title: Assessing histone demethylase inhibitors in cells: lessons learned.
abstract: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active 'probe' molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells.
  
  
  
  A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition.
  
  
  
  High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.
authors.forename: Stephanie B
authors.initials: SB
authors.lastname: Hatch
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.
authors.forename: Clarence
authors.initials: C
authors.lastname: Yapp
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.|Medical Faculty, Research and Drug Development Center, Federal University of Ceará, Rua Cel. Nunes de Melo n.1000-Rodolfo Teófilo, 60, Fortaleza, CE 430-270 Brazil.
authors.forename: Raquel C
authors.initials: RC
authors.lastname: Montenegro
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.
authors.forename: Pavel
authors.initials: P
authors.lastname: Savitsky
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.
authors.forename: Vicki
authors.initials: V
authors.lastname: Gamble
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.
authors.forename: Anthony
authors.initials: A
authors.lastname: Tumber
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.
authors.forename: Gian Filippo
authors.initials: GF
authors.lastname: Ruda
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London, SM2 5NG UK.
authors.forename: Vassilios
authors.initials: V
authors.lastname: Bavetsias
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.
authors.forename: Oleg
authors.initials: O
authors.lastname: Fedorov
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London, SM2 5NG UK.
authors.forename: Butrus
authors.initials: B
authors.lastname: Atrash
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London, SM2 5NG UK.
authors.forename: Florence
authors.initials: F
authors.lastname: Raynaud
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London, SM2 5NG UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: Lanigan
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London, SM2 5NG UK.
authors.forename: LeAnne
authors.initials: L
authors.lastname: Carmichael
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London, SM2 5NG UK.
authors.forename: Kathy
authors.initials: K
authors.lastname: Tomlin
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London, SM2 5NG UK.
authors.forename: Rosemary
authors.initials: R
authors.lastname: Burke
authors.affiliation: Epigenetics Discovery Performance Unit, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage, SG1 2NY UK.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Westaway
authors.affiliation: Epigenetics Discovery Performance Unit, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage, SG1 2NY UK.
authors.forename: Jack A
authors.initials: JA
authors.lastname: Brown
authors.affiliation: Epigenetics Discovery Performance Unit, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage, SG1 2NY UK.
authors.forename: Rab K
authors.initials: RK
authors.lastname: Prinjha
authors.affiliation: Hamon Center for Therapeutic Oncology Research, and Department of Pharmacology, UT Southwestern Medical Center at Dallas, Dallas, TX 75390 USA.
authors.forename: Elisabeth D
authors.initials: ED
authors.lastname: Martinez
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, NIHR Oxford Biomedical Research Unit, University of Oxford, Oxford, OX3 7LD UK.
authors.forename: Udo
authors.initials: U
authors.lastname: Oppermann
authors.affiliation: Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.
authors.forename: Chas
authors.initials: C
authors.lastname: Bountra
authors.affiliation: Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA UK.|Division of Cardiovascular Medicine, Radcliffe Department of Medicine, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN UK.
authors.forename: Akane
authors.initials: A
authors.lastname: Kawamura
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London, SM2 5NG UK.
authors.forename: Julian
authors.initials: J
authors.lastname: Blagg
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Brennan
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London, SM2 5NG UK.
authors.forename: Olivia
authors.initials: O
authors.lastname: Rossanese
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ UK.|Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ UK.|Buchmann Institute for Molecular Life Science, Goethe University Frankfurt, Riedberg Campus, Max-von-Laue-Straße 15, 60438 Frankfurt am Main, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Müller
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D055162:Biocatalysis; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells; D056466:Histone Demethylases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D008745:Methylation / Q000187:drug effects; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D017239:Paclitaxel / Q000633:toxicity; D010802:Phylogeny; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D006657:Histones; D020033:Protein Isoforms; D056466:Histone Demethylases; D017239:Paclitaxel
keywords: 2-Oxoglutarate oxygenases; Apoptosis; Cell proliferation; Chromatin; Epigenetics; Histone lysine demethylase; Immunofluorescence; Toxicity