1. Discovery of the SMYD3 Inhibitor BAY-6035 Using Thermal Shift Assay (TSA)-Based High-Throughput Screening
- Author
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Ingo Hartung, Masoud Vedadi, Magda Szewczyk, Volker Badock, Carlo Stresemann, Holger Steuber, Dalia Barsyte-Lovejoy, Shawna Organ, Norbert Schmees, Clara D. Christ, Detlef Stoeckigt, Steven Kennedy, Stefan Gradl, Cheryl H. Arrowsmith, Megha Abbey, Stephan Siegel, Andrea Haegebarth, Manfred Husemann, Fengling Li, Marcus Bauser, Joerg Weiske, Irene Chau, Viacheslav V. Trush, and Peter Brown
- Subjects
Models, Molecular ,0301 basic medicine ,Thermal shift assay ,High-throughput screening ,Antineoplastic Agents ,MAP3K2 ,Biochemistry ,Analytical Chemistry ,Small Molecule Libraries ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Drug Discovery ,Humans ,Binding site ,Protein kinase A ,Kinase ,Drug discovery ,Chemistry ,Hydrogen Bonding ,Isothermal titration calorimetry ,Histone-Lysine N-Methyltransferase ,High-Throughput Screening Assays ,030104 developmental biology ,030220 oncology & carcinogenesis ,Molecular Medicine ,Hydrophobic and Hydrophilic Interactions ,Protein Binding ,Biotechnology - Abstract
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
- Published
- 2021