Kinetics of nonpeptide antagonist binding to the human gonadotropin-releasing hormone receptor: Implications for structure–activity relationships and insurmountable antagonism

Numerous nonpeptide ligands have been developed for the human gonadotropin-releasing hormone (GnRH) receptor as potential agents for treatment of disorders of the reproductive-endocrine axis. While the equilibrium binding of these ligands has been studied in detail, little is known of the kinetics of their receptor interaction. In this study we evaluated the kinetic structure–activity relationships (SAR) of uracil-series antagonists by measuring their association and dissociation rate constants. These constants were measured directly using a novel radioligand, [3H] NBI 42902, and indirectly for unlabeled ligands. Receptor association and dissociation of [3H] NBI 42902 was monophasic, with an association rate constant of 93 ± 10 μM−1 min−1 and a dissociation rate constant of 0.16 ± 0.02 h−1 (t1/2 of 4.3 h). Four unlabeled compounds were tested with varying substituents at the 2-position of the benzyl group at position 1 of the uracil (–F, –SO(CH3), –SO2(CH3) and –CF3). The nature of the substituent did not appreciably affect the association rate constant but varied the dissociation rate constant >50-fold (t1/2 ranging from 52 min for –SO(CH3) to >43 h for –CF3). This SAR was poorly resolved in standard competition assays due to lack of equilibration. The functional consequences of the varying dissociation rate were investigated by measuring antagonism of GnRH-stimulated [3H] inositol phosphates accumulation. Slowly dissociating ligands displayed insurmountable antagonism (decrease of the GnRH Emax) while antagonism by more rapidly dissociating ligands was surmountable (without effect on the GnRH Emax). Therefore, evaluating the receptor binding kinetics of nonpeptide antagonists revealed SAR, not evident in standard competition assays, that defined at least in part the mode of functional antagonism by the ligands. These findings are of importance for the future definition of nonpeptide ligand SAR and for the identification of potentially useful slowly dissociating antagonists for the GnRH receptor.