Abstract
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 Kon and Koff 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 (Ki) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased Ki values over time (Ki30 min/Ki10 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 Ki value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the Ki 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.