Discovery and Molecular Basis of a Diverse Set of Polycomb Repressive Complex 2 Inhibitors Recognition by EED

Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, is crucial in gene regulation and has emerged as an important epigenetic drug target for cancer therapy. The WD40 domain-containing protein EED serves as the regulatory subunit of PRC2, binding to the tri-methylated lysine 27 of histone H3 (H3K27me3) and allosterically enhancing PRC2 activity. Recently, we introduced EED226, a novel PRC2 inhibitor that binds to the K27me3 pocket on EED and demonstrated strong antitumor efficacy in a xenograft mouse model. In this study, we identify and validate four additional EED binders, including EED162, the precursor to EED226. The crystal structures of these five compounds in complex with EED revealed a common deep pocket formed by significant conformational rearrangement of the aromatic cage residues (Y365, Y148, and F97) within the H3K27me3 binding pocket of EED. The width of this pocket is defined by the side chains of these rearranged residues. Additionally, all five compounds interact with Arg367 at the bottom of the pocket. Each compound also exhibits unique interactions with EED, highlighting the dynamic nature of the H3K27me3 pocket in accommodating various binders. These findings provide structural insights that could guide the rational design of new EED binders for inhibiting PRC2 complex activity.