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

Abstract

An increased appreciation of the importance of optimizing drugbinding 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., k off 5 10 minute 21 low-affinity "hits" through to slowly dissociating e.g., k off 5 0.01 minute 21 high-affinity "candidates"). For more rapidly dissociating ligands (e.g., k off 5 10 minute 21), the key to obtaining accurate kinetic parameters was to employ a tracer with a relatively fast off-rate (e.g., k off 5 1 minute 21) 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 lowaffinity competitors.

Perspective: Implications of Ligand–Receptor Binding Kinetics for Therapeutic Targeting of G Protein-Coupled Receptors

“…105 Similarly, for D 2 R, the slow k off of spiperone was less accurate in predicting the k off of fast dissociation ligands as compared to the relatively fast k off probe PPHT-red. 108 Moreover, the accuracy of kinetic measurements is dependent on the probe concentration. As such, the importance of using a suitable probe for ligand−receptor binding kinetics calls for careful consideration, and ideally one with a fast binding is required.…”

“…An interesting phenomenon was discovered for H 1 R, dopamine D 2 receptor (D 2 R), and A 1 AR, in which large differences in on- and off-rate were observed for a set of unlabeled ligands using two different probes. − The usage of [ 3 H]­mepyramine as a radioligand for H 1 R appeared to be easier to discriminate between unlabeled ligands with a fast k off than with [ 3 H]­levocetrizine. The reason for this was that levocetrizine had a 100-fold lower k off than mepyramine; therefore, the accuracy of both k on and k off of unlabeled ligands can be predicted better with mepyramine as a radioligand .…”

The Problems of Applying Classical Pharmacology Analysis to Modern In Vitro Drug Discovery Assays: Slow Binding Kinetics and High Target Concentration

“…7,52,53 It should noted that while measuring the rate constants is an effective way to measure affinity, kinetic binding assays require significant resources for assay development and data analysis. [59][60][61]…”

Fluorescent ligands: Bringing light to emerging GPCR paradigms

“…As the Motulsky and Mahan method is the most commonly used method to measure binding kinetics of unlabelled ligands, the above examples emphasise the importance of selecting a labelled probe with the appropriate kinetics for accurate determination of test compounds. It is also becoming clear that a probe with fast binding kinetics may be more generally applicable for measuring the kinetics of an unlabelled ligand (Bosma et al, ; Sykes, Jain, & Charlton, ). As radioligands are only subtly different from the parent pharmacophore, differences between radioligand probe binding kinetics are often less pronounced.…”

“…As demonstrated by the simulated data inFigure 3using the Motulsky and Mahan equations, binding rates of unlabelled ligands with significantly faster kinetics than the labelled ligand can be difficult to distinguish even with 10-fold difference in their k off values.As the Motulsky and Mahan method is the most commonly used method to measure binding kinetics of unlabelled ligands, the above examples emphasise the importance of selecting a labelled probe with the appropriate kinetics for accurate determination of test compounds. It is also becoming clear that a probe with fast binding kinetics may be more generally applicable for measuring the kinetics of an unlabelled ligand(Bosma et al, 2019;Sykes, Jain, & Charlton, 2019).As radioligands are only subtly different from the parent pharmacophore, differences between radioligand probe binding kinet-ics are often less pronounced. Fluorescent probes differ much more widely in their chemical structure as a result of using chemically diverse fluorophores and linkers.…”