The EED protein–protein interaction inhibitor A-395 inactivates the PRC2 complex

Abstract

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

WD40 repeat domain proteins: a novel target class?

“…3b). The compounds inhibit PRC2 in cells with low nanomolar IC 50s , and remain active against PRC2-mutated cell lines that are resistant to catalytic inhibitors currently in clinical trial in oncology 25,26 . EED also interacts with EZH2, and an EZH2-derived stapled helix targeting EED disrupts PRC2 in cells 37,38 .…”

“…OICR-9429 binds WDR5 with an affinity of 50 nM as measured by isothermal titration calorimetry, inhibits the co-immunoprecipitation of an MLL peptide with WDR5 with an IC50 of 223 nM, and elicits a clear phenotypic response in cellular assays at 5 μM 24 . A-395, a potent and novel protein-protein interaction inhibitor that binds EED to inhibit the PRC2 complex, was initially identified via a high-throughput, small molecule screen utilizing a thermal shift assay (TSA) with EED protein, and subsequently optimized by incorporating a strategic sp 3 -rich ring constraint and appending polar functionalities to increase potency while decreasing cLogP 25,94 . EED26, another EED-targeting chemical probe, was discovered via a high-throughput campaign to find compounds inhibiting the catalytic activity of the reconstituted PRC2 complex 26 .…”

“…Elucidating the mechanism of action of the screening hit enabled structure-guided fragmentation, regrowth and optimization into a potent, selective EED inhibitor 95,96 . A-395 and EED226 display target-based cellular activity and significant in vivo efficacy in mouse tumor models and are valuable chemical tools to further interrogate the role of the PRC2 complex in tumor initiation and maintenance 25,26 .…”

“…This suggests that achieving selectivity within this target class may not be as much of a challenge as for other protein classes that share binding pockets with similar chemical features, such as kinases or bromodomains. Indeed, the highly potent antagonists of EED do not bind to WDR5, nor does the potent WDR5 inhibitor OICR9429 bind to EED 24,25 .…”

“…Similarly, the geometry of the EED inhibitor-binding site is regulated by the conformation of W364, Y365 and R367. While the pocket is shallow in the apo and histone-bound states, conformational rearrangement of Y365 and adjacent residues (engaged either directly or indirectly in aromatic cage formation) produces a larger, deeper cavity that can accommodate small-molecule inhibitors (Fig 5d–f) 25,26,94–96 .…”