posted on 2024-03-11, 18:16authored byMiroslav Kosar, Roman C. Sarott, David A. Sykes, Alexander E. G. Viray, Rosa Maria Vitale, Nataša Tomašević, Xiaoting Li, Rudolf L. Z. Ganzoni, Bilal Kicin, Lisa Reichert, Kacper J. Patej, Uxía Gómez-Bouzó, Wolfgang Guba, Peter J. McCormick, Tian Hua, Christian W. Gruber, Dmitry B. Veprintsev, James A. Frank, Uwe Grether, Erick M. Carreira
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 Kd = 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 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.