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. These assays allowed to further characterize the interplay between EZH2 allosteric modulation by methylated histone tails (H3K27me3) and inhibitor binding, and to evaluate the impact of EZH2's clinically relevant mutant Y641N on drug target residence times. While all compounds in this study exhibited slower off-rates, those with clinical candidate status display significantly slower target residence times in wild type EZH2 and disease-related mutants. These inhibitors interact in a more entropy-driven fashion and show the most persistent effects in cellular washout and antiproliferative efficacy experiments. Our work provides mechanistic insights for the largest cohort of EZH2 inhibitors reported to date, demonstrating that-among several other binding parameters-target residence time is the best predictor of cellular efficacy.

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. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.

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. 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.

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. To address this, binding rate constants for a series of histamine H1 receptor (H1R) antagonists were determined using radioligands with either slow (low koff) or fast (high koff) 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.

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. Combining binding parameters with PK/ADME properties, we illustrate in silico and in cells how kinetic selectivity could be exploited as an optimization strategy. Furthermore, using bio- and chemoinformatics we uncovered structural features influencing rate constants. Our results underscore the value of binding kinetics information in rational drug design and provide a resource for future studies on this subject.

In vitro and in silico analysis of the effects of D2 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 D2 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 D2 receptor antagonists and signal transduction after dopamine and D2 receptor antagonist exposure were measured in vitro. These data were integrated by mechanistic modelling, taking into account competitive binding of endogenous dopamine and the antagonist, the turnover of the second messenger cAMP and negative feedback by PDE turnover. KEY RESULTS: The proposed signal transduction model successfully described the cellular cAMP response for 17 D2 receptor antagonists with widely different binding kinetics. Simulation of the response to fluctuating dopamine concentrations revealed that a significant effect of the target binding kinetics on the dynamics of the signalling only occurs at endogenous dopamine concentration fluctuations with frequencies below 1 min-1 . CONCLUSIONS AND IMPLICATIONS: Signal transduction and feedback are important determinants of the time course of drug effects. The effect of the D2 receptor antagonist dissociation rate constant (koff ) is limited to the maximal rate of fluctuations in dopamine signalling as determined by the dopamine koff and the cAMP turnover.

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. By using the interaction of the serine/threonine kinase haspin with 5-iodotubercidin (5-iTU) derivatives as a model for an archetypal active-state (type I) kinase-inhibitor binding mode, we demonstrate that inhibitor residence times markedly increase with the size and polarizability of the halogen atom. The halogen-aromatic π interactions in the haspin-inhibitor complexes were characterized by means of kinetic, thermodynamic, and structural measurements along with binding-energy calculations.

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. In this review, we highlight the contributions of the Kinetics for Drug Discovery (K4DD) Consortium, which targets major open questions related to binding kinetics in an industry-driven public-private partnership.

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. First we discuss structure-kinetic relationships (SKR), and how they can be influenced by factors such as conformational changes, molecular flexibility, hydrogen bonds, hydrophobicity, water molecules and (reversible-) covalent bonds. We then introduce the methodologies currently used for the investigation of BK parameters, briefly commenting on their strengths, weaknesses and future trends. Finally, we present our current perspective on the integration of BK in the drug discovery process, aiming to stimulate further thoughts on this important subject.

Characterization of 12 GnRH peptide agonists - a kinetic perspective.

BACKGROUND AND PURPOSE: Drug-target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin-releasing hormone (GnRH) receptor for the treatment of hormone-dependent diseases. Surprisingly, the kinetic receptor-binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor-binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. EXPERIMENTAL APPROACH: A novel radioligand-binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [(125) I]-triptorelin. In addition to radioligand-binding studies, a homogeneous time-resolved FRET Tag-lite™ method was developed as an alternative assay for the same purpose. KEY RESULTS: Two novel competition association assays were successfully developed and applied to determine the kinetic receptor-binding characteristics of 12 high-affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). CONCLUSIONS AND IMPLICATIONS: Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor.