A two-state model for the kinetics of competitive radioligand binding.

Abstract

Background and PurposeLigand–receptor binding kinetics is receiving increasing attention in the drug research community. The Motulsky and Mahan model, a one‐state model, offers a method for measuring the binding kinetics of an unlabelled ligand, with the assumption that the labelled ligand has no preference while binding to distinct states or conformations of a drug target. As such, the one‐state model is not applicable if the radioligand displays biphasic binding kinetics to the receptor.Experimental ApproachWe extended the Motulsky and Mahan model to a two‐state model, in which the kinetics of the unlabelled competitor binding to different receptor states (R1 and R2) can be measured. With this extended model, we determined the binding kinetics of unlabelled N‐5′‐ethylcarboxamidoadenosine (NECA), a representative agonist for the adenosine A1 receptor. Subsequently, an application of the model was exemplified by measuring the binding kinetics of other A1 receptor ligands. In addition, limitations of the model were investigated as well.Key ResultsThe kinetic rate constants of unlabelled NECA were comparable with the results of kinetic radioligand binding assays in which [3H]‐NECA was used. The model was further validated by good correlation between simulated results and the experimental data.ConclusionThe two‐state model is sufficient to analyse the binding kinetics of an unlabelled ligand, when a radioligand shows biphasic association characteristics. We expect this two‐state model to have general applicability for other targets as well.