We investigated the pharmacology of three novel compounds, Org 27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide), Org 27759 (3-ethyl-5-fluoro-1H-indole-2-carboxylic acid [2-94-dimethylamino-phenyl)-ethyl]-amide), and Org 29647 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic acid salt), at the cannabinoid CB₁ receptor. In equilibrium binding assays, the Org compounds significantly increased the binding of the CB₁ receptor agonist [³H]CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol], indicative of a positively cooperative allosteric effect. The same compounds caused a significant, but incomplete, decrease in the specific binding of the CB₁ receptor inverse agonist [³H]SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride], indicative of a limited negative binding cooperativity. Analysis of the data according to an allosteric ternary complex model revealed that the estimated affinity of each Org compound was not significantly different when the radioligand was [³H]CP 55,940 or [³H]SR 141716A. However, the estimated cooperatively factor for the interaction between modulator and radioligand was greater than 1 when determined against [³H]CP 55,940 and less than 1 when determined against [³H]SR 141716A. [³H]CP 55,940 dissociation kinetic studies also validated the allosteric nature of the Org compounds, because they all significantly decreased radioligand dissociation. These data suggest that the Org compounds bind allosterically to the CB₁ receptor and elicit a conformational change that increases agonist affinity for the orthosteric binding site. In contrast to the binding assays, however, the Org compounds behaved as insurmountable antagonists of receptor function; in the reporter gene assay, the guanosine 5′-O-(3-[³⁵S]thio)triphosphate binding assay and the mouse vas deferens assay they elicited a significant reduction in the E_max value for CB₁ receptor agonists. The data presented clearly demonstrate, for the first time, that the cannabinoid CB₁ receptor contains an allosteric binding site that can be recognized by synthetic small molecule ligands.

The uptake of 2-deoxyglucose into KB cells was stimulated about 2-fold by interleukin-1 (IL1), anisomycin or insulin-like growth factor-1 (IGF1). Stimulation by IL1 and anisomycin was prevented by SB 203580, a specific inhibitor of the mitogen-activated protein (MAP) kinase homologue termed ‘re-activating kinase’ [RK; also known as p38, p40 and CSBP (cytokine synthesis anti-inflammatory-drug-binding protein)], but was unaffected by PD 98059, a specific inhibitor of the activation of the classical MAP kinase pathway. In contrast, the stimulation of 2-deoxyglucose uptake by IGF1 was blocked by PD 98059 and unaffected by SB 203580. Consistent with these observations, IL1 and anisomycin were potent activators of MAP kinase-activated protein (MAPKAP) kinase-2, a physiological substrate of RK, whereas IGF1 was only a very weak activator of MAPKAP kinase-2. Conversely, IGF1 was a stronger activator of p42 MAP kinase than IL1 or anisomycin. These results imply that the activation of distinct MAP kinase pathways is required for the stimulation of glucose transport by IL1/anisomycin and IGF1 in KB cells, and suggest that the combined use of SB 203580 and PD 98059 is a powerful new approach to explore the roles of different MAP kinase cascades in cell regulation.

The measurement of receptor occupancy (RO) using positron emission tomography (PET) has been instrumental in guiding discovery and development of CNS directed therapeutics. We and others have investigated muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs) for the treatment of symptoms associated with neuropsychiatric disorders. In this article, we describe the synthesis, in vitro, and in vivo characterization of a series of central pyridine-related M4 PAMs that can be conveniently radiolabeled with carbon-11 as PET tracers for the in vivo imaging of an allosteric binding site of the M4 receptor. We first demonstrated its feasibility by mapping the receptor distribution in mouse brain and confirming that a lead molecule 1 binds selectively to the receptor only in the presence of the orthosteric agonist carbachol. Through a competitive binding affinity assay and a number of physiochemical properties filters, several related compounds were identified as candidates for in vivo evaluation. These candidates were then radiolabeled with 11C and studied in vivo in rhesus monkeys. This research eventually led to the discovery of the clinical radiotracer candidate [11C]MK-6884.