RT Journal Article
SR Electronic
T1 Label-Free Kinetics: Exploiting Functional Hemi-Equilibrium to Derive Rate Constants for Muscarinic Receptor Antagonists
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.115.100545
DO 10.1124/mol.115.100545
A1 Darren M Riddy
A1 Celine Valant
A1 Patricia Rueda
A1 William N Charman
A1 Patrick M Sexton
A1 Roger J Summers
A1 Arthur Christopoulos
A1 Christopher J Langmead
YR 2015
UL http://molpharm.aspetjournals.org/content/early/2015/08/03/mol.115.100545.abstract
AB Drug receptor kinetics is as a key component in drug discovery, development and efficacy. However, determining kinetic parameters has historically required direct radiolabelling or competition with a labelled tracer. Here we present a simple approach to determine the kinetics of competitive antagonists of GPCRs 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 ERK1/2 phosphorylation assays that have short incubation times and therefore prone to hemi-equilibrium 'behaviors', we have investigated a wide range of structurally and physico-chemically distinct muscarinic acetylcholine receptor (mAChR) antagonists. Using a combined operational/hemi-equilibrium model of antagonism to both simulate and analyse 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 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]-NMS binding, validating this approach as a rapid and simple method to functionally profile receptor kinetics of competitive antagonists in the absence of a labelled tracer.