title: Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform.
abstract: Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein-protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.
authors.affiliation: Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg 79104, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Wagner
authors.affiliation: Department of Urology and Center for Clinical Research, University Freiburg Medical Center, Freiburg 79106, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Greschik
authors.affiliation: Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg 79104, Germany.
authors.forename: Teresa
authors.initials: T
authors.lastname: Burgahn
authors.affiliation: Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg 79104, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Schmidtkunz
authors.affiliation: Department of Urology and Center for Clinical Research, University Freiburg Medical Center, Freiburg 79106, Germany.
authors.forename: Anne-Kathrin
authors.initials: AK
authors.lastname: Schott
authors.affiliation: Department of Urology and Center for Clinical Research, University Freiburg Medical Center, Freiburg 79106, Germany.
authors.forename: Joel
authors.initials: J
authors.lastname: McMillan
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius 02241, Lithuania.
authors.forename: Lina
authors.initials: L
authors.lastname: Baranauskienė
authors.affiliation: Department of Structural and Chemical Biology, Department of Oncological Sciences, Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029-6574, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Target Discovery Institute (TDI), Oxford OX3 7FZ, UK.
authors.forename: Oleg
authors.initials: O
authors.lastname: Fedorov
authors.affiliation: Department of Structural and Chemical Biology, Department of Oncological Sciences, Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029-6574, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
authors.affiliation: Structural Genomics Consortium, Botnar Research Center, NIHR Oxford BRU, University of Oxford, Oxford OX3 7LD, UK.
authors.forename: Udo
authors.initials: U
authors.lastname: Oppermann
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius 02241, Lithuania.
authors.forename: Daumantas
authors.initials: D
authors.lastname: Matulis
authors.affiliation: Department of Urology and Center for Clinical Research, University Freiburg Medical Center, Freiburg 79106, Germany German Cancer Consortium (DKTK), Freiburg, Germany BIOSS Centre of Biological Signalling Studies, University of Freiburg, 79106 Freiburg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Schüle
authors.affiliation: Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg 79104, Germany BIOSS Centre of Biological Signalling Studies, University of Freiburg, 79106 Freiburg, Germany German Cancer Research Centre (DKFZ), Heidelberg, Germany manfred.jung@pharmazie.uni-freiburg.de.
authors.forename: Manfred
authors.initials: M
authors.lastname: Jung
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D044127:Epigenesis, Genetic / Q000235:genetics*; D018922:HL-60 Cells; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000378:metabolism; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational / Q000502:physiology*
chemical_list: D018797:Cell Cycle Proteins; D006657:Histones; C413655:KDM4C protein, human; D008024:Ligands; D008869:Microtubule-Associated Proteins; D010750:Phosphoproteins; C104434:spindlin; D056484:Jumonji Domain-Containing Histone Demethylases; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine