Small-Molecule Inhibitors of the MLL1 CXXC Domain, an Epigenetic Reader of DNA Methylation

The CXXC domain is a reader of DNA methylation which preferentially binds to unmethylated CpG DNA motifs. Chromosomal translocations involving the MLL1 gene produce in-frame fusion proteins in which the N-terminal portion of the MLL1 protein harboring its CXXC domain is fused to the C-terminal portion of multiple partners. For the MLL-AF9 fusion, mutations which disrupt CXXC domain–DNA binding abrogate the ability to cause leukemia in mice. Based on this, we initiated an effort to develop small-molecule inhibitors of the MLL1 CXXC domain as a novel approach to therapy. We developed a fluorescence polarization-based assay for MLL CXXC domain–DNA binding and screened a library of Cys-reactive molecules. For the most potent hit from this screen, we have synthesized a library of analogs to explore the structure–activity relationship, defined the binding site using chemical shift perturbations in NMR spectra, and explored the selectivity of compounds across the CXXC domain family.