Investigating selectivity and bias for G protein subtypes and β-arrestins by synthetic cannabinoid receptor agonists at the cannabinoid CB1 receptor

The cannabinoid CB1 receptor (CB1) is a G protein-coupled receptor (GPCR) with widespread expression in the central nervous system. This canonically Gai/o-coupled receptor mediates the effects of A9-tetrahydrocannabinol (THC) and synthetic cannabinoid receptor agonists (SCRAs). Recreational use of SCRAs is associated with serious adverse health effects, making pharmacological research into these compounds a priority. Several studies have hypothesised that signalling bias may explain the different toxicological profiles between SCRAs and THC. Previous studies have focused on bias between G protein activation measured by cyclic adenosine monophosphate (cAMP) inhibition and beta-arrestin translocation. In contrast, the current study characterises bias between Ga subtypes of the Gai/o family and beta-arrestins; this method facilitates a more accurate assessment of ligand bias by assessing signals that have not undergone major amplification. We have characterised G protein dissociation and translocation of beta-arrestin 1 and 2 using real -time BRET reporters. The responses produced by each SCRA across the G protein subtypes tested were consistent with the responses produced by the reference ligand AMB-FUBINACA. Ligand bias was probed by applying the operational analysis to determine biases within the Gai/o family, and between G protein subtypes and beta-arrestins. Overall, these results confirm SCRAs to be balanced, high-efficacy ligands compared to the low efficacy ligand THC, with only one SCRA, 4CN-MPPBUT7IACA, demonstrating statistically significant bias in one pathway comparison (towards beta-arrestin 1 when compared with GaoA/oB). This suggests that the adverse effects caused by SCRAs are due to high potency and efficacy at CB1, rather than biased agonism.