TY - JOUR T1 - Investigating the influence of tracer kinetics on competition-kinetic association binding assays; identifying the optimal conditions for assessing the kinetics of low affinity compounds. JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.119.116764 SP - mol.119.116764 AU - David A Sykes AU - Palash Jain AU - Steven J Charlton Y1 - 2019/01/01 UR - http://molpharm.aspetjournals.org/content/early/2019/07/10/mol.119.116764.abstract N2 - There is an increased appreciation of the importance of optimizing drug-binding kinetics leading to the development of various different techniques for measuring the kinetics of unlabelled compounds. One popular approach is the competition-association kinetic binding method, first described by Aranyi (1980), then popularized by Motulsky and Mahan (1984). The kinetic characteristics of the tracer employed greatly affects the reliability of estimated kinetic parameters, a barrier to successfully introducing these kinetic assays earlier in the drug discovery process. Using a modeling and Monte Carlo simulation approach we identify the optimal tracer characteristics for determining the kinetics of the range of unlabeled ligands typically encountered during the different stages of a drug discovery program (i.e. rapidly-dissociating eg. koff = 10min-1 low-affinity "hits" through to slowly-dissociating eg. koff = 0.01min-1 high-affinity "candidates"). For more rapidly dissociating ligands (eg. koff = 10min-1) the key to obtaining accurate kinetic parameters was to employ a tracer with a relatively fast off-rate (eg. koff = 1min-1), or alternatively to increase the tracer concentration. Reductions in assay start-time ≤1sec, and read frequency ≤5sec significantly improved the reliability of curve fitting. Timing constraints are largely dictated by the method of detection, its inherent sensitivity (eg TR-FRET verses radiometric detection), and the ability to inject samples online. Furthermore we include data from TR-FRET experiments which validates this simulation approach confirming its practical utility. These insights into the optimal experimental parameters for development of competition-association assays provide a framework for identifying and testing novel tracers necessary for profiling unlabelled competitors, particularly rapidly-dissociating low affinity competitors.SIGNIFICANCE STATEMENT Optimizing the kinetics of drug binding can have significant clinical benefit, yet measuring binding kinetics at membrane proteins remains a complex endeavor. Competition association binding assays have proved useful in this regard, but to date there has been no systematic investigation into the optimal assay design for this methodology. In this article we have used a Monte Carlo simulation approach to identify the best conditions for measuring the kinetics of both slowly and rapidly dissociating compounds, providing clear guidance on assay design at each stage of the drug discovery process. Importantly, we highlight the difficulty of measuring the kinetics of rapidly dissociating compounds and the critical importance of rapidly equilibrating tracers, online injection capability and rapid read frequency. ER -