Concentration Dependence of the Unbound Partition Coefficient Kpuu and Its Application to Correct for Exposure-Related Discrepancies between Biochemical and Cellular Potency of KAT6A Inhibitors.

The unbound partition coefficient (Kpuu) allows the estimation of intracellular target exposure from free extracellular drug concentrations. Although the active mechanisms controlling Kpuu are saturable, Kpuu is commonly determined at a single concentration, which may not be appropriate in cases in which drug concentrations can largely vary, e.g., in plasma in vivo or in vitro IC50 assays. We examined the concentration dependence of Kpuu in vitro using KAT6A inhibitors with varying potency drop-off in ZR75-1 breast cancer cells to account for exposure-related discrepancies between cellular and biochemical IC50 Considering saturability resulted in a better quantitative bridge between both IC50 values and gave way to a simplified method to determine Kpuu that is suitable for the prediction of unbound cytosolic drug concentrations without the need to generate fu,cell estimates from binding studies in cell homogenates. As opposed to the binding method, which destroys cellular integrity, this approach provides an alternative fu,cell estimate and directly reflects the fraction of unbound drug in the cell cytosol based on Kp saturation (fu,cyto) of intact cells. In contrast to the binding method, prediction of intracellular KAT6A exposure with this more physiologic approach was able to bridge the average exposure gap between biochemical and cellular IC50 values from 73-fold down to only 5.4-fold. The concept of concentration-dependent Kpuu provides a solid rationale for early drug discovery to discriminate between pharmacology and target exposure-related IC50 discrepancies. The attractiveness of the approach also lies in the use of the same assay format for cellular IC50, fu,cyto, and the unbound partition coefficient based on fu,cyto (Kpuu,cyto) determination. SIGNIFICANCE STATEMENT: Examination of the yet-unexplored concentration dependence of the unbound partition coefficient led to a new experimental approach that resulted in more reliable predictions of intracellular target exposure and is well suited for routine drug discovery projects.

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.

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.

Functional and Structural Characterization of Bub3·BubR1 Interactions Required for Spindle Assembly Checkpoint Signaling in Human Cells.

The spindle assembly checkpoint (SAC) is an essential safeguarding mechanism devised to ensure equal chromosome distribution in daughter cells upon mitosis. The proteins Bub3 and BubR1 are key components of the mitotic checkpoint complex, an essential part of the molecular machinery on which the SAC relies. In the present work we have performed a detailed functional and biochemical characterization of the interaction between human Bub3 and BubR1 in cells and in vitro Our results demonstrate that genetic knockdown of Bub3 abrogates the SAC, promotes apoptosis, and inhibits the proliferation of human cancer cells. We also show that the integrity of the human mitotic checkpoint complex depends on the specific recognition between BubR1 and Bub3, for which the BubR1 Gle2 binding sequence motif is essential. This 1:1 binding event is high affinity, enthalpy-driven and with slow dissociation kinetics. The affinity, kinetics, and thermodynamic parameters of the interaction are differentially modulated by small regions in the N and C termini of the Gle2 binding domain sequence, suggesting the existence of "hotspots" for this protein-protein interaction. Furthermore, we show that specific disruption of endogenous BubR1·Bub3 complexes in human cancer cells phenocopies the effects observed in gene targeting experiments. Our work enhances the current understanding of key members of the SAC and paves the road for the pursuit of novel targeted cancer therapies based on SAC inhibition.

BAY 1125976, a selective allosteric AKT1/2 inhibitor, exhibits high efficacy on AKT signaling-dependent tumor growth in mouse models.

The PI3K-AKT-mTOR signaling cascade is activated in the majority of human cancers, and its activation also plays a key role in resistance to chemo and targeted therapeutics. In particular, in both breast and prostate cancer, increased AKT pathway activity is associated with cancer progression, treatment resistance and poor disease outcome. Here, we evaluated the activity of a novel allosteric AKT1/2 inhibitor, BAY 1125976, in biochemical, cellular mechanistic, functional and in vivo efficacy studies in a variety of tumor models. In in vitro kinase activity assays, BAY 1125976 potently and selectively inhibited the activity of full-length AKT1 and AKT2 by binding into an allosteric binding pocket formed by kinase and PH domain. In accordance with this proposed allosteric binding mode, BAY 1125976 bound to inactive AKT1 and inhibited T308 phosphorylation by PDK1, while the activity of truncated AKT proteins lacking the pleckstrin homology domain was not inhibited. In vitro, BAY 1125976 inhibited cell proliferation in a broad panel of human cancer cell lines. Particularly high activity was observed in breast and prostate cancer cell lines expressing estrogen or androgen receptors. Furthermore, BAY 1125976 exhibited strong in vivo efficacy in both cell line and patient-derived xenograft models such as the KPL4 breast cancer model (PIK3CAH1074R mutant), the MCF7 and HBCx-2 breast cancer models and the AKTE17K mutant driven prostate cancer (LAPC-4) and anal cancer (AXF 984) models. These findings indicate that BAY 1125976 is a potent and highly selective allosteric AKT1/2 inhibitor that targets tumors displaying PI3K/AKT/mTOR pathway activation, providing opportunities for the clinical development of new, effective treatments.

Affinity map of bromodomain protein 4 (BRD4) interactions with the histone H4 tail and the small molecule inhibitor JQ1.

Bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein family. It binds to acetylated histone tails via its tandem bromodomains BD1 and BD2 and forms a complex with the positive transcription elongation factor b, which controls phosphorylation of RNA polymerase II, ultimately leading to stimulation of transcription elongation. An essential role of BRD4 in cell proliferation and cancer growth has been reported in several recent studies. We analyzed the binding of BRD4 BD1 and BD2 to different partners and showed that the strongest interactions took place with di- and tetra-acetylated peptides derived from the histone 4 N-terminal tail. We also found that several histone 4 residues neighboring the acetylated lysines significantly influenced binding. We generated 10 different BRD4 BD1 mutants and analyzed their affinities to acetylated histone tails and to the BET inhibitor JQ1 using several complementary biochemical and biophysical methods. The impact of these mutations was confirmed in a cellular environment. Altogether, the results show that Trp-81, Tyr-97, Asn-140, and Met-149 play similarly important roles in the recognition of acetylated histones and JQ1. Pro-82, Leu-94, Asp-145, and Ile-146 have a more differentiated role, suggesting that different kinds of interactions take place and that resistance mutations compatible with BRD4 function are possible. Our study extends the knowledge on the contribution of individual BRD4 amino acids to histone and JQ1 binding and may help in the design of new BET antagonists with improved pharmacological properties.

A universal homogeneous assay for high-throughput determination of binding kinetics.

There is an increasing demand for assay technologies that enable accurate, cost-effective, and high-throughput measurements of drug-target association and dissociation rates. Here we introduce a universal homogeneous kinetic probe competition assay (kPCA) that meets these requirements. The time-resolved fluorescence energy transfer (TR-FRET) procedure combines the versatility of radioligand binding assays with the advantages of homogeneous nonradioactive techniques while approaching the time resolution of surface plasmon resonance (SPR) and related biosensors. We show application of kPCA for three important target classes: enzymes, protein-protein interactions, and G protein-coupled receptors (GPCRs). This method is capable of supporting early stages of drug discovery with large amounts of kinetic information.

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.

Label-free high-throughput screening via acoustic ejection mass spectrometry put into practice.

Acoustic droplet ejection-open port interface-mass spectrometry (ADE-OPI-MS) is a novel label-free analytical technique, promising to become a versatile readout for high-throughput screening (HTS) applications. The recent introduction of ADE-OPI-MS devices to the laboratory equipment market, paired with their compatibility with laboratory automation platforms, should facilitate the adoption of this technology by a broader community. Towards this goal, instrument robustness in the context of HTS campaigns - where up to millions of samples in complex matrices are tested in a short time frame - represents a major challenge, which explains the absence of detailed literature reports on this subject. Here, we present the results of our first fully automated HTS campaign, based on the ADE-OPI-MS technology, aiming to identify inhibitors of a metabolic enzyme in a >1 million compound library. The report encompasses the assay development and validation steps, as well as the adaptation for HTS requirements, where refinement of the capillary cleaning concept was crucial for final success. Altogether, our study unequivocally demonstrates the applicability of the ADE-OPI-MS technology for HTS-based drug discovery.

Differential analyte derivatization enables unbiased MALDI-TOF-based high-throughput screening: A proof-of-concept study for the discovery of catechol-o-methyltransferase inhibitors.

Recent advances in label-free high-throughput screening via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) offer unprecedented opportunities for the identification of novel chemical starting points in target-based drug discovery. A clear advantage of the technology is the possibility for label-free, direct quantification of analytes with high precision and robustness. Here we have expanded the range of analytes and biology that can be addressed via MALDI-TOF HTS, by developing a method based on post-reaction pyrylium-based derivatization to detect 3-methoxytyramine, the physiological enzyme product of the catechol-O-methyltransferase (COMT) enzyme. The introduction of pyrylium-type reagents as universal derivatization strategy under aqueous conditions for molecules containing primary amines represents a valuable addition to the toolbox of MALDI-TOF assay development. Characterization of COMT's enzymatic activity and inhibition by reference inhibitors, and comparison of the results obtained in our assay with data from previous mechanistic studies validated the performance of this new method. To address the problem of isobaric interference, a source of false results in MALDI-TOF assays measuring low molecular weight analytes, we devised a differential derivatization workflow which can potentially replace other counter- or orthogonal assays in future screening campaigns. Finally, we report on the first label-free HTS campaign for the identification of COMT inhibitors performed in miniaturized 1536-well microtiter plate format via MALDI-TOF MS analysis.

Acoustic Ejection Mass Spectrometry: A Fully Automatable Technology for High-Throughput Screening in Drug Discovery.

Acoustic droplet ejection (ADE)-open port interface (OPI)-mass spectrometry (MS) has recently been introduced as a versatile analytical method that combines fast and contactless acoustic sampling with sensitive and accurate electrospray ionization (ESI)-MS-based analyte detection. The potential of the technology to provide label-free measurements in subsecond analytical cycle times makes it an attractive option for high-throughput screening (HTS). Here, we report the first implementation of ADE-OPI-MS in a fully automated HTS environment, based on the example of a biochemical assay aiming at the identification of small-molecule inhibitors of the cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). First, we describe the optimization of the method to enable sensitive and accurate determination of enzyme activity and inhibition in miniaturized 1536-well microtiter plate format. Then we show both results from a validation single-concentration screen using a test set of 5500 compounds, and the subsequent concentration-response testing of selected hits in direct comparison with a previously established matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) readout. Finally, we present the development of an in-line OPI cleaning procedure aiming to match the instrument robustness required for large-scale HTS campaigns. Overall, this work points to critical method development parameters and provides guidance for the establishment of integrated ADE-OPI-MS as HTS-compatible technology for early drug discovery.

Characterization of the Menin-MLL Interaction as Therapeutic Cancer Target.

Inhibiting the interaction of menin with the histone methyltransferase MLL1 (KMT2A) has recently emerged as a novel therapeutic strategy. Beneficial therapeutic effects have been postulated in leukemia, prostate, breast, liver and in synovial sarcoma models. In those indications, MLL1 recruitment by menin was described to critically regulate the expression of disease associated genes. However, most findings so far rely on single study reports. Here we independently evaluated the pathogenic functions of the menin-MLL interaction in a large set of different cancer models with a potent and selective probe inhibitor BAY-155. We characterized the inhibition of the menin-MLL interaction for anti-proliferation, gene transcription effects, and for efficacy in several in vivo xenografted tumor models. We found a specific therapeutic activity of BAY-155 primarily in AML/ALL models. In solid tumors, we observed anti-proliferative effects of BAY-155 in a surprisingly limited fraction of cell line models. These findings were further validated in vivo. Overall, our study using a novel, highly selective and potent inhibitor, shows that the menin-MLL interaction is not essential for the survival of most solid cancer models. We can confirm that disrupting the menin-MLL complex has a selective therapeutic benefit in MLL-fused leukemia. In solid cancers, effects are restricted to single models and more limited than previously claimed.

Discovery and Characterization of BAY 1214784, an Orally Available Spiroindoline Derivative Acting as a Potent and Selective Antagonist of the Human Gonadotropin-Releasing Hormone Receptor as Proven in a First-In-Human Study in Postmenopausal Women.

The growth of uterine fibroids is sex hormone-dependent and commonly associated with highly incapacitating symptoms. Most treatment options consist of the control of these hormonal effects, ultimately blocking proliferative estrogen signaling (i.e., oral contraceptives/antagonization of human gonadotropin-releasing hormone receptor [hGnRH-R] activity). Full hGnRH-R blockade, however, results in menopausal symptoms and affects bone mineralization, thus limiting treatment duration or demanding estrogen add-back approaches. To overcome such issues, we aimed to identify novel, small-molecule hGnRH-R antagonists. This led to the discovery of compound BAY 1214784, an orally available, potent, and selective hGnRH-R antagonist. Altering the geminal dimethylindoline core of the initial hit compound to a spiroindoline system significantly improved GnRH-R antagonist potencies across several species, mandatory for a successful compound optimization in vivo. In a first-in-human study in postmenopausal women, once daily treatment with BAY 1214784 effectively lowered plasma luteinizing hormone levels by up to 49%, at the same time being associated with low pharmacokinetic variability and good tolerability.

Treating Cancer by Spindle Assembly Checkpoint Abrogation: Discovery of Two Clinical Candidates, BAY 1161909 and BAY 1217389, Targeting MPS1 Kinase.

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.

Identification and characterization of novel small molecules as potent inhibitors of the plasmodial calcium-dependent protein kinase 1.

Malaria remains a major killer in many parts of the world. Recently, there has been an increase in the role of public-private partnerships inciting academic and industrial scientists to merge their expertise in drug-target validation and in the early stage of drug discovery to identify potential new medicines. There is a need to identify and characterize new molecules showing high efficacy, low toxicity with low propensity to induce resistance in the parasite. In this context, we have studied the structural requirements of the inhibition of PfCDPK1. This is a calcium-dependent protein kinase expressed in Plasmodium falciparum, which has been genetically confirmed as essential for survival. A primary screening assay has been developed. A total of 54000 compounds were tested, yielding two distinct chemical series of nanomolar small molecule inhibitors. The most potent members of each series were further characterized through enzymatic and biophysical analyses. Dissociation rates of the inhibitor-kinase complexes were shown to be key parameters to differentiate both series. Finally, a homology-based model of the kinase core domain has been built which allows rational design of the next generation of inhibitors.

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.

Inhibition of BUB1 Kinase by BAY 1816032 Sensitizes Tumor Cells toward Taxanes, ATR, and PARP Inhibitors In Vitro and In Vivo.

PURPOSE: The catalytic function of BUB1 is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors and plays only a minor role in spindle assembly checkpoint activation. Here, we present the identification and preclinical pharmacologic profile of the first BUB1 kinase inhibitor with good bioavailability. EXPERIMENTAL DESIGN: The Bayer compound library was screened for BUB1 kinase inhibitors and medicinal chemistry efforts to improve target affinity and physicochemical and pharmacokinetic parameters resulting in the identification of BAY 1816032 were performed. BAY 1816032 was characterized for kinase selectivity, inhibition of BUB1 signaling, and inhibition of tumor cell proliferation alone and in combination with taxanes, ATR, and PARP inhibitors. Effects on tumor growth in vivo were evaluated using human triple-negative breast xenograft models. RESULTS: The highly selective compound BAY 1816032 showed long target residence time and induced chromosome mis-segregation upon combination with low concentrations of paclitaxel. It was synergistic or additive in combination with paclitaxel or docetaxel, as well as with ATR or PARP inhibitors in cellular assays. Tumor xenograft studies demonstrated a strong and statistically significant reduction of tumor size and excellent tolerability upon combination of BAY 1816032 with paclitaxel or olaparib as compared with the respective monotherapies. CONCLUSIONS: Our findings suggest clinical proof-of-concept studies evaluating BAY 1816032 in combination with taxanes or PARP inhibitors to enhance their efficacy and potentially overcome resistance.