Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors

Abstract

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

Convulsant Effects of Abused Synthetic Cannabinoids JWH-018 and 5F-AB-PINACA Are Mediated by Agonist Actions at CB1 Receptors in Mice

“…The present results would seem to be inconsistent with the notion that partial CB1R agonism (as produced by increased anandamide signaling) would be protective against PTZ-elicited convulsions, as neither acute THC treatment nor downregulation and desensitization of CB1Rs attenuated the convulsant effects of PTZ. So-called medical marijuana products suggested to treat epilepsy typically contain both THC and the phytocannabinoid cannabidiol (CBD), but it is perhaps important to note that CBD exhibits extremely weak affinity for CB1Rs and CB2Rs (McPartland, et al, 2007;Rosenthaler et al, 2014), although recent evidence suggests it may possess pharmacologically relevant affinity for an allosteric site on the CB2R (Martínez-Pinilla et al, 2017).…”

Cannabidiol inhibits sucrose self‐administration by CB1 and CB2 receptor mechanisms in rodents

“…CBD has been shown to have multiple acting targets including CB1 and CB2 receptors, GPR55 receptor, 5‐HT 1A receptor, GPR3, GPR6, and GPR12, μ and δ opioid receptors, vallinoid receptor 1 (TRPV1), and peroxisome proliferator–activated receptor γ (PPAR γ) . However, more recent studies suggest that CBD is a potent allosteric modulator of CB1 and CB2 receptors with nanomolar binding affinity . These new findings suggest that CB1 and CB2 receptors could be important targets in the pharmacological action produced by CBD.…”

Adreno–melatonin receptor complexes control ion homeostasis and intraocular pressure ‐ their disruption contributes to hypertensive glaucoma

“…The human non‐pigmented ciliary epithelial cell line, 59HCE, was kindly supplied by Dr. Coca‐Prados (Yale University) and grown in 5% heat‐inactivated FBS high‐glucose DMEM (Gibco/Invitrogen, Carlsbad, CA, USA). HEK‐293T cells [] were grown in 5% FBS‐DMEM (Gibco/Invitrogen) as previously described (Martínez‐Pinilla et al, ; Navarro et al, ).…”