Enabling Lead Discovery for Histone Lysine Demethylases by High-Throughput RapidFire Mass Spectrometry

Author:

Hutchinson Sue E.1,Leveridge Melanie V.2,Heathcote Michelle L.2,Francis Peter3,Williams Laura1,Gee Michelle2,Munoz-Muriedas Jordi3,Leavens Bill4,Shillings Anthony1,Jones Emma1,Homes Paul1,Baddeley Stuart2,Chung Chun-wa3,Bridges Angela1,Argyrou Argyrides1

Affiliation:

1. Departments of Biological Reagents and Assay Development

2. Screening and Compound Profiling

3. Computational and Structural Chemistry

4. Analytical Chemistry, Platform Technology and Science, GlaxoSmithKline, Stevenage, UK

Abstract

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe2+, O2, 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.

Publisher

Elsevier BV

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