title: Development of homogeneous nonradioactive methyltransferase and demethylase assays targeting histone H3 lysine 4.
abstract: Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z' factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.
authors.affiliation: PerkinElmer,1744 William Street, Suite 600, Montreal, Quebec, Canada, H3J 1R4.
authors.forename: Nancy
authors.initials: N
authors.lastname: Gauthier
authors.forename: Mireille
authors.initials: M
authors.lastname: Caron
authors.forename: Liliana
authors.initials: L
authors.lastname: Pedro
authors.forename: Mathieu
authors.initials: M
authors.lastname: Arcand
authors.forename: Julie
authors.initials: J
authors.lastname: Blouin
authors.forename: Anne
authors.initials: A
authors.lastname: Labonté
authors.forename: Claire
authors.initials: C
authors.lastname: Normand
authors.forename: Valérie
authors.initials: V
authors.lastname: Paquet
authors.forename: Anja
authors.initials: A
authors.lastname: Rodenbrock
authors.forename: Marjolaine
authors.initials: M
authors.lastname: Roy
authors.forename: Nathalie
authors.initials: N
authors.lastname: Rouleau
authors.forename: Lucille
authors.initials: L
authors.lastname: Beaudet
authors.forename: Jaime
authors.initials: J
authors.lastname: Padrós
authors.forename: Roberto
authors.initials: R
authors.lastname: Rodriguez-Suarez
mesh_terms: D019921:Biotinylation; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D044127:Epigenesis, Genetic / Q000187:drug effects; D056466:Histone Demethylases / Q000037:antagonists & inhibitors*; D011495:Histone-Lysine N-Methyltransferase / Q000037:antagonists & inhibitors*; D006657:Histones / Q000378:metabolism; D007118:Immunoassay / Q000379:methods; D008239:Lysine / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D054852:Small Molecule Libraries
chemical_list: D004791:Enzyme Inhibitors; D006657:Histones; D010455:Peptides; D054852:Small Molecule Libraries; D056466:Histone Demethylases; C495195:KDM1A protein, human; D011495:Histone-Lysine N-Methyltransferase; C540312:SETD7 protein, human; D008239:Lysine