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. 2021 Dec 22;7(1):875-899.
doi: 10.1021/acsomega.1c05519. eCollection 2022 Jan 11.

In Silico and In Vitro Studies for Benzimidazole Anthelmintics Repurposing as VEGFR-2 Antagonists: Novel Mebendazole-Loaded Mixed Micelles with Enhanced Dissolution and Anticancer Activity

Ayman Abo Elmaaty  1 , Khaled M Darwish  2 , Amani Chrouda  3   4 , Amira A Boseila  5 , Mohamed A Tantawy  6   7 , Sameh S Elhady  8 , Afzal B Shaik  9 , Muhamad Mustafa  10 , Ahmed A Al-Karmalawy  11
Affiliations

In Silico and In Vitro Studies for Benzimidazole Anthelmintics Repurposing as VEGFR-2 Antagonists: Novel Mebendazole-Loaded Mixed Micelles with Enhanced Dissolution and Anticancer Activity

Ayman Abo Elmaaty et al. ACS Omega. .

Abstract

Cancer is a leading cause of death worldwide and its incidence is unfortunately anticipated to rise in the next years. On the other hand, vascular endothelial growth factor receptor 2 (VEGFR-2) is highly expressed in tumor-associated endothelial cells, where it affects tumor-promoting angiogenesis. Therefore, VEGFR-2 is considered one of the most promising therapeutic targets for cancer treatment. Furthermore, some FDA-approved benzimidazole anthelmintics have already shown potential anticancer activities. Therefore, repurposing them against VEGFR-2 can provide a rapid and effective alternative that can be implicated safely for cancer treatment. Hence, 13 benzimidazole anthelmintic drugs were subjected to molecular docking against the VEGFR-2 receptor. Among the tested compounds, fenbendazole (FBZ, 1), mebendazole (MBZ, 2), and albendazole (ABZ, 3) were proposed as potential VEGFR-2 antagonists. Furthermore, molecular dynamics simulations were carried out at 200 ns, giving more information on their thermodynamic and dynamic properties. Besides, the anticancer activity of the aforementioned drugs was tested in vitro against three different cancer cell lines, including liver cancer (HUH7), lung cancer (A549), and breast cancer (MCF7) cell lines. The results depicted potential cytotoxic activity especially against both HUH7 and A549 cell lines. Furthermore, to improve the aqueous solubility of MBZ, it was formulated in the form of mixed micelles (MMs) which showed an enhanced drug release with better promising cytotoxicity results compared to the crude MBZ. Finally, an in vitro quantification for VEGFR-2 concentration in treated HUH7 cells has been conducted based on the enzyme-linked immunosorbent assay. The results disclosed that FBZ, MBZ, and ABZ significantly (p < 0.001) reduced the concentration of VEGFR-2, while the lowest inhibition was achieved in MBZ-loaded MMs, which was even much better than the reference drug sorafenib. Collectively, the investigated benzimidazole anthelmintics could be encountered as lead compounds for further structural modifications and thus better anticancer activity, and that was accomplished through studying their structure-activity relationships.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Graphical representation for the repurposing of benzimidazole anthelmintic drugs as VEGFR-2 antagonists.
Figure 2
Figure 2
Chemical structures of FDA-approved benzimidazole-based anthelmintic agents; fenbendazole 1, mebendazole 2, albendazole 3, ricobendazole 4, cyclobendazole 5, oxibendazole 6, oxfendazole 7, dribendazole 8, parbendazole 9, bendazole 10, tiabendazole 11, triclabendazole 12, flubendazole 13, as well as the crystalline benzimidazole-urea inhibitor (14) as potent VEGFR-2 inhibitor.
Figure 3
Figure 3
Crystal structure of VEGFR-2 (PDB: 2OH4) with a benzimidazole urea inhibitor shows its essential binding interactions.
Figure 4
Figure 4
rmsd trajectory analysis of the examined anthelmintic compounds and reference inhibitor in bound with VEGFR-2 target across the 200 ns explicit MD runs. (A) Protein’s backbone-rmsds; (B) ligand–protein complex backbone-rmsds; and (C) only ligand backbone-rmsds (Å), along MD timeframe (ns).
Figure 5
Figure 5
Global stability profiles of obtained Rg and SASA tones of the examined anthelmintic compounds and reference inhibitor complexed with VEGFR-2 target across the 200 ns explicit MD run. (A) Complex Rg (Å); (B) complex SASA tones (nm2), along MD timeframe (ns).
Figure 6
Figure 6
Difference RMSF analysis across VEGFR-2 residues bound to the investigated anthelmintic compounds and reference ligand across the 200 ns MD runs. The protein’s backbone-ΔRMSFs were determined considering independent 200 ns MD run for holo VEGFR-2 states (in complex with investigated ligands or crystalline reference inhibitor, GIG) against the unliganded/apo state (PDB ID: 1VR2). Trajectories of ΔRMSF are illustrated as functional residue numbers (residues 814-up to-1169).
Figure 7
Figure 7
Conformational and intermolecular distance analysis of simulated ligand–protein complexes. (A) FBZ; (B) MBZ; (C) ABZ; and (D) GIG. Upper panels are overlaid snapshots of the ligand-VEGFR-2 complexes at 0 and 200 ns of MD runs. The VEGFR-2 proteins are illustrated in red and green 3D representation (cartoon) relative to the last and initially extracted frames, respectively. Both ligands (represented as sticks) and polar hydrogen bond interactions (dashed lines) are colored in correspondence to their respective extracted frames. Lower panels represent the heatmap representation of the time evolution of intermolecular distances between binding ligand and protein residues functionalities during the whole MD simulation. Polar interaction (hydrogen bonding) distances were measured between the designated interacting donor-H···acceptor, while the distances of hydrophobic interactions were measured from the nearest interacting atom of the ligand to the Cα of a particular residue. The values of the intermolecular distances were conditionally formatted through a color scale from 0 Å (green) and up to 10 Å (white) using the Microsoft EXCEL spreadsheets.
Figure 7
Figure 7
Conformational and intermolecular distance analysis of simulated ligand–protein complexes. (A) FBZ; (B) MBZ; (C) ABZ; and (D) GIG. Upper panels are overlaid snapshots of the ligand-VEGFR-2 complexes at 0 and 200 ns of MD runs. The VEGFR-2 proteins are illustrated in red and green 3D representation (cartoon) relative to the last and initially extracted frames, respectively. Both ligands (represented as sticks) and polar hydrogen bond interactions (dashed lines) are colored in correspondence to their respective extracted frames. Lower panels represent the heatmap representation of the time evolution of intermolecular distances between binding ligand and protein residues functionalities during the whole MD simulation. Polar interaction (hydrogen bonding) distances were measured between the designated interacting donor-H···acceptor, while the distances of hydrophobic interactions were measured from the nearest interacting atom of the ligand to the Cα of a particular residue. The values of the intermolecular distances were conditionally formatted through a color scale from 0 Å (green) and up to 10 Å (white) using the Microsoft EXCEL spreadsheets.
Figure 8
Figure 8
Binding-free energy/residue decomposition illustrating the protein residue contribution at ligand–protein complex ΔGTotalbinding calculation. (A) FBZ; (B) MBZ; (C) ABZ; and (D) GIG.
Figure 9
Figure 9
(A) Size and size distribution of the micelles, (B) zeta potential of the MBZ-loaded MMs.
Figure 10
Figure 10
TEM micrograph of MBZ-loaded MMs. Scale bar in 200 (A) and 100 nm (B).
Figure 11
Figure 11
Dissolution profiles of MBZ drug and MBZ-loaded MMs in 0.1 N HCl medium.
Figure 12
Figure 12
Cytotoxic effect of the tested compounds against different cancer cell lines. (A) HUH7% cell viability upon treatment with a series of tested compounds, (B) A549% cell viability upon treatment with a series of tested compounds. (C) MCF7% cell viability upon treatment with a series of tested compounds. Concentrations were used starting from 1 to 1000 μM for 48 h, and the cytotoxicity effect was detected by the MTT assay (n = 3).
Figure 13
Figure 13
Schematic diagram illustrating the concentration of VEGFR-2 in HUH7 cells treated with the IC50 of tested drugs after 48 h using ELISA technique (n = 3), *p < 0.05, ***p < 0.001 compared to control.
Figure 14
Figure 14
SAR study of the tested benzimidazole anthelmintics as VEGFR-2 antagonists.
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