Quantitative
Analysis of Histone Demethylase Probes
Using Fluorescence Polarization
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Abstract
We
previously reported methylstat as a selective inhibitor of jumonji
C domain-containing histone demethylases (JHDMs). Herein, we describe
the synthesis of a fluorescent analogue of methylstat and its application
as a tracer in fluorescence polarization assays. Using this format,
we have evaluated the binding affinities of several known JHDM probes,
as well as the native cofactor and substrate of JHDM1A. This fluorophore
allowed a highly robust and miniaturized competition assay sufficient
for high-throughput screening