title: Enabling lead discovery for histone lysine demethylases by high-throughput RapidFire mass spectrometry.
abstract: A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.
authors.affiliation: Departments of Biological Reagents and Assay Development, Platform Technology and Science, GlaxoSmithKline,Stevenage, SG1 2NY, UK.
authors.forename: Sue E
authors.initials: SE
authors.lastname: Hutchinson
authors.forename: Melanie V
authors.initials: MV
authors.lastname: Leveridge
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Heathcote
authors.forename: Peter
authors.initials: P
authors.lastname: Francis
authors.forename: Laura
authors.initials: L
authors.lastname: Williams
authors.forename: Michelle
authors.initials: M
authors.lastname: Gee
authors.forename: Jordi
authors.initials: J
authors.lastname: Munoz-Muriedas
authors.forename: Bill
authors.initials: B
authors.lastname: Leavens
authors.forename: Anthony
authors.initials: A
authors.lastname: Shillings
authors.forename: Emma
authors.initials: E
authors.lastname: Jones
authors.forename: Paul
authors.initials: P
authors.lastname: Homes
authors.forename: Stuart
authors.initials: S
authors.lastname: Baddeley
authors.forename: Chun-Wa
authors.initials: CW
authors.lastname: Chung
authors.forename: Angela
authors.initials: A
authors.lastname: Bridges
authors.forename: Argyrides
authors.initials: A
authors.lastname: Argyrou
mesh_terms: D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D044127:Epigenesis, Genetic / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D056466:Histone Demethylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D056484:Jumonji Domain-Containing Histone Demethylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D015125:Oxyquinoline / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D011725:Pyridines; C055293:2,4-pyridinedicarboxylic acid; D015125:Oxyquinoline; D056466:Histone Demethylases; D056484:Jumonji Domain-Containing Histone Demethylases; C521694:KDM4D protein, human; C549811:KDM4E protein, human; C494424:KDM4A protein, human; D008239:Lysine