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.

Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics.

The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.