title: Screening for lysine-specific demethylase-1 inhibitors using a label-free high-throughput mass spectrometry assay.
abstract: Posttranslational modifications on the N terminus of histone H3 act in a combinatorial fashion to control epigenetic responses to extracellular stimuli. Lysine-specific demethylase-1 (LSD1) represents an emerging epigenetic target class for the discovery of novel antitumor therapies. In this study, a high-throughput mass spectrometry (HTMS) assay was developed to measure LSD1-catalyzed demethylation of lysine-4 on several H3 substrates. The assay leverages RapidFire chromatography in line with a triple stage quadrupole detection method to measure multiple LSD1 substrate and product reactions from an assay well. This approach minimizes artifacts from fluorescence interference and eliminates the need for antibody specificity to methylated lysines. The assay was robust in a high-throughput screen of a focused library consisting of more than 56,000 unique chemical scaffolds with a median Z' of 0.76. Validated hits from the primary screen were followed up by successive rounds of virtual and HTMS screening to mine for related structures in a parent library consisting of millions of compounds. The screen resulted in the rapid discovery of multiple chemical classes amenable to medicinal chemistry optimization. This assay was further developed into a generic platform capable of rapidly screening epigenetic targets that use the N-terminal tail of histone H3 as a substrate.
authors.affiliation: Department of Lead Discovery, Biological Mass Spectrometry Screening Group, Amgen Inc., Cambridge, MA 02142, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Plant
authors.forename: Tom
authors.initials: T
authors.lastname: Dineen
authors.forename: Alan
authors.initials: A
authors.lastname: Cheng
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Long
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.forename: Kurt A
authors.initials: KA
authors.lastname: Morgenstern
mesh_terms: D004305:Dose-Response Relationship, Drug; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D056466:Histone Demethylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D013194:Staining and Labeling; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D056466:Histone Demethylases; C495195:KDM1A protein, human