Structure-Guided Development of Small-Molecule PRC2 Inhibitors Targeting EZH2–EED Interaction

Abstract

Disruption of EZH2−embryonic ectoderm development (EED) protein−protein interaction (PPI) is a new promising cancer therapeutic strategy. We have previously reported the discovery of astemizole, a small-molecule inhibitor targeting the EZH2−EED PPI. Herein, we report the cocrystal structure of EED in complex with astemizole at 2.15 Å. The structure elucidates the detailed binding mode of astemizole to EED and provides a structure-guided design for the discovery of a novel EZH2−EED interaction inhibitor, DC-PRC2in-01, with an affinity K d of 4.56 μM. DC-PRC2in-01 destabilizes the PRC2 complex, thereby leading to the degradation of PRC2 core proteins and the decrease of global H3K27me3 levels in cancer cells. The proliferation of PRC2-driven lymphomas cells is effectively inhibited, and the cell cycle is arrested in the G0/G1 phase. Together, these data demonstrate that DC-PRC2in-01 could be an effective chemical probe for investigating the PRC2-related physiology and pathology and providing a promising chemical scaffold for further development.

Polycomb Repressive Complex 2 Modulation through the Development of EZH2–EED Interaction Inhibitors and EED Binders

“…72 Very recently, Luo's group has provided the cocrystal structure of the EED−1 complex clarifying its binding mode: 1 is able to bind to the bottom of the WD40-repeat domain of EED, thus hampering its binding to EZH2, but differently from EED inhibitors that bind to the H3K27me3 binding pocket at the top of WD40repeat domain of EED. 72 In the same work, a first SAR investigation on 1 revealed that a cyclic homology study led to a significant improvement in potency with compound 5b that was the most potent (IC 50 = 4.21 μM) among the novel analogues. Importantly, this new compound proved the ability to destabilize the PRC2 complex, leading to the degradation of EED, EZH2, and SUZ12 proteins, 72 differently from the EED inhibitors, that contrarily stabilize the PRC2.…”

“…72 In the same work, a first SAR investigation on 1 revealed that a cyclic homology study led to a significant improvement in potency with compound 5b that was the most potent (IC 50 = 4.21 μM) among the novel analogues. Importantly, this new compound proved the ability to destabilize the PRC2 complex, leading to the degradation of EED, EZH2, and SUZ12 proteins, 72 differently from the EED inhibitors, that contrarily stabilize the PRC2. 83,87 The EED−1 crystal structure highlighted that the EZH2− EED interaction interface could be targeted by small molecules and built the basis for future structure-based optimization and development of novel chemical entities able to display such a mechanism of action.…”

“…71 Very recently, Luo's group published the cocrystal structure of EED in complex with 1 at 2.15 Å (residues 40−441) (PDB 7KXT). 72 The structure (Figure 5B) elucidates the detailed binding mode of 1 to EED and provides insights for a structure-guided drug design that led to a novel EZH2−EED interaction inhibitor, DC-PRC2in-01 (5b). The compound 5b, bearing a pyrrolidine moiety instead of the piperidine as in 1, displayed a K D value versus EED protein of 5 μM, measured by surface plasmon resonance (SPR), and an IC 50 of 4 μM, determined by fluorescence polarization competitive binding assay.…”

“…61 Moreover, the benzimidazole moiety establishes interactions with hydrophobic residues Leu318, Met336, and Leu353, whereas the 4-methoxyphenethyl moiety engages in hydrophobic interactions with Leu246, Phe372, Trp373, and Gln374. 72 The compound 1 scaffold was optimized according to two lines of intervention: (1) the N-benzyl and the terminal phenethyl moiety were modified with functional groups aiming to reinforce the hydrophobic interactions or to establish novel polar contacts within the connectivity of the original structure (3 and 4); (2) the distance between the nitrogen atoms of the 4-aminopiperidine linker was optimized through cyclic homology and ring-opening study of the piperidine moiety (5) (Figure 5C).…”

“…Notably, 5b dose-dependently and specifically reduced the levels of H3K27me3 with respect to other H3 methyl marks, thus demonstrating the efficacy of the EZH2−EED interaction inhibitors in evoking epigenetic effect. 72 Compound 5b dosedependently impaired cell viability of Pfeiffer, KARPAS422, SU-DHL-4, and DB cell lines, harboring EZH2 heterozygous mutations, with IC 50 values below 10 μM, by blocking the cell cycle in G0/G1 phase.…”