Supplementary Methods: Synthesis and characterization of BAY-8002; Tritium labeling of AZD3965 and BAY-8002; MCT1 and MCT4 14C-lactate uptake measurements in the X. laevis oocytes expression System

The status of industrial Medicinal Chemistry was discussed with European Medicinal Chemistry Leaders from large to mid-sized pharma and CRO companies as well as biotechs. The chemical modality space has expanded recently from small molecules to address new challenging targets. Besides the classical SAR/SPR optimization of drug molecules also their 'greenness' has increasing importance. The entire pharma discovery ecosystem has developed significantly. Beyond pharma and academia new key players such as Biotech and integrated CROs as well as Digital companies have appeared and are now to a large extend fueled by VC money. Digitalization is happening everywhere but surprisingly did not change speed and success rates of projects so far. Future Medicinal Chemists will still have to be excellent synthetic chemists but in addition they must be knowledgeable in new computational areas such as data sciences. Their ability to collaborate and to work in teams is key.

Supplemental Figure S1: A, SLC16A1 (MCT1), SLC16A7 (MCT2), SLC16A8 (MCT3) and SLC16A3 (MCT4) mRNA expression levels and Effects of BAY-8002 on lactate Export and ECAR. Supplemental Figure S2: Binding affinity of the two MCT1 inhibitors BAY-8002 and AZD3965. Supplemental Figure S3: supplemental cell panel analysis. Supplemental Figure S4: subset analysis of MCT4 low expressing cells from the cell panel Analysis. Supplemental Figure S5: Intratumor 2/3-PG and 6PG levels after treatment with AZD3965

Supplemental Table S1: Selectivity profile of BAY-8002. Supplemental Table S2: Summary table of functional and proliferation IC50 data for BAY-8002 and AZD3965. Supplemental Table S3: Responses to BAY-8002 and AZD3965 of cell lines with acquired resistance to MCT1 inhibition.

Objectives The aim of this report was to characterize the key physicochemical, pharmacokinetic (PK), and magnetic resonance imaging (MRI) properties of gadoquatrane (BAY 1747846), a newly designed tetrameric, macrocyclic, extracellular gadolinium-based contrast agent (GBCA) with high relaxivity and stability. Materials and Methods The r1-relaxivities of the tetrameric gadoquatrane at 1.41 and 3.0 T were determined in human plasma and the nuclear magnetic relaxation dispersion profiles in water and plasma. The complex stability was analyzed in human serum over 21 days at pH 7.4 at 37 degrees C and was compared with the linear GBCA gadodiamide and the macrocyclic GBCA (mGBCA) gadobutrol. In addition, zinc transmetallation assay was performed to investigate the kinetic inertness. Protein binding and the blood-to-plasma ratio were determined in vitro using rat and human plasma. The PK profile was evaluated in rats (up to 7 days postinjection). Magnetic resonance imaging properties were investigated using a glioblastoma (GS9L) rat model. Results The new chemical entity gadoquatrane is a macrocyclic tetrameric Gd complex with one inner sphere water molecule per Gd (q = 1). Gadoquatrane showed high solubility in buffer (1.43 mol Gd/L, 10 mM Tris-HCl, pH 7.4), high hydrophilicity (logP -4.32 in 1-butanol/water), and negligible protein binding. The r1-relaxivity of gadoquatrane in human plasma per Gd of 11.8 mM(-1)center dot s(-1) (corresponding to 47.2 mM(-1)center dot s(-1) per molecule at 1.41 T at 37 degrees C, pH 7.4) was more than 2-fold (8-fold per molecule) higher compared with established mGBCAs. Nuclear magnetic relaxation dispersion profiles confirmed the more than 2-fold higher r1-relaxivity in human plasma for the clinically relevant magnetic field strengths from 0.47 to 3.0 T. The complex stability of gadoquatrane at physiological conditions was very high. The observed Gd release after 21 days at 37 degrees C in human serum was below the lower limit of quantification. Gadoquatrane showed no Gd3+ release in the presence of zinc in the transmetallation assay. The PK profile (plasma elimination, biodistribution, recovery) was comparable to that of gadobutrol. In MRI, the quantitative evaluation of the tumor-to-brain contrast in the rat glioblastoma model showed significantly improved contrast enhancement using gadoquatrane compared with gadobutrol at the same Gd dose administered (0.1 mmol Gd/kg body weight). In comparison to gadoterate meglumine, similar contrast enhancement was reached with gadoquatrane with 75% less Gd dose. In terms of the molecule dose, this was reduced by 90% when compared with gadoterate meglumine. Because of its tetrameric structure and hence lower number of molecules per volume, all prepared formulations of gadoquatrane were iso-osmolar to blood. Conclusions The tetrameric gadoquatrane is a novel, highly effective mGBCA for use in MRI. Gadoquatrane provides favorable physicochemical properties (high relaxivity and stability, negligible protein binding) while showing essentially the same PK profile (fast extracellular distribution, fast elimination via the kidneys in an unchanged form) to established mGBCAs on the market. Overall, gadoquatrane is an excellent candidate for further clinical development.

Mitochondria are key regulators of energy supply and cell death. Generation of ATP within mitochondria occurs through oxidative phosphorylation (OXPHOS), a process which utilizes the four complexes (complex I-IV) of the electron transport chain and ATP synthase. Certain oncogenic mutations (e.g., LKB1 or mIDH) can further enhance the reliance of cancer cells on OXPHOS for their energetic requirements, rendering cells sensitive to complex I inhibition and highlighting the potential value of complex I as a therapeutic target. Herein, we describe the discovery of a potent, selective, and species cross-reactive complex I inhibitor. A high-throughput screen of the Bayer compound library followed by hit triaging and initial hit-to-lead activities led to a lead structure which was further optimized in a comprehensive lead optimization campaign. Focusing on balancing potency and metabolic stability, this program resulted in the identification of BAY-179, an excellent in vivo suitable tool with which to probe the biological relevance of complex I inhibition in cancer indications.

Abstract CSNK1A1 is a serine/threonine kinase involved in multiple cellular processes, including cell division, beta catenin signaling, and TP53 activation. Inhibition of CSNK1A1 has previously been validated as a therapeutic strategy in hematologic malignancy, and degradation of CSNK1A1 protein is the downstream mechanism of action for lenalidomide in 5q- myelodysplasia (Krönke, et al. Nature. 2015.). However, lenalidomide is inactive in most solid tumor models, thus limiting the study of CSNK1A1 inhibition in other contexts. Analysis of genetic loss-of-function data from the Cancer Dependency Map reveals multiple sensitive models, including lineage-specific enrichment in colorectal and gastric cancer. In an academic-industry collaboration, we a) developed first-in-class potent and selective ATP-competitive CSNK1A1 small molecule inhibitors with preclinical anti-cancer efficacy in vivo, and b) identified FAM83 expression as a key determinant of inhibitor sensitivity. We identified a tetrahydro-pyrrolopyridinone scaffold that was subsequently optimized to yield BAY-888 (CSNK1A1 IC50 4 nM @ 10 μM ATP; 63 nM @ 1 mM ATP) and BAY-204 (CSNK1A1 IC50 2 nM @ 10 μM ATP; 12 nM @ 1 mM ATP). The crystal structure of CSNK1A1 in complex with BAY-888 confirmed compound binding in the ATP binding pocket. Across the PRISM barcoded cell line panel of more than 500 solid tumor cell lines, inhibitors phenocopy the CSNK1A1 shRNA knockdown profile. To determine downstream mediators of CSNK1A1 inhibitor sensitivity, we performed co-IP mass spectrometry following CSNK1A1 pulldown and global phosphoproteomic assays following inhibitor treatment. We identified multiple interacting proteins that are also phosphorylation targets, including FAM83 family members. FAM83 was recently reported to mediate the subcellular localization of CSNK1A1 (Fulcher, et al. Sci Signal. 2018.). Excitingly, the baseline expression of FAM83B and FAM83H correlates with inhibitor and shRNA cell line sensitivity. Modulation of FAM83H expression altered CSNK1A1 localization and sensitivity to CSNK1A1 inhibition. BAY-888 and BAY-204 are orally bioavailable and were evaluated in multiple murine cell line xenograft models. We observed promising efficacy in DLBCL (TMD8) in vivo as well as in multiple FAM83-high solid tumor models, including colorectal (HCT116 and HT29), gastric (IM95), and urothelial cancer (KU19-19). We identified RPS6 phosphorylation as one of the PD biomarkers correlating with efficacy in vivo. In summary, CSNK1A1 is a promising target with anti-tumor efficacy and achievable therapeutic index in preclinical models of FAM83-high solid tumors. Citation Format: Steven M. Corsello, Huajia Zhang, Rajesha Rupaimoole, Volker K. Schulze, Clara Lemos, Kasia B. Handing, Douglas L. Orsi, Mrinal Shekhar, Ulrike Sack, Sven Christian, Wilhelm Bone, Ranad Humeidi, William Colgan, Stephanie Hoyt, Andrew Cherniack, Jens Schroder, Stefan Kaulfuss, Krzysztof Brzezinka, Oliver von Ahsen, Anne Mengel, Roman C. Hillig, Detlev Suelzle, Jeremie Mortier, Caitlin Harrington, Rohith Nagari, Justyna Wierzbinska, Derek Chiang, Georg Beckmann, Meagan Olive, Namrata Udeshi, Annie Apffel, Steven Carr, Philip Lienau, Christian Lechner, Ulf Boemer, Alisha Caliman, David McKinney, Florence Wagner, Dominik Mumberg, Marcus Bauser, Andrea Haegebarth, Knut Eis, Ashley Eheim, Todd R. Golub. Discovery of potent and selective CSNK1A1 inhibitors for solid tumor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3588.

Objectives The aim of this report was to characterize the key physicochemical, pharmacokinetic (PK), and magnetic resonance imaging (MRI) properties of gadoquatrane (BAY 1747846), a newly designed tetrameric, macrocyclic, extracellular gadolinium-based contrast agent (GBCA) with high relaxivity and stability. Materials and Methods The r1-relaxivities of the tetrameric gadoquatrane at 1.41 and 3.0 T were determined in human plasma and the nuclear magnetic relaxation dispersion profiles in water and plasma. The complex stability was analyzed in human serum over 21 days at pH 7.4 at 37°C and was compared with the linear GBCA gadodiamide and the macrocyclic GBCA (mGBCA) gadobutrol. In addition, zinc transmetallation assay was performed to investigate the kinetic inertness. Protein binding and the blood-to-plasma ratio were determined in vitro using rat and human plasma. The PK profile was evaluated in rats (up to 7 days postinjection). Magnetic resonance imaging properties were investigated using a glioblastoma (GS9L) rat model. Results The new chemical entity gadoquatrane is a macrocyclic tetrameric Gd complex with one inner sphere water molecule per Gd ( q = 1). Gadoquatrane showed high solubility in buffer (1.43 mol Gd/L, 10 mM Tris-HCl, pH 7.4), high hydrophilicity (logP −4.32 in 1-butanol/water), and negligible protein binding. The r1-relaxivity of gadoquatrane in human plasma per Gd of 11.8 mM −1 ·s −1 (corresponding to 47.2 mM −1 ·s −1 per molecule at 1.41 T at 37°C, pH 7.4) was more than 2-fold (8-fold per molecule) higher compared with established mGBCAs. Nuclear magnetic relaxation dispersion profiles confirmed the more than 2-fold higher r1-relaxivity in human plasma for the clinically relevant magnetic field strengths from 0.47 to 3.0 T. The complex stability of gadoquatrane at physiological conditions was very high. The observed Gd release after 21 days at 37°C in human serum was below the lower limit of quantification. Gadoquatrane showed no Gd 3+ release in the presence of zinc in the transmetallation assay. The PK profile (plasma elimination, biodistribution, recovery) was comparable to that of gadobutrol. In MRI, the quantitative evaluation of the tumor-to-brain contrast in the rat glioblastoma model showed significantly improved contrast enhancement using gadoquatrane compared with gadobutrol at the same Gd dose administered (0.1 mmol Gd/kg body weight). In comparison to gadoterate meglumine, similar contrast enhancement was reached with gadoquatrane with 75% less Gd dose. In terms of the molecule dose, this was reduced by 90% when compared with gadoterate meglumine. Because of its tetrameric structure and hence lower number of molecules per volume, all prepared formulations of gadoquatrane were iso-osmolar to blood. Conclusions The tetrameric gadoquatrane is a novel, highly effective mGBCA for use in MRI. Gadoquatrane provides favorable physicochemical properties (high relaxivity and stability, negligible protein binding) while showing essentially the same PK profile (fast extracellular distribution, fast elimination via the kidneys in an unchanged form) to established mGBCAs on the market. Overall, gadoquatrane is an excellent candidate for further clinical development.

SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase that was initially described as an H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has been reported to methylate and regulate several nonhistone proteins relevant to cancer, including mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition, overexpression of SMYD3 has been linked to poor prognosis in certain cancers, suggesting SMYD3 as a potential oncogene and attractive cancer drug target. Here we report the discovery of a novel SMYD3 inhibitor. We performed a thermal shift assay (TSA)-based high-throughput screening (HTS) with 410,000 compounds and identified a novel benzodiazepine-based SMYD3 inhibitor series. Crystal structures revealed that this series binds to the substrate binding site and occupies the hydrophobic lysine binding pocket via an unprecedented hydrogen bonding pattern. Biochemical assays showed substrate competitive behavior. Following optimization and extensive biophysical validation with surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC), we identified BAY-6035, which shows nanomolar potency and selectivity against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibits methylation of MAP3K2 by SMYD3 in a cellular mechanistic assay with an IC50 <100 nM. Moreover, we describe a congeneric negative control to BAY-6035. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe that will foster the exploration of the biological role of SMYD3 in diseased and nondiseased tissues.

PIP4K2A is an insufficiently studied type II lipid kinase that catalyzes the conversion of phosphatidylinositol-5-phosphate (PI5P) into phosphatidylinositol 4,5-bisphosphate (PI4,5P2). The involvement of PIP4K2A/B in cancer has been suggested, particularly in the context of p53 mutant/null tumors. PIP4K2A/B depletion has been shown to induce tumor growth inhibition, possibly due to hyperactivation of AKT and reactive oxygen species-mediated apoptosis. Herein, we report the identification of the novel potent and highly selective inhibitors BAY-091 and BAY-297 of the kinase PIP4K2A by high-throughput screening and subsequent structure-based optimization. Cellular target engagement of BAY-091 and BAY-297 was demonstrated using cellular thermal shift assay technology. However, inhibition of PIP4K2A with BAY-091 or BAY-297 did not translate into the hypothesized mode of action and antiproliferative activity in p53-deficient tumor cells. Therefore, BAY-091 and BAY-297 serve as valuable chemical probes to study PIP4K2A signaling and its involvement in pathophysiological conditions such as cancer.

The Front Cover illustrates a novel inhibitor covalently bound to KRASG12C with an unprecedented mode of binding (image made with LigandScout). This discovery is the outcome of a computationally driven workflow based on the enumeration of novel chemical matter and stepwise prioritization. The most promising compounds were synthesized, biochemically tested, and co-crystallized with KRASG12C. More information can be found in the Communication by Jérémie Mortier, Duy Nguyen, Clara D.Christ et al.

Due to its frequent mutations in multiple lethal cancers, KRAS is one of the most-studied anticancer targets nowadays. Since the discovery of the druggable allosteric binding site containing a G12C mutation, KRAS(G12C) has been the focus of attention in oncology research. We report here a computationally driven approach aimed at identifying novel and selective KRAS(G12C) covalent inhibitors. The workflow involved initial enumeration of virtual molecules tailored for the KRAS allosteric binding site. Tools such as pharmacophore modeling, docking, and free-energy perturbations were deployed to prioritize the compounds with the best profiles. The synthesized naphthyridinone scaffold showed the ability to react with G12C and inhibit KRAS(G12C). Analogues were prepared to establish structure-activity relationships, while molecular dynamics simulations and crystallization of the inhibitor-KRAS(G12C) complex highlighted an unprecedented binding mode.

DHODH is a key enzyme in the biosynthesis of pyrimidines and recent studies have renewed interest in this old anti-cancer target. Here, we disclose the discovery of 4-triazolosalicylamides as inhibitors of DHODH and their structure activity relationship (SAR). The hit cluster was discovered during a phenotypic high throughput screen (HTS) of 2.5 million compounds where proliferation of H460 lung cancer cells was used as read-out. DHODH was successfully identified as the molecular target by comparing the activity profile of the hits in a panel of cell lines to a set of inhibitors with known pharmacological activity. The hit compounds showed good cellular potency but had undesirable DMPK properties. Interestingly, the compounds are non-ionizable in contrast to many other DHODH inhibitors and show no potency shift from biochemical to cellular assays. Structural modifications lead to compounds with sub-nanomolar potency in cellular assays and increased metabolic stability enabling the proof of concept in vivo xenograft experiments. Further optimization guided by lipophilicity efficiency and identification of metabolic hot spots resulted in molecules with low clearance and improved solubility. BAY 2402234 was selected as the clinical candidate after side by side comparison of a number of promising compounds. It shows great oral bioavailability, target engagement in all preclinical species tested, induces differentiation in AML models, and has excellent activity in a variety of leukemia models. A clinical phase I study has been initiated in patients with myeloid malignancies. (NCT03404726) Citation Format: Stefan N. Gradl, Thomas Mueller, Steven Ferrara, Sherif El Sheikh, Andreas Janzer, Han-Jie Zhou, Anders Friberg, Judith Guenther, Martina Schaefer, Timo Stellfeld, Knut Eis, Michael Kroeber, Duy Nguyen, Claudia Merz, Michael Niehues, Detlef Stoeckigt, Sven Christian, Katja Zimmermann, Pascal Lejeune, Michael Bruening, Hanna Meyer, Vera Puetter, David T. Scadden, David B. Sykes, Henrik Seidel, Ashley Eheim, Martin Michels, Andrea Haegebarth, Marcus Bauser. Discovery of BAY 2402234 by phenotypic screening: A human Dihydroorotate Dehydrogenase (DHODH) inhibitor in clinical trials for the treatment of myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2.

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.

DLBCL is the most common type of non-Hodgkin lymphoma. It is an aggressive and fast growing tumor with two major molecular subtypes: germinal center (GCB) and activated B-cell like (ABC). While the majority of patients are 60 years or older, DLBCL can occur at any age. Despite a cure rate of around 50% the need for novel therapies remains high, especially for relapsed/refractory DLBCL patients not eligible for stem cell transplant. DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. We recently discovered its role in AML differentiation (Sykes et al 2016, Cell) and we are investigating the novel DHODH inhibitor BAY 2402234 in an ongoing phase I study in myeloid malignancies (NCT03404726). Further screening of non-leukemia cancer types identified DLBCL as highly responsive to DHODH inhibition in preclinical studies. Here, we disclose for the first time the functional preclinical characterization of BAY 2402234 in DLBCL. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro it potently inhibits proliferation of DLBCL cell lines in the sub-nanomolar to low-nanomolar range. The anti-proliferative effects can be rescued by uridine supplementation which bypasses DHODH via the salvage pathway and demonstrates the on-target specificity of the inhibitor. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in subcutaneous models derived from patient-derived xenograft (PDX) and cell lines representing various DLBCL subtypes, including GCB and ABC. Dose dependent target engagement and drug exposure of BAY 2402234 could be observed by increases in plasma dihydroorotate levels and unbound plasma drug levels after treatment with the inhibitor. Based on preclinical data presented herein we plan to start clinical investigations of BAY 2402234 in patients with DLBCL in early 2019. Citation Format: Ashley Eheim, Sven Christian, Hanna Meyer, Detlef Stoeckigt, Claudia Merz, Katja Zimmermann, Marcus Bauser, Andrea Haegebarth, Steven Ferrara, David B. Sykes, David T. Scadden, Stefan Gradl, Andreas Janzer. BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of diffuse large B-cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3597.

Abstract The lactate transporter SLC16A1/monocarboxylate transporter 1 (MCT1) plays a central role in tumor cell energy homeostasis. In a cell-based screen, we identified a novel class of MCT1 inhibitors, including BAY-8002, which potently suppress bidirectional lactate transport. We investigated the antiproliferative activity of BAY-8002 in a panel of 246 cancer cell lines and show that hematopoietic tumor cells, in particular diffuse large B-cell lymphoma cell lines, and subsets of solid tumor models are particularly sensitive to MCT1 inhibition. Associated markers of sensitivity were, among others, lack of MCT4 expression, low pleckstrin homology like domain family A member 2, and high pellino E3 ubiquitin protein ligase 1 expression. The antitumor effect of MCT1 inhibition was less pronounced on tumor xenografts, with tumor stasis being the maximal response. BAY-8002 significantly increased intratumor lactate levels and transiently modulated pyruvate levels. In order to address potential acquired resistance mechanisms to MCT1 inhibition, we generated MCT1 inhibitor–resistant cell lines and show that resistance can occur by upregulation of MCT4 even in the presence of sufficient oxygen, as well as by shifting energy generation toward oxidative phosphorylation. These findings provide insight into novel aspects of tumor response to MCT1 modulation and offer further rationale for patient selection in the clinical development of MCT1 inhibitors. Mol Cancer Ther; 17(11); 2285–96. ©2018 AACR.

Recent literature has both suggested and questioned MTH1 as a novel cancer target. BAY-707 was just published as a target validation small molecule probe for assessing the effects of pharmacological inhibition of MTH1 on tumor cell survival, both in vitro and in vivo.(1) In this report, we describe the medicinal chemistry program creating BAY-707, where fragment based methods were used to develop a series of highly potent and selective MTH1 inhibitors. Using structure-based drug design and rational medicinal chemistry approaches, the potency was increased over 10,000 times from the fragment starting point while maintaining high ligand efficiency and drug-like properties.

The key learnings of the utilization of sulfoximines, sulfondiimines and sulfonimidamides in drug discovery at Bayer AG are shared.

Abstract SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase (PKMT) which was initially described as H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has recently been reported to methylate and regulate several non-histone cancer relevant proteins such as mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition overexpression of SMYD3 has been linked to poor prognosis in certain cancers, thus supporting a possible oncogenic role for SMYD3 and making it an attractive target for anticancer drug development. Here we report the discovery of a novel potent and selective SMYD3 inhibitor series. We performed a thermal shift assay based (TSA) high throughput screening followed by extensive biophysical validation resulting in identification of a benzodiazepine-based SMYD3 inhibitor series. The co-crystallization structures revealed that this series binds to the substrate binding site and occupies the hydrophobic pocket for lysine binding using an unprecedented hydrogen bond pattern. The competitive behavior of the inhibitor in biochemical assays was consistent with the binding mode observed in the crystal structure. Further optimization generated BAY-6035, which showed improved nanomolar potency and was selective against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibited methylation of MAP3K2 by SMYD3 in a cellular assay with similar potency. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe and will foster the exploration of the biologic role of SMYD3 in diseased and non-diseased tissues. Citation Format: Stefan Gradl, Holger Steuber, Jörg Weiske, Norbert Schmees, Stephan Siegel, Detlef Stoeckigt, Clara D. Christ, Fengling Li, Shawna Organ, Dalia Barsyte-Lovejoy, Magdalena M. Szewczyk, Steven Kennedy, Viacheslav Trush, Masoud Vedadi, Cheryl H. Arrowsmith, Peter J. Brown, Manfred Husemann, Amaury E. Fernandez-Montalvan, Volker Badock, Marcus Bauser, Andrea Haegebarth, Ingo V. Hartung, Carlo Stresemann. Discovery and characterization of BAY-6035, a novel benzodiazepine-based SMYD3 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1646.

The AMP-activated protein kinase (AMPK) is a sensor of the energy status in the cells, playing a key role in controlling their metabolism. For many years, AMPK was mainly perceived as a tumor suppressor in agreement with being a component of the LKB1 tumor suppressor cascade, which inhibits mTORC1. However, in the last few years, some studies suggested that AMPK might actually exert a pro-tumorigenic role in certain contexts. For instance, Liu and colleagues demonstrated that dysregulated MYC expression renders tumor cells sensitive to AMPK depletion (Liu et al, 2012. Nature). The authors showed that, due to their increased anabolism, MYC-dependent cells rely on AMPK to restore ATP levels and to prevent an energy crisis that results in apoptosis and cell death. Here we report the discovery of a new lead structure for the inhibition of AMPK by biochemical high throughput screening. The optimization of this lead structure towards potency and selectivity led to the probe compound BAY-3827 and the use of this tool compound to evaluate the therapeutic potential of AMPK inhibition in MYC-dependent tumors. To demonstrate a cellular effect of BAY-3827 an HRTF® assay (Homogeneous Time Resolved Fluorescence, cisbio) for phospho-Acetyl-CoA carboxylase (p-ACC, Ser79), a direct substrate of AMPK, was used. ACC phosphorylation was strongly inhibited by BAY-3827 in COLO 320DM and IMR-32 cells. However, despite its high potency, BAY-3827 failed to inhibit the proliferation of cells with dysregulated c-MYC or N-MYC. In conclusion, we have identified a potent and selective AMPK inhibitor. Despite demonstrated inhibition of AMPK kinase activity, BAY-3827 treatment did not translate into antiproliferative activity in MYC-dependent cells. While we could not confirm our initial hypothesis, one might speculate that inhibition of AMPK might be of therapeutic utility in other biological contexts. Therefore, the availability of potent and selective inhibitors, as described here, will contribute to further insight into the potential of AMPK inhibition as a therapeutic target in cancer. Citation Format: Clara Lemos, Volker K. Schulze, Benjamin Bader, Clara D. Christ, Hans Briem, Oliver Politz, Florian Prinz, Simon Holton, Tobias Heinrich, Julien Lefranc, Philip Lienau, Arne Scholz, Franz von Nussbaum, Carl Friedrich Nising, Dominik Mumberg, Marcus Bauser, Andrea Hagebarth. BAY-3827, a selective inhibitor of AMPK for the evaluation of the role of AMPK in Myc-dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5873.