title: Development of homogeneous luminescence assays for histone demethylase catalysis and binding.
abstract: Covalent modifications to histones play important roles in chromatin dynamics and the regulation of gene expression. The JumonjiC (JmjC)-containing histone demethylases (HDMs) catalyze the demethylation of methylated lysine residues on histone tails. Here we report the development of homogeneous luminescence-based assay methods for measuring the catalytic activity and the binding affinities of peptides to HDMs. The assays use amplified luminescent proximity homogeneous assay (ALPHA) technology, are sensitive and robust, and can be used for small molecule inhibitor screening of HDMs. We have profiled known inhibitors of JMJD2E and demonstrate a correlation between the inhibitor potencies determined by the ALPHA and other types of assays. Although this study focuses on the JMJD2E isoform, the catalytic turnover and binding assays described here can be used in studies on other HDMs. The assays should be useful for the development of small molecule inhibitors selective for HDM isoforms.
authors.affiliation: Department of Chemistry and Oxford Centre for Integrative Systems Biology, University of Oxford, UK.
authors.forename: Akane
authors.initials: A
authors.lastname: Kawamura
authors.forename: Anthony
authors.initials: A
authors.lastname: Tumber
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Rose
authors.forename: Oliver N F
authors.initials: ON
authors.lastname: King
authors.forename: Michelle
authors.initials: M
authors.lastname: Daniel
authors.forename: Udo
authors.initials: U
authors.lastname: Oppermann
authors.forename: Tom D
authors.initials: TD
authors.lastname: Heightman
authors.forename: Christopher
authors.initials: C
authors.lastname: Schofield
mesh_terms: D055162:Biocatalysis; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D020033:Protein Isoforms; D056484:Jumonji Domain-Containing Histone Demethylases; C549811:KDM4E protein, human