A miniaturized mode-of-action profiling platform enables high throughput characterization of the molecular and cellular dynamics of EZH2 inhibition.

The market approval of Tazemetostat (TAZVERIK) for the treatment of follicular lymphoma and epithelioid sarcoma has established "enhancer of zeste homolog 2" (EZH2) as therapeutic target in oncology. Despite their structural similarities and common mode of inhibition, Tazemetostat and other EZH2 inhibitors display differentiated pharmacological profiles based on their target residence time. Here we established high throughput screening methods based on time-resolved fluorescence energy transfer, scintillation proximity and high content analysis microscopy to quantify the biochemical and cellular binding of a chemically diverse collection of EZH2 inhibitors.

Dual targeting of the androgen receptor and PI3K/AKT/mTOR pathways in prostate cancer models improves antitumor efficacy and promotes cell apoptosis.

Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy.

Considerations for improved performance of competition association assays analysed with the Motulsky-Mahan's "kinetics of competitive binding" model.

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.

Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor.

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.

Binding Kinetics Survey of the Drugged Kinome.

Target residence time is emerging as an important optimization parameter in drug discovery, yet target and off-target engagement dynamics have not been clearly linked to the clinical performance of drugs. Here we developed high-throughput binding kinetics assays to characterize the interactions of 270 protein kinase inhibitors with 40 clinically relevant targets. Analysis of the results revealed that on-rates are better correlated with affinity than off-rates and that the fraction of slowly dissociating drug-target complexes increases from early/preclinical to late stage and FDA-approved compounds, suggesting distinct contributions by each parameter to clinical success.

In vitro and in silico analysis of the effects of D receptor antagonist target binding kinetics on the cellular response to fluctuating dopamine concentrations.

Background And Purpose: Target binding kinetics influence the time course of the drug effect (pharmacodynamics) both (i) directly, by affecting the time course of target occupancy, driven by the pharmacokinetics of the drug, competition with endogenous ligands and target turnover, and (ii) indirectly, by affecting signal transduction and homeostatic feedback. For dopamine D receptor antagonists, it has been hypothesized that fast receptor binding kinetics cause fewer side effects, because part of the dynamics of the dopaminergic system is preserved by displacement of these antagonists.

Experimental Approach: Target binding kinetics of D receptor antagonists and signal transduction after dopamine and D receptor antagonist exposure were measured in vitro.

Halogen-Aromatic π Interactions Modulate Inhibitor Residence Times.

Prolonged drug residence times may result in longer-lasting drug efficacy, improved pharmacodynamic properties, and "kinetic selectivity" over off-targets with high drug dissociation rates. However, few strategies have been elaborated to rationally modulate drug residence time and thereby to integrate this key property into the drug development process. Herein, we show that the interaction between a halogen moiety on an inhibitor and an aromatic residue in the target protein can significantly increase inhibitor residence time.

Kinetics for Drug Discovery: an industry-driven effort to target drug residence time.

A considerable number of approved drugs show non-equilibrium binding characteristics, emphasizing the potential role of drug residence times for in vivo efficacy. Therefore, a detailed understanding of the kinetics of association and dissociation of a target-ligand complex might provide crucial insight into the molecular mechanism-of-action of a compound. This deeper understanding will help to improve decision making in drug discovery, thus leading to a better selection of interesting compounds to be profiled further.

Binding kinetics in drug discovery - A current perspective.

The impact of target binding kinetics (BK) on the clinical performance of therapeutic agents is presently a topic of intense debate in drug discovery. While retrospective studies suggest that BK is a differentiating parameter in marketed medicines, it is yet unclear how this information could be used to prioritize drug candidates during lead optimization. Motivated by the question whether BK can be understood and rationally optimized, we review the most relevant literature in the field, with special focus on selected examples from our organization.