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.