Flipping the GPCR Switch: Structure–Based Development of Selective Cannabinoid Receptor 2 Inverse Agonists

We report the structure-based design of cannabinoid receptor type 2 (CB2R)-selective inverse agonists (S)-1 and (R)-1, which were derived from privileged agonist HU-308 by introduction of a phenyl group at the gem-dimethylheptyl sidechain. Epimer (R)-1 exhibits high affinity for CB2R with Kd = 39 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, the fluorescent probes, for the first time, retain their inverse agonist functionality, high affinity, and selectivity for CB2R independent of linker and fluorophore substitution. Ligands (S)-1, (R)-1, and their derivatives act as inverse agonists in CB2R-mediated cAMP as well as G protein recruitment assays, and do not trigger β-arrestin–receptor association. Furthermore, no receptor activation was detected in live cell ERK1/2 phosphorylation and Ca2+-release assays. Confocal fluorescence imaging experiments with (R)-7 (Alexa488) and (R)-9 (Alexa647) probes employing BV-2 microglial cells visualized CB2R expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch, Trp2586.48, and thereby stabilize CB2R in its inactive state. The present study serves as a blueprint for the rational design of GPCR ligands beyond CB2R with a tailored functional response.