Insight into the Inhibitory Mechanism of Embryonic Ectoderm Development Subunit by Triazolopyrimidine Derivatives as Inhibitors through Molecular Dynamics Simulation

Abstract

Inhibition of the Embryonic Ectoderm Development (EED) subunit in Polycomb Repressive Complex 2 (PRC2) can inhibit tumor growth. In this paper, we selected six experimentally designed EED competitive Inhibitors of the triazolopyrimidine derivatives class. We investigated the difference in the binding mode of the natural substrate to the Inhibitors and the effects of differences in the parent nuclei, heads, and tails of the Inhibitors on the inhibitory capacity. The results showed that the binding free energy of this class of Inhibitors was close to or lower compared to the natural substrate, providing an energetic basis for competitive inhibition. For the Inhibitors, the presence of a strong negatively charged group at the 6-position of the parent nucleus or the 8'-position of the head would make the hydrogen atom on the head imino group prone to flip, resulting in the vertical movement of the parent nucleus, which significantly decreased the inhibitory ability. When the 6-position of the parent nucleus was a nonpolar group, the parent nucleus would move horizontally, slightly decreasing the inhibitory ability. When the 8'-position of the head was methylene, it formed an intramolecular hydrophobic interaction with the benzene ring on the tail, resulting in a significant increase in inhibition ability.

Keywords: EED; MM/PBSA; inhibitory mechanism; molecular dynamics simulation; triazolopyrimidine derivatives.

Figure 1

Conformation of the core subunit in PRC2 protein (PDBID:6c24).

Figure 2

Conformations of the EED protein. (a) The EED top surface contains H3K27me3 (PDBID:3IIW), where the four light blue residues represent aromatic cage residues and the pink portion is the natural substrate (b) and the EED bottom surface contains the N-terminal structural domain of the EZH2 subunit (PDBID:7QK4).

Figure 3

The structures of the six Inhibitors include the ring’s atom numbering and corresponding IC₅₀ value.

Figure 4

The binding position of inhibitors and the formation of hydrogen bonds in EED. (a,b) The binding positions of the Inhibitors in the EED, and the circular magnification frame detail the comparison of the poses of the six Inhibitors, where the atoms marked in dark red are oxygen atoms. (c) The square magnification frame shows the hydrogen bonding formed between the Inhibitors and the surrounding residues after docking (in the case of inhibitor 2, which had the highest number of hydrogen bonds), where the red dotted lines represent hydrogen bonds and the portion labelled green is the residues that form hydrogen bonds with Inhibitor 2.

Figure 5

RMSD diagram of the C_α atomic skeleton of six complex systems and EED-ARL^me3SA with simulated time.

Figure 6

Free energy landscape and sampling of seven complex systems.

Figure 7

Complexes overlap the diagram of six samples, where the atoms marked in dark red are oxygen atoms. The square magnification frame is the overlap of six Inhibitors, and the red rectangular frame shows the location of the parent nucleus in Inhibitors 1, 4, and 6.

Figure 8

Intramolecular interactions diagrams and dihedral angle diagrams. (a) The hydrogen atom on the head imino group of the Inhibitors reversal and intramolecular interactions, where green represents the Inhibitor 1, yellow represents the Inhibitor 3, light blue represents the Inhibition 5 and red dashed lines represent intramolecular interactions. (b) The change of dihedral angle H₁-N₂-C₃-N₄ of the six complex systems with the simulation time during the whole simulation process.

Figure 9

(a) Schematic calculation of Gaussian optimization for Inhibitor 3–Inhibitor 3′. (b) Schematic calculation of Gaussian optimization for Inhibitor 5–Inhibitor 5′.

Figure 10

Interaction between aromatic cage residues and Inhibitors, where subfigures (a–c) are residues Tyr148, Trp364, Tyr365 interacting with the Inhibitor and subfigures (d–f) are residues Phe97 interacting with the inhibitor. The yellow dashed lines represent π-π stacking interaction, and the grey dashed lines represent hydrophobic interactions.

Figure 11

Locations of the four residues interacting with the parent nucleus of the Inhibitors. The green dashed lines represent salt bridges, the blue dashed lines represent the electrostatic attraction, the pink dashed lines represent the electrostatic repulsion and the distances in the figure represent average distances during the simulation.

Figure 12

Locations of the six residues interacting with the head and tail of the Inhibitors. The red dashed lines represent the hydrogen bond, the yellow dashed lines represent π-π stacking interaction, the gray dashed lines represent hydrophobic interactions, the blue dashed lines represent the electrostatic attraction, the pink dashed lines represent the electrostatic repulsion and the distances in the figures represent average distances during the simulation.

Figure 13

Comparison of ARL^me3SA and Inhibitor binding poses and interaction of ARL^me3SA with its surrounding residues. The green portion is ARL^me3SA and the gold portion is the Inhibitor 4. The light blue residues represent the negatively charged region, the pink residues represent the positively charged region, and the yellow residues represent the aromatic cage.