Investigating the Influence of Tracer Kinetics on Competition-Kinetic Association Binding Assays: Identifying the Optimal Conditions for Assessing the Kinetics of Low-Affinity Compounds

Sykes, David A.; Jain, Palash; Charlton, Steven J.

Abstract

An increased appreciation of the importance of optimizing drug-binding kinetics has lead to the development of various techniques for measuring the kinetics of unlabeled compounds. One approach is the competition-association kinetic binding method first described in the 1980s. 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, e.g., koff = 10 minute−1 low-affinity “hits” through to slowly dissociating e.g., koff = 0.01 minute−1 high-affinity “candidates”). For more rapidly dissociating ligands (e.g., koff = 10 minute−1), the key to obtaining accurate kinetic parameters was to employ a tracer with a relatively fast off-rate (e.g., koff = 1 minute−1) or, alternatively, to increase the tracer concentration. Reductions in assay start-time ≤1second and read frequency ≤5 seconds significantly improved the reliability of curve fitting. Timing constraints are largely dictated by the method of detection, its inherent sensitivity (e.g., TR-FRET versus radiometric detection), and the ability to inject samples online. Furthermore, we include data from TR-FRET experiments that validate 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 unlabeled competitors, particularly rapidly dissociating low-affinity competitors.

Citation

Sykes, D. A., Jain, P., & Charlton, S. J. (2019). Investigating the Influence of Tracer Kinetics on Competition-Kinetic Association Binding Assays: Identifying the Optimal Conditions for Assessing the Kinetics of Low-Affinity Compounds. Molecular Pharmacology, 96(3), 378-392. https://doi.org/10.1124/mol.119.116764

• Journal Article Type: Article
• Acceptance Date: Jun 22, 2019
• Online Publication Date: Jul 10, 2019
• Publication Date: 2019-09
• Deposit Date: Oct 17, 2019
• Journal: Molecular Pharmacology
• Print ISSN: 0026-895X
• Electronic ISSN: 1521-0111
• Publisher: American Society for Pharmacology and Experimental Therapeutics
• Peer Reviewed: Peer Reviewed
• Volume: 96
• Issue: 3
• Pages: 378-392
• DOI: https://doi.org/10.1124/mol.119.116764
• Keywords: Molecular Medicine; Pharmacology
• Public URL: https://nottingham-repository.worktribe.com/output/2852677
• Publisher URL: http://molpharm.aspetjournals.org/content/96/3/378