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Covalent modifications, such as methylation and demethylation of lysine residues in histones, play important roles in chromatin dynamics and the regulation of gene expression. The lysine demethylases (KDMs) catalyze the demethylation of lysine residues on histone tails and are associated with diverse human diseases, including cancer, and are therefore proposed as targets for the therapeutic modulation of gene transcription. High-throughput assays have been developed to find inhibitors of KDMs, most of which are fluorescence-based assays. Here we report the development of a coupled scintillation proximity assay (SPA) for 3 KDMs: KDM1A (LSD1), KDM3A (JMJD1A), and KDM4A (JMJD2A). In this assay methylated peptides are first demethylated by a KDM, and a protein methyltransferase (PMT) is added to methylate the resulting peptide with tritiated S-(5'-adenosyl)-l-methionine. The enzyme activities were optimized and kinetic parameters were determined. These robust coupled assays are suitable for screening KDMs in 384-well format (Z' factors of 0.70-0.80), facilitating discovery of inhibitors in the quest for cancer therapeutics.

Original publication

DOI

10.1016/j.ab.2014.06.023

Type

Journal article

Journal

Anal Biochem

Publication Date

15/10/2014

Volume

463

Pages

54 - 60

Keywords

KDM1A (LSD1), KDM3A (JMJD1A), KDM4A (JMJD2A), Lysine demethylases, Protein methyltransferases, Scintillation proximity assay, Enzyme Assays, Enzyme Inhibitors, Histone Demethylases, Humans, Jumonji Domain-Containing Histone Demethylases, Kinetics, Lysine, Methylation, Protein Binding, Protein Methyltransferases