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Epigenetics: Tracing Histone Demethylase Inhibitors

SBKB [doi:10.1038/sbkb.2012.176]
Technical Highlight - December 2013
Short description: The fluorescent analog of a histone demethylase inhibitor enables quantitative binding assays.

The fluorescent tracer methylstatfluor can be used to measure quantitative binding of Jumonji histone demethylase inhibitors by fluorescence polarization. Reprinted with permission from American Chemical Society. 1

Histone methylation can have profound and heritable effects on gene expression, affecting cellular functions from proliferation to differentiation. Misregulation of this mark can lead to disease, including cancer and neurological disorders. An important family of demethylases known as Jumonji C domain-containing histone demethylases (JHDMs) regulates methyl group turnover at a broad variety of histone lysine residues.

One strategy to probe the function of such enzymes is to develop small-molecule inhibitors that can complement genetic tools and offer therapeutic potential. A difficulty with small-molecule inhibitors lies in how to determine their selectivity among demethylases. Current methods measure half maximal inhibitory concentration (IC50), which cannot always be compared across enzymes or assays. Wang and colleagues previously generated the specific inhibitor methylstat using structure-guided synthesis of linker-joined substrate and cofactor mimics. The authors have now synthesized a fluorescent analog of methylstat (methylstatfluor) that can be used in fluorescence polarization (FP) assays to derive dissociation constants as a broadly comparable measurement of binding.

Methylstatfluor was synthesized by modifying a dual amine analog with fluorescein isothiocyanate. The molecule shares the fluorescence properties of fluorescein and binds JHDM1A with high affinity (Kd = 9.3 nM) in FP assays. Unlike methylstat, it does not bind JMJD2A and JMJD3, representatives from two other demethylase classes, suggesting that selective inhibitors may be obtained by modifying the secondary amine of methylstat.

The researchers optimized assay buffer conditions based on JHDM1A binding to methylstatfluor, finding that Ni2+ can replace Fe2+ for more stable binding and a larger dynamic range. Using the probe as a tracer to derive the binding of nonfluorescent inhibitors in an optimized FP competition assay produced IC50 and dissociation constants for the cofactor α-ketoglutarate and its mimics 2,4-dicarboxylic acid and N-oxalyl glycine, as well as for the substrate dimethylated histone 3 lysine 36. The assay also confirmed that methylstat is a competitive ligand.

This new tracer allows the measurement of binding to the JHDM active site for the first time. Miniaturizing the assay to fit a 384-well plate format, the researchers have set the stage for high-throughput screening of demethylase inhibitors.

Tal Nawy


  1. W. Xu et al. Quantitative analysis of histone demethylase probes using fluorescence polarization.
    J. Med. Chem. 56, 5198-5202 (2013). doi:10.1021/jm3018628

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