title: High-throughput TR-FRET assays for identifying inhibitors of LSD1 and JMJD2C histone lysine demethylases.
abstract: Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.
authors.affiliation: Amgen, Inc., Cambridge, MA 02142, USA. vyu@amgen.com
authors.forename: Violeta
authors.initials: V
authors.lastname: Yu
authors.forename: Tanja
authors.initials: T
authors.lastname: Fisch
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Long
authors.forename: Jin
authors.initials: J
authors.lastname: Tang
authors.forename: Josie Han
authors.initials: JH
authors.lastname: Lee
authors.forename: Markus
authors.initials: M
authors.lastname: Hierl
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.forename: Peter
authors.initials: P
authors.lastname: Yakowec
authors.forename: Ralf
authors.initials: R
authors.lastname: Schwandner
authors.forename: Renee
authors.initials: R
authors.lastname: Emkey
mesh_terms: D000595:Amino Acid Sequence; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D056466:Histone Demethylases / Q000037:antagonists & inhibitors*; D007118:Immunoassay / Q000379:methods; D056484:Jumonji Domain-Containing Histone Demethylases / Q000037:antagonists & inhibitors*; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; C413655:KDM4C protein, human; D010455:Peptides; D056466:Histone Demethylases; D056484:Jumonji Domain-Containing Histone Demethylases; C495195:KDM1A protein, human; D008239:Lysine