Newly Synthesized Anticancer Purine Derivatives Inhibiting p-EIF4E Using Surface-Modified Lipid Nanovesicles

Abstract

Translation of mRNA is one of the processes adopted by cancer cells to maintain survival via phosphorylated (p)-eIF4E overexpression. Once p-eIF4E binds to the cap structure of mRNA, it advocates a nonstop translation process. In this regard, 15 new-based GMP analogs were synthesized to target eIF4E and restrain its binding to cap mRNA. The compounds were tested against three types of cancer cell lines: Caco-2, HepG-2, MCF-7, and normal kidney cells (Vero cells). Most of the compounds showed high potency against breast cancer cells (MCF-7), characterized by the highest cancer type for overexpression of p-eIF4E. Compound 4b was found to be the most active against three cell lines, colon (Caco-2), hepatic (HepG-2), and breast (MCF-7), with positive IC₅₀ values of 31.40, 27.15, and 21.71 μM, respectively. Then, chitosan-coated niosomes loaded with compound 4b (Cs/4b-NSs) were developed (as kinetically enhanced molecules) to improve the anticancer effects further. The prepared Cs/4b-NSs showed pronounced cytotoxicity compared to the free 4b against Caco2, Hepg2, and MCF-7 with IC₅₀ values of 16.15, 26.66, and 6.90 μM, respectively. Then, the expression of both the phosphorylated and nonphosphorylated western blot techniques was conducted on MCF-7 cells treated with the most active compounds (based on the obtained IC₅₀ values) to determine the total protein expression of both eIF4E and p-eIF4e. Interestingly, the selected most active compounds displayed 35.8-40.7% inhibition of p-eIF4E expression when evaluated on MCF-7 compared to Ribavirin (positive control). CS/4b-NSs showed the best inhibition (40.7%). The findings of the present joint in silico molecular docking, simulation dynamic studies, and experimental investigation suggest the potential use of niosomal nanovesicles as a promising nanocarrier for the targeted delivery of the newly synthesized compound 4b to eukaryotic initiation factor 4E. These outcomes support the possible use of Cs/4b-NSs in targeted cancer therapy.

Figure 1

Cellular mechanism of translation initiation.

Figure 2

Scheme diagram depicting the synthesis of the new chemical compounds.

Figure 3

Docking interaction between eIf4E (3U7X) and different ligands: (A) 2D binding mode of m7GTP, (B) 2D binding mode of Ribavirin, (C) 2D binding mode of 4b, and (D) 3D binding mode of 4b. Blue color ribbons represent beta sheets, red color ribbons represent helix, gray color ribbons represent turns, and green color ribbons represent residues less than 10 Å.

Figure 4

Results of dynamic simulation studies: (A) total energy vs time, (B) RMSD vs conformations, and (C) RMSF vs residue index. (D) Color map.

Figure 5

Ramachandran plot representation of torsional energy conformations for the interaction between 4b and eIF4E.

Figure 6

(A) TEM image for Cs/4b-NSs and (B) time-dependent release % of 4b from Cs/4b-NSs at 37 °C into pH 5.5 (triangle) and pH 7.4 (square).

Figure 7

(A) IC₅₀expressed by the novel compounds with evident activity against Caco-2, HepG-2, and MCF-7 cells. Data are represented as the mean ± SD (n = 3). Statistical analysis was carried out using one-way ANOVA test followed by a Tukey posthoc test (P ranged from <0.05 to <0.0001). The symbols mean significant differences compared to (*) rapamycin and (#) Ribavirin. (B) Collective dose–response curves of novel compounds with evident activity against MCF-7 cells. Data are represented as the mean ± SD (n = 3). Compounds with IC₅₀ over 100 were not represented within curves. (C) Collective dose–response curves of novel compounds with evident activity against HepG-2 cells. Data are represented as the mean ± SD (n = 3). Compounds with IC₅₀ over 100 were not represented within curves. (D) Collective dose–response curves of novel compounds with evident activity against Caco-2 cells. Data are represented as the mean ± SD (n = 3). Compounds with IC₅₀ values over 100 were not represented within curves.

Figure 8

Total protein expression of both eIF4E and p-eIF4E on treated MCF-7 cells. (A) Western blot depicting the protein expression levels of eIF4E and p-eIF4E. (B) Relative protein expression levels normalized to beta-actin control. Data are presented as the mean ± SD (n = 3). Statistical analysis was carried out using one-way ANOVA test followed by Tukey posthoc test (P < 0.0001). The symbols mean a significant difference as compared with (*) MCF-7 control, (#) Ribavirin, (@) 3c, (&) 4f, (α) Cs/4b-NSs, and (μ) 3b.