Scintillation proximity assay as a high-throughput method to identify slowly dissociating nonpeptide ligand binding to the GnRH receptor

Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as K-on and K-off are time and labor intensive, prompting the development of a screening assay to identify slowly dissociating compounds for follow-up rate constant determination. The authors measured inhibition binding constants (K) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased K, values over time (Ki(30 min)/Ki (10 h) > 6). They used scintillation proximity assay technology to perform these binding experiments because this homogeneous assay does not have a fixed termination end point as does filtration binding, permitting successive readings to be taken from the same assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound's K-i value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the K-i ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants.