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

Background and purpose: Ligand-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 approach: We 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 results: The kinetic rate constants of unlabelled NECA were comparable with the results of kinetic radioligand binding assays in which [³ H]-NECA was used. The model was further validated by good correlation between simulated results and the experimental data.

Conclusion: The 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.