Drug receptor kinetics is as a key component in drug discovery, development, and efficacy; however, determining kinetic parameters has historically required direct radiolabeling or competitionwith a labeled tracer. Here we present a simple approach to determining the kinetics of competitive antagonists of G protein-coupled receptors by exploiting the phenomenon of hemi-equilibrium, the state of partial re-equilibration of agonist, antagonist, and receptor in some functional assays. Using functional [Ca2+]i-flux and extracellular kinases 1 and 2 phosphorylation assays that have short incubation times and therefore are prone to hemi-equilibrium "behaviors," we investigated a wide range of structurally and physicochemically distinct muscarinic acetylcholine receptor antagonists. Using a combined operational and hemi-equilibrium model of antagonism to both simulate and analyze data, we derived estimates of association and dissociation rates for the test set of antagonists, identifying both rapidly dissociating (4-DAMP, himbacine) and slowly dissociating (tiotropium, glycopyrrolate) ligands. The results demonstrate the importance of assay incubation time and the degree of receptor reserve in applying the analytical model. There was an excellent correlation between estimates of antagonist pKB, kon, and koff from functional assays and those determined by competition kinetics using whole-cell [3H]N-methylscopolamine binding, validating this approach as a rapid and simple method to functionally profile receptor kinetics of competitive antagonists in the absence of a labeled tracer.
Riddy, D. M., Valant, C., Rueda, P., Charman, W. N., Sexton, P. M., Summers, R. J., Christopoulos, A., & Langmead, C. J. (2015). Label-free kinetics: exploiting functional hemi-equilibrium to derive rate constants for muscarinic receptor antagonists. Molecular Pharmacology, 88(4), 779-790. https://doi.org/10.1124/mol.115.100545