Virtual Screening and Characterization of AN‐329/11627212 as a Novel Inhibitor Disrupting the EED‐H3K27me3 Interaction

The key epigenetic regulator polycomb repressive complex 2 (PRC2) is dysregulated in multiple cancers. While most therapeutic strategies target its catalytic EZH2 subunit, inhibition of its allosteric EED subunit offers a potential strategy for overcoming emerging drug resistance. Here, we identify and characterize AN-329/11627212, a novel EED-targeting compound, through structure-based virtual screening. Biochemical and biophysical analyses confirm its direct binding to EED. Strikingly, x-ray crystallography reveals that AN-329/11627212 induces dissociation of the pre-bound EED-H3K27me3 complex, consistent with a "hit-and-run" inhibitory mechanism. Detailed molecular docking revealed that AN-329/11627212 embeds itself within EED's allosteric H3K27me3-binding pocket. Functionally, AN-329/11627212 exhibited anti-proliferative activity in PRC2-dependent cancer cell lines, most potently against HCT116 colon cancer cells. Our work identifies AN-329/11627212 as a novel chemotype that acts by dissociating the EED-H3K27me3 complex, providing a foundation for developing next-generation anti-tumor agents targeting PRC2.