Table 1.
Data collection and statistics for the structure of EED -inhibitor complexes.
Fig 1.
Identification of allosteric PRC2 inhibitors through EED binding.
a. Flowchart of EEDi identification and validation from PRC2 high throughput screening. b. Chemical structures of five identified SAM non-competitive inhibitors. The IC50 values were determined using an EED-H3K27me3 AlphaScreen competition binding assay.
Fig 2.
EED inhibitors compete with H3K27me3 peptide in both enzymatic and binding assays.
a. EED inhibitors compete with H3K27me3 in NCP based PRC2 enzymatic assays. The assay was carried out at 1 x and 10 x Kact for the stimulatory H3K27me3 peptide and the concentration of SAM and nucleosome were kept at Km. Inhibitors demonstrated significantly reduced potency at a higher concentration of H3K27me3 peptide. b. EED inhibitors compete with H3K27me3 in EED-H3K27me3 AlphaScreen binding assay (competition mode). All compounds reduced the AlphaScreen signal in a dose dependent-manner.
Fig 3.
EED inhibitors are non-competitive with SAM or H3K27me0 peptide.
a. EED inhibitors are non-competitive with SAM. Enzymatic assays were carried out at 1 x and 10 x SAM with H3K27me0 in excess. There is no IC50 shift when increasing SAM concentration. b. EED inhibitors are non-competitive with H3K27me0. Enzymatic assays were carried out at 1 x and 10 x H3K27me0 with SAM in excess. There is no IC50 shift when increasing H3K27me0 concentration. c. Binding affinity determination of EED210 to EED by ITC. The stoichiometry of binding between EED210 and EED was approximately 1:1 molar ratio with N = 0.78. The enthalpy change is -20.14 ±5.29 Kcal/mol and the entropy change is -46.5cal/mol/deg. d. Concentration dependent SPR analysis of EED210 binding to EED (residues 40–441).
Fig 4.
The crystal structures of H3K27me3 competitive inhibitors binding to EED.
a. Structures of EED-EZH2 peptide in complex with EED396, EED666, EED709, EED162 and EED210. The five structures are aligned and shown in the same view. The EZH2 peptide is highlighted as red cylinder. b. A representative highlight of the conformational change of Arg367, Trp364, and Tyr365, in comparison of the EED666 bound (Arg 367 in green, Tyr365 in blue, and Trp364 in red) in and H3K27me3 bound EED structures (top); below, comparison of EED666-bound EED pocket (right) with H3K27me3-bound pocket (left); EED is shown as a surface and colored white. H3K27me3 peptide is shown as ball-and-stick in green color; for clarity, only the surface of residues Arg367 (green), Tyr365 (blue) and Trp364(red) are highlighted. c. The dynamic conformational changes of Arg367, Tyr365, Tyr148 and Phe97 in inhibitor bound EED structures.
Fig 5.
The molecular basis of H3K27me3 competitive inhibitors recognition by EED.
a. Electrostatic surface potentials of EED666 binding pocket (left); Blue—positive charge, red—negative charge; The blue, black and pink dashed circles indicate the deep pocket, aromatic packing region and the edge of pocket, respectively; Five compounds are aligned to underline the common binding features (right). Chemical groups embedded in the deep pocket are colored blue; chemical groups packed against Tyr365, Tyr148 and Phe97 are colored black; chemical groups located in the edge of the pockets are colored pink. Chemical groups involved in three binding regions are divided by two dashed lines. b. Binding mode of each co-structure. Interacting residues in EED are labeled and shown as sticks. Water molecules are shown as red sphere. Yellow dashed lines are hydrogen bonds.