Allosteric Modulation of the Cannabinoid CB₁ Receptor

Abstract

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 CB 1 receptor. In equilibrium binding assays, the Org compounds significantly increased the binding of the CB 1 receptor agonist, indicative of a positively cooperative allosteric effect. The same compounds caused a significant, but incomplete, decrease in the specific binding of the CB 1 receptor inverse agonist 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 CB 1 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 5Ј-O-(3-[35 S]thio)triphosphate binding assay and the mouse vas deferens assay they elicited a significant reduction in the E max value for CB 1 receptor agonists. The data presented clearly demonstrate, for the first time, that the cannabinoid CB 1 receptor contains an allosteric binding site that can be recognized by synthetic small molecule ligands.Mammalian tissues express at least two types of cannabinoid receptor, CB 1 and CB 2 , both G protein-coupled (for review, see Howlett et al., 2002). CB 1 receptors are found predominantly at central and peripheral nerve terminals where they mediate inhibition of transmitter release. Endogenous ligands for these receptors also exist. These "endocannabinoids" are all eicosanoids, prominent examples including arachidonoylethanolamide (anandamide) and 2-arachidonoyl glycerol, both of which are synthesized on demand, removed from their sites of action by tissue uptake processes and metabolized by intracellular enzymes (Pertwee and Ross,

Anti-inflammatory lipoxin A 4 is an endogenous allosteric enhancer of CB 1 cannabinoid receptor

“…Kinetic dissociation-binding assays allow evaluating the influence of a given substance on the dissociation kinetics of a preformed orthosteric ligand-receptor complex. As the dissociation kinetic is not altered if the interacting ligands recognize the same binding site, this assay is considered the method of choice for measuring allosteric modulation (9). The binding of [ 3 H]CP55940 was displaced by an excessive amount of WIN55,212 and followed over time.…”

“…In addition to our previous study showing that aspirin-triggered LXA 4 enhances AEA effects (22), here we show that endogenous LXA 4 contributes to CB 1 -mediated effects as a positive allosteric modulator, with physiological relevance for endocannabinoid-dependent regulation of brain functions and potential therapeutic utility. Allosteric modulation of CB 1 receptor was originally described using synthetic compounds (9). The "Org" compounds (Org27596 and Org29647) and PSNCBAM-1 (31) enhance affinity and reduce efficacy of cannabinoid agonists acting at the orthosteric site of CB 1 receptors (9).…”

“…Two synthetic compounds, Org27596 and Org29647, enhance the affinity and reduce the efficacy of CB 1 agonists, suggesting the existence of an allosteric binding site at CB 1 receptor (9). However, the existence of endogenous allosteric cannabinoid modulators has not yet been proved.…”

PUFA-derived endocannabinoids: an overview

“…This association is greatly affected by cholesterol content; indeed, membrane cholesterol enrichment in both primary and immortalised cell lines reduces the binding to CB 1 ; instead cholesterol depletion modifies anandamide-induced endocytosis of CB 1 , which apparently loses the ability to be directed towards the lysosomal compartment (33) . Importantly, the existence on the CB 1 cannabinoid receptors of an allosteric binding site that can be recognised by synthetic small molecules was reported for the first time by our group (34) . Whether the CB 2 receptor, such as CB 1 , possesses an allosteric binding site, warrants further investigation.…”

The Effective Application of Biased Signaling to New Drug Discovery

“…Thus the agonist K A reflects the change in the natural affinity of the receptor for the signaling protein in the absence versus the presence of an agonist in the form of the allosteric parameter a (Stockton et al, 1983; Ehlert, 1988) Applying Biased Signaling to Drug Discovery and the change in the efficacy of interaction between the receptor and signaling protein in the absence or presence of the agonist (denoted b in the functional allosteric model in Ehlert, 2005;Kenakin, 2005;and Price et al, 2005; denoted as B in Ehlert, 1988). Under these circumstances, the transducer coefficient is unique to the allosteric vector made up of agonist/ receptor/signaling protein and is thus cell type and system independent.…”