Amaury Ernesto Fernández-Montalván

Quantification of Histone H3 Lys27 Trimethylation (H3K27me3) by High-Throughput Microscopy Enables Cellular Large-Scale Screening for Small-Molecule EZH2 Inhibitors

EZH2 inhibition can decrease global histone H3 lysine 27 trimethylation (H3K27me3) and thereby reactivates silenced tumor suppressor genes. Inhibition of EZH2 is regarded as an option for therapeutic cancer intervention. To identify novel small-molecule (SMOL) inhibitors of EZH2 in drug discovery, trustworthy cellular assays amenable for phenotypic high-throughput screening (HTS) are crucial. We describe a reliable approach that quantifies changes in global levels of histone modification marks using high-content analysis (HCA). The approach was validated in different cell lines by using small interfering RNA and SMOL inhibitors. By automation and miniaturization from a 384-well to 1536-well plate, we demonstrated its utility in conducting phenotypic HTS campaigns and assessing structure-activity relationships (SAR). This assay enables screening of SMOL EZH2 inhibitors and can advance the mechanistic understanding of H3K27me3 suppression, which is crucial with regard to epigenetic therapy. We observed that a decrease in global H3K27me3, induced by EZH2 inhibition, comprises two distinct mechanisms: (1) inhibition of de novo DNA methylation and (II) inhibition of dynamic, replication-independent H3K27me3 turnover. This report describes an HCA assay for primary HTS to identify, profile, and optimize cellular active SMOL inhibitors targeting histone methyltransferases, which could benefit epigenetic drug discovery.

Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524

The kinase Bub1 functions in the spindle assembly checkpoint (SAC) and in chromosome congression, but the role of its catalytic activity remains controversial. Here, we use two novel Bub1 inhibitors, BAY-320 and BAY-524, to demonstrate potent Bub1 kinase inhibition both in vitro and in intact cells. Then, we compared the cellular phenotypes of Bub1 kinase inhibition in HeLa and RPE1 cells with those of protein depletion, indicative of catalytic or scaffolding functions, respectively. Bub1 inhibition affected chromosome association of Shugoshin and the chromosomal passenger complex (CPC), without abolishing global Aurora B function. Consequently, inhibition of Bub1 kinase impaired chromosome arm resolution but exerted only minor effects on mitotic progression or SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of Paclitaxel, impairing both chromosome segregation and cell proliferation. These findings are relevant to our understanding of Bub1 kinase function and the prospects of targeting Bub1 for therapeutic applications. DOI: http://dx.doi.org/10.7554/eLife.12187.001

Characterization of 12 GnRH peptide agonists - a kinetic perspective.

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.

Abstract 3524: BAY 1238097, a novel BET inhibitor with strong efficacy in hematological tumor models

Several BET inhibitors with strong anti-tumor efficacy have been described in the last few years. Most of them are derived from diazepine and azepine scaffolds, but more recently quinazolinones and isoxazoles chemotypes have also been described. We have identified a novel scaffold with strong BET inhibitory activity. Here we describe the biochemical characteristics of BAY 1238097 and its anti-proliferative activity in acute myeloid leukemia (AML) and multiple myeloma (MM) models. In vitro, BAY 1238097 showed strong inhibitory activity (IC50 In vivo, BAY 1238097 showed strong efficacy in the AML models THP-1, MOLM-13 and KG-1, with T/C between 13 and 20%. Overall, the compound was well tolerated at MTD, with body weight losses of 5-9% at nadir. BAY 1238097 was also active in MM models. Efficacy was observed against a human IGH-cyclin D1 translocated MOLP-8 model with a T/C of 3%, whereas the standard-of-care agents bortezomib and lenalidomide were inactive or poorly active. In this model, BAY 1238097 was well tolerated at 10 mg/kg applied over 14 days, with no body weight loss measured. BAY 1238097 was also active against the FGFR/MMSET translocated model NCIH929, with 19% T/C versus 49% T/C for the standard-of-care lenalidomide. The compound was well tolerated applied at 12 mg/kg for 9 days (maximum body weight loss 6%). Gene expression profiling performed in liver, blood and duodenum of rats treated daily with 2 mg/kg BAY 1238097 for 14 days demonstrated substantial effects on genes involved in cell proliferation and in the immune response in vivo. Altogether, BAY 1238097 is a potent inhibitor of BET binding to histones and has strong anti-proliferative activity in different AML and MM models through down-regulation of c-Myc levels and its downstream transcriptome. Initiation of clinical studies with BAY 1238097 is planned for early 2015. Citation Format: Pascale Lejeune, Tatsuo Sugawara, Kathy A. Gelato, Heidrun Ellinger-Ziegelbauer, Amaury E. Fernandez-Montalvan, Norbert Schmees, Stephan Siegel, Hilmar Weinmann, Volker Gekeler, Annette O. Walter, Matthias Ocker, Stuart Ince, Bernard Haendler. BAY 1238097, a novel BET inhibitor with strong efficacy in hematological tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3524. doi:10.1158/1538-7445.AM2015-3524

In vitro and in silico analysis of the effects of D2 receptor antagonist target binding kinetics on the cellular response to fluctuating dopamine concentrations

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.

Abstract 287: BAY 1816032, a novel BUB1 kinase inhibitor with potent antitumor activity

The spindle assembly checkpoint represents a highly conserved surveillance mechanism which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. Non-catalytic functions of the mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) were reported to be essential for spindle assembly checkpoint activation. In contrast, the catalytic function of BUB1 plays a minor role in spindle assembly checkpoint activation but is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors. Here, we disclose for the first time the structure and functional characterization of a novel, first-in-class Bub1 kinase inhibitor. Medicinal chemistry efforts resulted in BAY 1816032 featuring high potency, long target residence time and good oral bioavailablity. It inhibits BUB1 enzymatic activity with an IC50 of 7 nanomol/L, shows slow dissociation kinetics resulting in a long target residence time of 87 min, and an excellent selectivity on a panel of 395 kinases. Mechanistically BAY 1816032 abrogated nocodazole-induced Thr-120 phosphorylation of the major BUB1 target protein histone H2A in HeLa cells with an IC50 of 29 nanomol/L, induced lagging chromosomes and mitotic delay. Persistent lagging chromosomes and missegregation were observed upon combination with low concentrations of paclitaxel. Single agent BAY 1816032 inhibited proliferation of various tumor cell lines with a median IC50 of 1.4 micromol/L and demonstrated synergy or additivity with paclitaxel or docetaxel in almost all cell lines evaluated (minimal combination index 0.3). In tumor xenograft studies BAY 1816032 only marginally inhibited tumor growth as single agent upon oral administration, however, upon combination with paclitaxel or docetaxel a strong and statistically significant reduction of tumor size as compared to the respective monotherapy was observed. Intratumoral levels of phospho-Thr120 H2A were found to be strongly reduced, and no hints on drug-drug interactions were found. In line with the good tolerability in xenograft studies, no relevant findings from non-GLP 2 weeks toxicological studies in rat and dog were reported. Our findings validate the innovative concept of interference with mitotic checkpoints and justify clinical proof of concept studies evaluating BUB1 inhibitor BAY 1816032 in combination with taxanes in order to enhance their efficacy and potentially overcome resistance. Citation Format: Gerhard Siemeister, Anne Mengel, Wilhelm Bone, Jens Schroder, Sabine Zitzmann-Kolbe, Hans Briem, Amaury E. Fernandez-Montalvan, Simon Holton, Ursula Monning, Oliver von Ahsen, Sandra Johanssen, Arwed Cleve, Marion Hitchcock, Kirstin Meyer, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. BAY 1816032, a novel BUB1 kinase inhibitor with potent antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 287. doi:10.1158/1538-7445.AM2017-287

Abstract 2725: Probing mitotic functions of BUB1 kinase using the small molecule inhibitors BAY-320 and BAY-524

The maintenance of correct chromosome number (euploidy) during cell division is ensured by a highly conserved surveillance mechanism termed ‘spindle assembly checkpoint’ which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. The mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) was reported to contribute to both chromosome congression and checkpoint function, yet the role of BUB1 catalytic activity in these processes remains a matter of debate. To differentiate between catalytic and non-catalytic functions of BUB1 we compared phenotypes provoked by BUB1 protein depletion with specific BUB1 kinase inhibition using two novel small molecule inhibitors of BUB1, termed BAY-320 and BAY-524. BAY-320 and BAY-524 were highly potent and selective ATP-competitive inhibitors of BUB1 kinase activity with IC50 values in the single digit nanomolar range (at 10 micromolar ATP concentration). By monitoring phosphorylation of Thr120 in histone H2A, we showed that both compounds acted as potent BUB1 kinase inhibitors both biochemically and in human cells. We found that BUB1 inhibition substantially altered the chromosomal association of Shugoshin and the chromosomal passenger complex without major effects on global Aurora B function. Consequently, inhibition of BUB1 kinase clearly impaired chromosome arm resolution but, in stark contrast to depletion of BUB1 protein, only had a minor effect on cell cycle and SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of paclitaxel, synergistically affecting chromosome segregation and cell proliferation. These findings are highly relevant to both our understanding of BUB1 kinase function during mitosis and the prospects of BUB1 as a target of anti-cancer therapies. In this regard, BAY-320 and BAY-524 are first-in-class inhibitors of BUB1 kinase and their potential utility as anti-cancer agents is being explored. Citation Format: Anna P. Baron, Conrad von Schubert, Fabien Cubizolles, Gerhard Siemeister, Marion Hitchcock, Anne Mengel, Jens Schroder, Amaury Fernandez-Montalvan, Martin Lange, Franz von Nussbaum, Dominik Mumberg, Erich Nigg. Probing mitotic functions of BUB1 kinase using the small molecule inhibitors BAY-320 and BAY-524. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2725.

Abstract 4714: In vivo efficacy of BET inhibitor BAY 1238097 in preclinical models of melanoma and lung cancer

Bromodomain and extra terminal domain (BET) family proteins which bind to acetylated lysines of histones and regulate gene transcription are promising targets in oncology. We have previously shown that the BET inhibitor BAY 1238097 possesses strong activity in a number of hematological models, both in vitro and in vivo (AACR 2015). Here we show that BAY 1238097 is also able to inhibit the growth of solid tumors such as melanoma and lung tumors. BAY 1238097 was evaluated in vitro against a panel of melanoma cell lines and was effective in BRAF wild-type as well as BRAF mutant models. When given to C57BL/6 mice at the maximum tolerated dose (MTD) of 15 mg/kg, p.o., qd, BAY 1238097 showed efficacy against the B16/F10 syngeneic model with a T/C of 31% on day 12 post tumor inoculation (T/C≤42% = active according to NCI criteria). In this study, dacarbazine was less active than BAY 1238097 with a T/C of 44%. This was also the case in a patient-derived melanoma model resistant to dacarbazine where BAY 1238097 was found active with 39% T/C. Potent efficacy of BAY 1238097 was also observed against the human LOX-IMVI model in SCID mice either using a daily dose of 15 mg/kg or a q3d schedule at 45 mg/kg (MTD), leading to 10% and 13% T/C, respectively on day 12. Dose-dependent down-regulation of the MYC oncogene was demonstrated ex vivo in B16/F10 tumors as well as in mice peripheral blood mononuclear cells (PBMCs) with maximal effect observed between 3 to 6h post oral treatment with BAY1238097. In addition, strong up-regulation of HEXIM1 mRNA was observed in tumors and PBMCs from the mice. BAY 1238097 was also evaluated in a number of in vitro and in vivo studies in lung cancer cell lines. It was strongly active in KRAS mutant non-small cell lung cancer (NSCLC) (DV-90, NCI-H1373, LCLC-97TM1) and small cell lung cancer (SCLC) models (NCI-H69, NCI-H146, NCI-H526) with IC50 values below 1 μM. Down-regulation of c-Myc protein expression upon treatment with 1 μM was observed in all three sensitive NSCLC models. In the NCI-H1373 model, an induction of caspase 3/7 activity was observed upon 24h of treatment in vitro. In the same model implanted in vivo, a strong reduction of tumor growth was observed following daily oral application of BAY 1238097 (12 mg/kg). The treatment was well tolerated with a T/C of 16% at day 15 after start of treatment. A similar antitumor activity was obtained by applying the compound intravenously twice a week at 100 mg/kg and ex vivo analysis revealed time-dependent regulation of MYC and HEXIM1 after treatment. In vivo efficacy was not limited to NSCLC as daily oral application of BAY 1238097 (10 mg/kg) to the SCLC xenograft model NCI-H526 resulted in high efficacy with 7% T/C on day 21. Taken together, these positive results in preclinical models of melanoma and lung tumors warrant further evaluation of BET inhibition in solid tumors in the clinic. Citation Format: Olaf Klingbeil, Bernard Haendler, Antje Stresemann, Claudia Merz, Annette Walter, Amaury Ernesto Fernandez-Montalvan, Matthias Ocker, Stephan Siegel, Pascale Lejeune. In vivo efficacy of BET inhibitor BAY 1238097 in preclinical models of melanoma and lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4714.

Abstract 4691: Identification and characterization of BRPF1 bromodomain inhibitors

BRPF (bromodomain and PHD finger-containing) proteins represent a subgroup of the bromodomain protein family with three closely related members, namely BRPF1, -2, and -3. They are found in different histone acetyltransferase complexes and regulate development and cell differentiation. Despite their high similarity, they have unique, non-redundant functions. Knock-out of BRPF1 in mice leads to embryonic lethality at E9.5 due to vascular defects whereas BRPF2 deficiency leads to embryonic lethality at E15.5 due to anemia. BRPF proteins contain multiple functional regions including a PZP domain which binds to unmodified H3K4, a PWWP domain which binds to H3K36me3 and a single bromodomain which recognizes acetyl-lysines in histones. BRPF1 is overexpressed in a variety of tumors, suggesting a role in cell proliferation. As first steps towards understanding the role of BRPF1, we performed knock-down studies in different tumor cell lines and initiated the search for potent and selective inhibitors of the BRPF1 bromodomain. BRPF1-specific siRNAs were designed and used for gene silencing experiments in bladder and ovarian cancer cell lines. Following BRPF1 knock-down, marked inhibition of proliferation was observed in the CAL-29, 5637 and JMSU-1 bladder cancer cell lines, and in the ES-2 and OVCAR-8 ovarian cancer cell lines. In order to further delineate the function of BRPF1, we sought to identify selective BRPF1 bromodomain inhibitors. A collection of 5000 compounds was selected by virtual screening based on the BRPF1 bromodomain crystal structure (4LC2) and tested in a biochemical TR-FRET assay for inhibition of the interaction between BRPF1 bromodomain and an acetylated histone H4-derived peptide. Two potent hit clusters (IC50 20 μM). This binding and selectivity profile was confirmed in cellular NanoBRET assays where IC50 values for BAY-140 and BAY-496 were 1300 and 810 nM for BRPF1 bromodomain, 3600 and 2310 nM for BRPF2 bromodomain, and >10 μM for full-length BRD4. In vitro proliferation assays performed with these compounds showed single digit micromolar inhibition for the ovarian cancer ES-2, small cell lung cancer DMS-53 and acute myeloid leukemia THP-1 cell lines. However, no anti-proliferative activity was observed in a number of other tumor cell lines derived from a variety of hematological and solid tumors. Altogether, these data show that inhibiting BRPF1 bromodomain has only a limited anti-proliferative effect on tumor cell lines, suggesting that other regions than the bromodomain of the protein account for the anti-proliferative effects seen in knock-down experiments. Citation Format: Bernard Haendler, Lea Bouche, Stephan Siegel, Amaury E. Fernandez-Montalvan, Tatsuo Sugawara, Julia Meier, Stefan Knapp, Vicki Gamble. Identification and characterization of BRPF1 bromodomain inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4691.

Abstract 4703: The BET inhibitor BAY 1238097 shows efficacy in BRAF wild-type and mutant melanoma models

BET proteins recognize histone acetylation marks and play an essential role in transcription elongation. BRD4, the best studied family member, binds to the regulatory regions of oncogenes such as MYC, thereby controlling its expression and that of the downstream transcriptome. Following the identification of the first selective inhibitors, BET proteins have been shown to play essential roles in hematological and, more recently, in solid tumors. Here we studied the efficacy of the novel BET inhibitor BAY 1238097 in melanoma models. BAY 1238097 was a potent BET inhibitor with IC50 values of 47 and 295 nM for BRD4 BD1 and BD2, respectively. In NanoBRET assays, the interaction between BRD4, BRD3 or BRD2 with histone H4 was blocked in intact cells with IC50 values of 65 nM, 294 nM and 642 nM, respectively. In addition to its strong anti-proliferative activity in several hematological tumor models, BAY 1238097 was also effective in several melanoma cell lines with GI50 values below 500 nM in BRAF wild-type (CHL-1, COLO-792, B16F10, IPC-298, MeWo) as well as BRAF mutant (A375, G-361, SK-MEL-30, LOX-IMVI, SK-MEL-5, MEL-HO) models. Resistant cell lines with GI50 values above 10 μM were identified in both the BRAF wild-type and mutant groups. ChIP analysis performed in BRAF wild-type CHL-1 cells showed that BAY 1238097 displaced BRD4 from regulatory regions of oncogenes such as MYC, leading to loss of expression. The oxygen consumption rates of the melanoma cell lines were measured and we found that cell lines sensitive to BAY 1238097 depended on oxidative phosphorylation rather than glycolysis for energy production. In vivo efficacy was determined for BAY 1238097 in three patient-derived melanoma models harboring the wild-type BRAF gene. Daily, oral treatment with 7.5 mg/kg BAY 1238097 led to significantly reduced tumor growth (39% T/C) in one model. Interestingly, this model was resistant to dacarbazine given i.p. at 100 mg/kg daily. No biologically significant anti-tumor activity was observed for the two other models treated with the same conditions (62% and 70% T/C, respectively). Altogether the results show BAY 1238097 to be a potent inhibitor of BRD4 binding to histones. The compound has strong anti-proliferative activity in different melanoma models, regardless of the BRAF mutation status but related to the metabolic activity. One out of three patient-derived melanoma models with wild-type BRAF responded to BAY 1238097 treatment in vivo. Future studies will help to better characterize the impact of BET inhibition on melanoma. Citation Format: Bernard Haendler, Kathy A. Gelato, Laura Schockel, Tatsuo Sugawara, Pascale Lejeune, Heidrun Ellinger-Ziegelbauer, Amaury E. Fernandez-Montalvan, Simon Holton, Stephan Siegel, Melanie Heroult, Annette Walter, Stuart Ince, Matthias Ocker. The BET inhibitor BAY 1238097 shows efficacy in BRAF wild-type and mutant melanoma models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4703.