Enabling Lead Discovery for Histone Lysine Demethylases by High-Throughput RapidFire Mass Spectrometry
| Autor: | Peter Francis, Bill Leavens, Michelle L. Heathcote, Melanie Leveridge, Chun-wa Chung, Paul Homes, Argyrides Argyrou, Jordi Munoz-Muriedas, Michelle Gee, Laura Williams, Stuart M. Baddeley, Sue Hutchinson, Anthony Shillings, Emma J. Jones, Angela Bridges |
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| Rok vydání: | 2012 |
| Předmět: |
Jumonji Domain-Containing Histone Demethylases
Pyridines High-throughput screening Drug Evaluation Preclinical Peptide Mass spectrometry Biochemistry Mass Spectrometry Epigenesis Genetic Substrate Specificity Analytical Chemistry Histone H3 Epigenetics Enzyme Inhibitors Histone Demethylases chemistry.chemical_classification Dose-Response Relationship Drug Lysine Substrate (chemistry) Oxyquinoline High-Throughput Screening Assays Kinetics Enzyme chemistry Molecular Medicine Peptides Biotechnology |
| Zdroj: | SLAS Discovery. 17:39-48 |
| ISSN: | 2472-5552 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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