Drug-target binding kinetics are suggested to be important parameters for the prediction of in vivo drug-efficacy. For G protein-coupled receptors (GPCRs), the binding kinetics of ligands are typically determined using association binding experiments in competition with radiolabelled probes, followed by analysis with the widely used competitive binding kinetics theory developed by Motulsky and Mahan. Despite this, the influence of the radioligand binding kinetics on the kinetic parameters derived for the ligands tested is often overlooked. To address this, binding rate constants for a series of histamine H 1 receptor (H 1 R) antagonists were determined using radioligands with either slow (low k off ) or fast (high k off ) dissociation characteristics. A correlation was observed between the probe-specific datasets for the kinetic binding affinities, association rate constants and dissociation rate constants. However, the magnitude and accuracy of the binding rate constant-values was highly dependent on the used radioligand probe. Further analysis using recently developed fluorescent binding methods corroborates the finding that the Motulsky-Mahan methodology is limited by the employed assay conditions. The presented data suggest that kinetic parameters of GPCR ligands depend largely on the characteristics of the probe used and results should therefore be viewed within the experimental context and limitations of the applied methodology.
Inhibition of monopolar spindle 1 (MPS1) kinase represents a novel approach to cancer treatment: instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached/misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series “triazolopyridines” and “imidazopyrazines”. The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10-fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy models.
Background and Purpose: Target engagement dynamics can influence drugs' pharmacological effects. Kinetic parameters for drug:target interactions are often quantified by evaluating competition association experiments-measuring simultaneous protein binding of labelled tracers and unlabelled test compounds over time-with Motulsky-Mahan's "kinetics of competitive binding" model. Despite recent technical improvements, the current assay formats impose practical limitations to this approach. This study aims at the characterisation, understanding and prevention of these experimental constraints, and associated analytical challenges. Experimental Approach: Monte Carlo simulations were used to run virtual kinetic and equilibrium tracer binding and competition experiments in both normal and perturbed assay conditions. Data were fitted to standard equations derived from the mass action law (including Motulsky-Mahan's) and to extended versions aiming to cope with frequently observed deviations of the canonical traces. Results were compared to assess the precision and accuracy of these models and identify experimental factors influencing their performance. Key Results: Key factors influencing the precision and accuracy of the Motulsky-Mahan model are the interplay between compound dissociation rates, measurement time and interval frequency, tracer concentration and binding kinetics and the relative abundance of equilibrium complexes in vehicle controls. Experimental results produced recommendations for better design of tracer characterisation experiments and new strategies to deal with systematic signal decay.Conclusions and Implications: Our data advances our comprehension of the Motulsky-Mahan kinetics of competitive binding models and provides experimental design recommendations, data analysis tools, and general guidelines for its practical application to in vitro pharmacology and drug screening. ---This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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