Identification and Characterization of Small Molecule Inhibitors of a Plant Homeodomain Finger

Abstract

A number of histone-binding domains are implicated in cancer through improper binding of chromatin. In a clinically reported case of acute myeloid leukemia (AML), a genetic fusion protein between nucleoporin 98 and the third plant homeodomain (PHD) finger of JARID1A drives an oncogenic transcriptional program that is dependent on histone binding by the PHD finger. By exploiting the requirement for chromatin binding in oncogenesis, therapeutics targeting histone readers may represent a new paradigm in drug development. In this study, we developed a novel small molecule screening strategy that utilizes HaloTag technology to identify several small molecules that disrupt binding of the JARID1A PHD finger to histone peptides. Small molecule inhibitors were validated biochemically through affinity pull downs, fluorescence polarization, and histone reader specificity studies. One compound was modified through medicinal chemistry to improve its potency while retaining histone reader selectivity. Molecular modeling and site-directed mutagenesis of JARID1A PHD3 provided insights into the biochemical basis of competitive inhibition.

Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes

“…However, the histone H3K4me3 tail is bound in a wide and shallow binding site of the PHD finger. This binding site is not easily amenable to the design of conventional small molecule inhibitors, and only a few groups have reported progress in this regard (23,24).…”

Synthetic trimethyllysine receptors that bind histone 3, trimethyllysine 27 (H3K27me3) and disrupt its interaction with the epigenetic reader protein CBX7

“…The flexible synthesis also offers the chance to identify agents with selectivities for other post-translational modification states, 30 and that would potentially disrupt other classes of reader protein interactions. Given the extreme rarity of chemical agents of any kind that can disrupt the complexes of methyllysine-reader proteins, [45][46][47][48][49] these compounds, though unconventional in their approach, represent a jumping off point for new bioorganic and biochemical studies of post-translational methylation pathways.…”

Small-Molecule Modulation of Methyl-Lysine-Mediated Interactions

“…It may be telling that this exception revealed a unique dimerization mechanism of small-molecule binding which effectively enhanced the available binding pocket and thereby ligand affinity. Other reported small-molecule ligands for KMe domains are rare and generally have micromolar affinities for their reader target (Wagner et al 2012), perhaps not a surprising result given the relatively low affinity of KMe readers for their cognate peptides. These facts point to the need for additional hit discovery strategies and should encourage efforts to target KMe readers with multiple recognition pockets and alternative ligand sites that have different chemical features (Spruijt et al 2013;Ruthenburg et al 2011;Arita et al 2012;Rothbart et al 2013).…”

“…Denu and co-workers developed an assay to screen for inhibitors of the third PHD domain (PHD3) of JARID1A using 96-well polystyrene plates activated with a HaloTag ligand for covalent capture of a HaloTag fusion of JARID1A PHD3 (Wagner et al 2012). JARID1A PHD3 then binds a biotinylated H3K4Me 3 ligand, and the interaction can be detected via chemiluminescence catalyzed by streptavidin-conjugated horseradish peroxidase (HRP).…”

“…In contrast, disulfram was determined to inhibit JARID1A PHD3 and other PHD fingers through ejection of zinc instead of acting as a ligand (Wagner et al 2012). This work adds to the growing number of tools available for screening KMe readers, while suggesting new structural motifs for possible antagonists and demonstrating the tractability of PHD domains for future probe development.…”