Discovery of New Inhibitors of eEF2K from Traditional Chinese Medicine Based on In Silico Screening and In Vitro Experimental Validation

Abstract

Eukaryotic elongation factor 2 kinase (eEF2K) is a highly conserved α kinase and is increasingly considered as an attractive therapeutic target for cancer as well as other diseases. However, so far, no selective and potent inhibitors of eEF2K have been identified. In this study, pharmacophore screening, homology modeling, and molecular docking methods were adopted to screen novel inhibitor hits of eEF2K from the traditional Chinese medicine database (TCMD), and then cytotoxicity assay and western blotting were performed to verify the validity of the screen. Resultantly, after two steps of screening, a total of 1077 chemicals were obtained as inhibitor hits for eEF2K from all 23,034 compounds in TCMD. Then, to verify the validity, the top 10 purchasable chemicals were further analyzed. Afterward, Oleuropein and Rhoifolin, two reported antitumor chemicals, were found to have low cytotoxicity but potent inhibitory effects on eEF2K activity. Finally, molecular dynamics simulation, pharmacokinetic and toxicological analyses were conducted to evaluate the property and potential of Oleuropein and Rhoifolin to be drugs. Together, by integrating in silico screening and in vitro biochemical studies, Oleuropein and Rhoifolin were revealed as novel eEF2K inhibitors, which will shed new lights for eEF2K-targeting drug development and anticancer therapy.

Keywords: anticancer; eEF2K inhibitors; molecular docking; pharmacophore screening; traditional Chinese medicine.

Figure 1

The structures of eEF2K-related chemicals. (A) Molecular structures of the training set. (B) Molecular structures of the test set, in which 2, 3, 12, 13, 16, and 18 are inactive compounds.

Figure 2

Virtual screening based on the pharmacophore modeling. (A) The structure of the best pharmacophore model (08), wherein green features represent hydrogen bond acceptor, blue features represent hydrophobic features and orange features represent ring aromatic, respectively. (B) The pharmacophore-based virtual screening process.

Figure 3

Homology modeling and evaluation. (A) The 3D structures of the template 6nx4_A, 5ks5_A, 3lkm_A, and 3pdt_A from the top left to the bottom right, respectively. (B) eEF2K 3D homology model (M0007). (C) Ramachandran map analysis of eEF2K 3D homology model.

Figure 4

Molecular docking to screen chemicals interaction with eEF2K. (A) The defined inhibitor binding site to eEF2K protein, indicated by the circle. (B,C) The position of A484954 (B) and Compound 34 (C) in the binding pocket of eEF2K. (D) The work flow of molecular docking.

Figure 5

The predicted position of Rhoifolin and Oleuropein in the binding pocket of eEF2K. (A,B) The predicted binding mode of Rhoifolin (A) and Oleuropein (B) with eEF2K. (C,D) Ligand interactions of Rhoifolin (C) and Oleuropein (D) with eEF2K residues. (E,F) 2D interaction diagram of Rhoifolin (E) and Oleuropein (F) with eEF2K residues.

Figure 6

Effects of the selected compounds on cell viability and eEF2K activity. A484954 was used as a positive control. (A,B) CCK-8 assay of different concentrations of Rhoifolin and Oleuropein on Hela cells viability under serum-free condition (A) or HBSS condition (B), respectively (n = 4). (C,D) Western blotting analysis of treatments of different concentrations of Rhoifolin and Oleuropein on indicated protein levels under serum-free condition (C) or HBSS condition (D), respectively.

Figure 7

MD simulation analyses of eEF2K complexed to A484954, Oleuropein, and Rhoifolin. (A) RMSD values. (B) RMSF values. (C) Rg values. All analysis were performed with Gromacs.