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

We report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses 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 side chain. Epimer (R)-1 exhibits high affinity for CB₂R with K_d = 39.1 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, for the first time these fluorescent probes 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 ERK_1/2 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 Trp258^6.48 and thereby stabilize CB₂R in its inactive state.

We report a generalizable strategy for structure-based agonist-to-inverse-agonist functional transformation and probe development by ligand modification that modulates the GPCR toggle switch of CB₂R.