title: A scintillation proximity assay for histone demethylases.
abstract: Covalent modifications, such as methylation and demethylation of lysine residues in histones, play important roles in chromatin dynamics and the regulation of gene expression. The lysine demethylases (KDMs) catalyze the demethylation of lysine residues on histone tails and are associated with diverse human diseases, including cancer, and are therefore proposed as targets for the therapeutic modulation of gene transcription. High-throughput assays have been developed to find inhibitors of KDMs, most of which are fluorescence-based assays. Here we report the development of a coupled scintillation proximity assay (SPA) for 3 KDMs: KDM1A (LSD1), KDM3A (JMJD1A), and KDM4A (JMJD2A). In this assay methylated peptides are first demethylated by a KDM, and a protein methyltransferase (PMT) is added to methylate the resulting peptide with tritiated S-(5'-adenosyl)-l-methionine. The enzyme activities were optimized and kinetic parameters were determined. These robust coupled assays are suitable for screening KDMs in 384-well format (Z' factors of 0.70-0.80), facilitating discovery of inhibitors in the quest for cancer therapeutics.
authors.affiliation: Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
authors.forename: Wenyu
authors.initials: W
authors.lastname: Yu
authors.affiliation: Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
authors.forename: Mohammad S
authors.initials: MS
authors.lastname: Eram
authors.affiliation: Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
authors.forename: Taraneh
authors.initials: T
authors.lastname: Hajian
authors.affiliation: Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Szykowska
authors.affiliation: Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: Burgess-Brown
authors.affiliation: Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada; Department of Pharmacology and Toxicology, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Masoud
authors.initials: M
authors.lastname: Vedadi
authors.affiliation: Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada. Electronic address: peterj.brown@utoronto.ca.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Brown
mesh_terms: D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D056466:Histone Demethylases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D011485:Protein Binding; D011496:Protein Methyltransferases / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D056466:Histone Demethylases; D056484:Jumonji Domain-Containing Histone Demethylases; C530247:KDM3A protein, human; C495195:KDM1A protein, human; C494424:KDM4A protein, human; D011496:Protein Methyltransferases; D008239:Lysine
keywords: KDM1A (LSD1); KDM3A (JMJD1A); KDM4A (JMJD2A); Lysine demethylases; Protein methyltransferases; Scintillation proximity assay