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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

ChemMedChem 16(7), 1116 - 1125 (2021). doi:10.1002/cmdc.202100018, Protein arginine N-methyl transferase 4 (PRMT4) asymmetricallydimethylates the arginine residues of histone H3 and nonhistoneproteins. The overexpression of PRMT4 in several cancershas stimulated interest in the discovery of inhibitors as biologicaltools and, potentially, therapeutics. Although severalPRMT4 inhibitors have been reported, most display poorselectivity against other members of the PRMT family of methyltransferases. Herein, we report the structure-based design of anew class of alanine-containing 3-arylindoles as potent andselective PRMT4 inhibitors, and describe key structure–activityrelationships for this class of compounds., Published by Wiley-VCH, Weinheim [u.a.]

Immune checkpoint blockade using antibodies targeting the cell surface expressed proteins CTLA-4, PD-1 and PD-L1 has revolutionized cancer care and its clinical impact in several indications has prompted a search for complementary immunostimulatory approaches that can further increase the efficacy of these drugs. Mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1; HPK1), a serine/threonine kinase expressed exclusively in hematopoietic cell lineages, mediates a negative feedback signal downstream of T-cell receptor stimulation. Its activity is enhanced by PGE2 and TGFβ, factors commonly present in the tumor microenvironment. Mice deficient for MAP4K1 or expressing a kinase-dead variant of MAP4K1 exhibit enhanced T-cell function, including increased anti-tumor immunity. We developed the small molecule inhibitor BAY-405 that displays potent nanomolar MAP4K1 inhibition in biochemical and cellular assays, good kinase selectivity, and in vivo exposure after oral dosing. Pharmacological inhibition of MAP4K1 enhances T-cell immunity and overcomes the suppressive impact of PGE2, TGFβ and CD4+ T-regulatory cells. Single agent treatment of tumor-bearing mice results in suppression of tumor outgrowth in several syngeneic models. This is accompanied by an increase in the anti-tumor T-cell response, dependent on an intact T-cell compartment, while not involving direct anti-tumor cytotoxicity. Inhibition of MAP4K1 in conjunction with PD-L1 blockade results in further suppression of tumor outgrowth. Moreover, we found that MAP4K1 is expressed in both PD-L1-high and PD-L1-low human cancers. In summary, our data show that selective inhibition of MAP4K1 by means of small molecule drugs may be used to expand the patient population responding to immune checkpoint inhibition. Citation Format: Gabriele Leder, Rafael Carretero, Jeffrey Mowat, Sandra Berndt, Roland Neuhaus, Nuria Aiguabella Font, Ulf Boemer, Oliver von Ahsen, Uwe Eberspaecher, Judith Guenther, Mareike Grees, Corinna Link, Barbara Nicke, Daniel Baumann, Martina Schaefer, Mine Oezcan-Wahlbrink, Nicolas D. Werbeck, Ingo Hartung, Bertolt Kreft, Rienk Offringa. Enhancement of anti-tumor T-cell immunity by means of an oral small molecule targeting the intracellular immune checkpoint MAP4K1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1722.

Re-constitution of anti-tumor T-cell responses by clinically-approved immune checkpoint inhibitors (ICIs) targeting CTLA4 or PD-1/PD-L1 represents a breakthrough cancer therapy. Nevertheless, a substantial number of patients do not benefit from these new therapeutic modalities chiefly due to local immunosuppression in the tumor microenvironment. In addition, the long circulation time of ICIs restricts options to modify dosing regimens for management of adverse effects. Oral small molecule inhibitors as next generation immune-oncology agents may - in contrast to antibodies - allow targeting of intracellular targets for a defined duration of time. This will permit a fine-tuning of efficacy versus tolerability in single-agent treatment as well as in combination with approved ICIs. The overexpression of indole dioxygenase (IDO1) and tryptophan dioxygenase (TDO2) by many tumors results in increased metabolism of tryptophan (TRP) into kynurenine (KYN), which induces immunosuppression via activation of the aryl hydrocarbon receptor (AhR). Inhibition of AhR was proposed to restore T-cell function and induce tumor rejection. However, it was expected that identification of selective lead candidates for AhR inhibition would be challenging due to the known affinity of the AhR ligand binding site for polyaromatic ligands such as 2,3,7,8-tetrachlorodibenzodioxine (TCDD). A library of 4 million compounds was screened in a cell-based HTS campaign. A thorough hit reduction process was performed based on stringent filter parameters for lead-likeness. This process delivered a hit set of significant chemical diversity. Out of several compound classes with drug-like properties, 1,3-diaryl-pyrazin-6-one-5-carboxylic amides were selected as a preferred lead series. A comprehensive SAR exploration, including in vitro mechanistic and functional validation, was performed. Lead optimization was strongly emphasized on improving lipophilicity efficiency (LLE) to balance potency with a viable PK and CYP450 interaction profile. Several candidates suitable for in vivo profiling were identified and BAY-218 was advanced to in-depth pharmacodynamic and pharmacokinetic in vivo assessments. BAY-218 showed mono-therapeutic efficacy that was comparable to ICI treatment and further therapeutic improvement was achieved by combination with an anti-PD-L1 antibody. We were able to characterize 1,3-diaryl-pyrazin-6-one-5-carboxylic amides as a new and unprecedented class of AhR inhibitors as well as identify the key substitutions that contribute to the overall compound profile. Citation Format: Norbert Schmees, Ilona Gutcher, Ulrike Roehn, Horst Irlbacher, Detlef Stoeckigt, Benjamin Bader, Christina Kober, Lars Roese, Rafael Carretero, Iris Oezen, Ludiwg Zorn, Michael Platten, Ingo Volker Hartung, Bertolt Kreft, Hilmar Weinmann. Identification of BAY-218, a potent and selective small-molecule AhR inhibitor, as a new modality to counteract tumor immunosuppression [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 4454.

Tumor cells co-opt multiple pathways in order to evade attack by infiltrating immune cells. One such mechanism is the upregulation of indole-2,3-dioxygenase (IDO1) and/or tryptophan-2,3-dioxygenase (TDO2), both of which are first-step, rate-limiting enzymes degrading tryptophan to the immunosuppressive metabolites kynurenine (KYN) and kynurenic acid (KA). KYN and KA bind and activate the aryl hydrocarbon receptor (AhR), which is expressed in many cell types and is well known for its immunosuppressive effects. Targeting of the AhR with an inhibitor may therefore provide a novel immunotherapeutic approach for enhancing anti-tumoral immune responses and treating cancer. Here we describe the identification and functional immune characterization of BAY-218, a novel, selective and potent AhR small molecule inhibitor. Mechanistically, BAY-218 inhibited AhR nuclear translocation, dioxin response element (DRE)-luciferase reporter expression and AhR-regulated target gene expression induced by both exogenous and endogenous AhR ligands. In vitro, BAY-218 rescued TNFα production from KA-suppressed LPS-treated primary human monocytes. Furthermore, BAY-218 enhanced T cell cytokine production in a human mixed lymphocyte reaction (MLR) and a mouse antigen-specific bone-marrow-derived dendritic cell (BMDC)-OT-I T cell co-culture. In the MLR, BAY-218 increased anti-PD1 antibody-mediated IL-2 and IFNγ secretion, while an IDO inhibitor did not, indicating that BAY-218 is able to block AhR activation mediated by ligands outside of the IDO-KYN pathway. In vivo, BAY-218 enhanced anti-tumoral immune responses and reduced tumor growth in the syngeneic mouse tumor models CT26 and B16-OVA. FACS analysis of leukocytes infiltrating B16-OVA tumors demonstrated that administration of BAY-218 increased the frequency of tumor-infiltrating CD8+ T cells and NK cells while decreasing GR1+ myeloid cells and CD206+M2 macrophages. Furthermore, BAY-218 enhanced therapeutic efficacy of an anti-PD-L1 antibody in the CT26 model. In summary, AhR inhibition with BAY-218 stimulates pro-inflammatory monocyte and T cell responses in vitro and drives anti-tumor immune responses, resulting in decreased tumor growth, in vivo. Thus, inhibiting AhR represents a novel immunotherapeutic approach for blocking AhR-mediated tumor-associated immunosuppression. Citation Format: Ilona Gutcher, Christina Kober, Lars Roese, Julian Roewe, Norbert Schmees, Florian Prinz, Matyas Gorjanacz, Ulrike Roehn, Benjamin Bader, Horst Irlbacher, Detlef Stoeckigt, Rafael Carretero, Katharina Sahm, Iris Oezen, Hilmar Weinmann, Ingo V. Hartung, Bertolt Kreft, Michael Platten. Blocking tumor-associated immune suppression with BAY-218, a novel, selective aryl hydrocarbon receptor (AhR) inhibitor [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 1288.

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.

With the exception of the bromodomain and extra-terminal domain BET subgroup, little is known about the role of bromodomain (BD) containing proteins in cancer so that there is a dire need for chemical probes addressing other family members. The bromodomain and PHD-finger (BRPF) family encompasses three paralogs, BRPF1, BRPF2 and BRPF3, which are all found in histone acetyltransferase (HAT) complexes. BRPF2 is a scaffold protein and its knock-out leads to embryonic lethality at E15.5, potentially due to its role in embryonic stem cell differentiation. Here we present the structure-activity relationship (SAR) and characterization of the first selective BRPF2 chemical probe BAY-299, with additionnal strong activity at TAF1, a major component of the basal transcription initiation complex TFIID. BAY-299 shows in vitro activity for BRPF2 (IC50 = 67 nM) and TAF1 second bromodomain (BD2; IC50 = 8 nM) in the TR-FRET assay, as well as in the cellular NanoBRET assay [IC50 (BRPF2) = 575 nM; IC50 (TAF1 BD2) = 825 nM]. To the best of our knowledge BAY-299 is the only disclosed inhibitor showing BRPF2 selectivity over its two paralogues BRPF1 and BRPF3. It belongs to the 1,3-benzimidazolone scaffold and bears a novel substitution which is responsible for its high BRPF2 selectivity and also for its inactivity on BET BDs. The dual inhibitory properties of BAY-299 against BRPF2 and TAF1 make it an ideal research tool for further investigation of these two proteins in physiological and pathological processes. Citation Format: Lea Bouche, Clara D. Christ, Stephan Siegel, Cynthia Tallant, Amaury E. Fernández-Montalván, Kilian V. Huber, Verra Pütter, Susanne Müller, Oleg Fedorov, Antonius ter Laak, Tatsuo Sugawara, Detlef Stöckigt, Julia Meier, Simon J. Holton, Ingo V. Hartung, Bernard Haendler. BAY-299, a novel chemical probe for in-depth analysis of the function of the bromodomain proteins BRPF2 and TAF1 [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 980. doi:10.1158/1538-7445.AM2017-980

ATAD2 (ATPase family AAA-domain containing protein 2, also called ANCCA) is an epigenetic regulator that binds to chromatin through its bromodomain (BD), a motif specialized for acetyl-lysine recognition. ATAD2 directly associates with multiple transcription factors such as ERα, AR, E2F, and Myc; hence, ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors. Furthermore, we have recently reported a novel role for ATAD2 during DNA replication, uncovering interactions between ATAD2 and histone acetylation marks on newly synthesized histone H4. High expression of ATAD2 strongly correlates with poor patient prognosis in multiple tumor types, including gastric, endometrial, hepatocellular, ovarian, breast and lung cancers. However, the exact function of ATAD2 in these tumor types remains unclear. A more thorough validation of ATAD2 as a therapeutic target is hampered by the lack of isoform-selective, potent and cellularly active ATAD2 inhibitors. A systematic assessment of crystal structures of BD-containing protein family predicted that development of selective inhibitors of ATAD2 would be challenging. In line with this prediction, only limited progress in developing lead compounds targeting ATAD2 has been reported so far. A few notable exceptions relied on fragments as starting points, however, their weak potency, insufficient selectivity against other BDs, permeability limitations or modest cellular activity have curbed their further development towards drug candidates. Here we embarked on a novel strategy to identify ATAD2 inhibitors: 11 different DNA-encoded libraries adding up to 67 billion unique encoded compounds were combined and incubated with ATAD2 BD followed by two rounds of affinity-mediated selection. This approach provided with several series of binders, for which specific target engagement of their SMOL moiety upon off-DNA synthesis was confirmed in biochemical and biophysical assays. Several rounds of potency optimization led to the identification of BAY-850, a highly potent and ATAD2 (isoform A) mono-selective inhibitor, which holds an amine substituted 3-(2-furyl)benzamide core. This compound shows - as revealed by size exclusion chromatography and native mass spectrometry - a novel mode of action for a BD inhibitor based on specific target dimerization. In a cellular fluorescence recovery after photobleaching (FRAP) assay BAY-850 displaced wild-type ATAD2 from the chromatin to the same extent as the genetic mutagenesis of ATAD2 BD. In contrast, chemically very similar inactive control compounds showed no major effects on ATAD2 association with the chromatin. These results qualify BAY-850 as the first biologically active ATAD2 isoform A-specific chemical probe, which will enable further elucidation of the cancer biology of this intriguing protein. Citation Format: Amaury E. Fernández-Montalván, Markus Berger, Benno Kuropka, Seong Joo Koo, Volker Badock, Joerg Weiske, Simon J. Holton, Apirat Chaikuad, Laura Díaz-Sáez, James Bennett, Oleg Federov, Kilian Huber, Paolo Centrella, Matthew A. Clark, Christoph E. Dumelin, Eric A. Sigel, Holly S. Soutter, Dawn M. Troast, Ying Zhang, John W. Cuozzo, Anthony D. Keefe, Didier Roche, Vincent Rodeschini, Jan Hübner, Hilmar Weinmann, Ingo V. Hartung, Matyas Gorjanacz. Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action [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 5084. doi:10.1158/1538-7445.AM2017-5084

The vasodilatory properties of nitric oxide (NO) have been utilized in pharmacotherapy for more than 130 years. Still today, NO-donor drugs are important in the management of cardiovascular diseases. However, inhaled NO or drugs releasing NO and organic nitrates are associated with noteworthy therapeutic shortcomings, including resistance to NO in some disease states, the development of tolerance during long-term treatment, and nonspecific effects, such as post-translational modification of proteins. The beneficial actions of NO are mediated by stimulation of soluble guanylate cyclase (sGC), a heme-containing enzyme which produces the intracellular signaling molecule cyclic guanosine monophosphate (cGMP). Recently, two classes of compounds have been discovered that amplify the function of sGC in a NO-independent manner, the so-called sGC stimulators and sGC activators. The most advanced drug, the sGC stimulator riociguat, has successfully undergone Phase III clinical trials for different forms of pulmonary hypertension.