While equilibrium binding affinities and in vitro functional antagonism of CB1 receptor antagonists have been studied in detail, little is known on the kinetics of their receptor interaction. In... Show moreWhile equilibrium binding affinities and in vitro functional antagonism of CB1 receptor antagonists have been studied in detail, little is known on the kinetics of their receptor interaction. In this study, we therefore conducted kinetic assays for nine 1-(4,5-diarylthiophene-2-carbonyl)-4-phenylpiperidine-4-carboxamide derivatives and included the CB1 antagonist rimonabant as a comparison. For this we newly developed a dual-point competition association assay with [3H]CP55940 as the radioligand. This assay yielded Kinetic Rate Index (KRI) values from which structure-kinetics relationships (SKR) of hCB1 receptor antagonists could be established. The fast dissociating antagonist 6 had a similar receptor residence time (RT) as rimonabant, i.e. 19 and 14 min, respectively, while the slowest dissociating antagonist (9) had a very long RT of 2222 min, i.e. pseudo-irreversible dissociation kinetics. In functional assays, 9 displayed insurmountable antagonism, while the effects of the shortest RT antagonist 6 and rimonabant were surmountable. Taken together, this study shows that hCB1 receptor antagonists can have very divergent RTs, which are not correlated to their equilibrium affinities. Furthermore, their RTs appear to define their mode of functional antagonism, i.e. surmountable vs. insurmountable. Finally, based on the recently resolved hCB1 receptor crystal structure, we propose that the differences in RT can be explained by a different binding mode of antagonist 9 from short RT antagonists that is able to displace unfavorable water molecules. Taken together, these findings are of importance for future design and evaluation of potent and safe hCB1 receptor antagonists. Show less
