthosteric agonists and have promoted the search for CB1R positive allosteric modulators (PAMs) with the promise of improved drug-like pharmacology and enhanced safety over typical CB1R agonists. In this study, we describe the synthesis and in vitro and ex vivo pharmacology of the novel allosteric CB1R modulator GAT211 (racemic) and its resolved enantiomers, GAT228 (R) and GAT229 (S). GAT211 engages CB1R allosteric site(s), enhances the binding of the orthosteric full agonist [H]CP55,490, and reduces the binding of the orthosteric antagonist/inverse agonist [H]SR141716A. GAT211 displayed both PAM and agonist activity in HEK293A and Neuro2a cells expressing human recombinant CB1R (hCB1R) and in mouse-brain membranes rich in native CB1R. GAT211 also exhibited a strong PAM effect in isolated vas deferens endogenously expressing CB1R. Each resolved and crystallized GAT211 enantiomer showed a markedly distinctive pharmacology as a CB1R allosteric modulator. In all biological systems examined, GAT211's allosteric agonist activity resided with the R-(+)-enantiomer (GAT228), whereas its PAM activity resided with the S-(-)-enantiomer (GAT229), which lacked intrinsic activity. These results constitute the first demonstration of enantiomer-selective CB1R positive allosteric modulation and set a precedent whereby enantiomeric resolution can decisively define the molecular pharmacology of a CB1R allosteric ligand.