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. 2023 Jan 19;18(2):159-176.
doi: 10.4103/1735-5362.367795. eCollection 2023 Apr.

A comparative study of the arazyme-based fusion proteins with various ligands for more effective targeting cancer therapy: an in-silico analysis

Rezvan Mehrab  1 , Hamid Sedighian  2 , Fattah Sotoodehnejadnematalahi  1 , Raheleh Halabian  2 , Abbas Ali Imani Fooladi  2
Affiliations

A comparative study of the arazyme-based fusion proteins with various ligands for more effective targeting cancer therapy: an in-silico analysis

Rezvan Mehrab et al. Res Pharm Sci. .

Abstract

Background and purpose: Recently, the use of immunotoxins for targeted cancer therapy has been proposed, to find new anticancer drugs with high efficacy on tumor cells with minimal side effects on normal cells. we designed and compared several arazyme (AraA)-based fusion proteins with different ligands to choose the best-targeted therapy for interleukin 13 receptor alpha 2 (IL13Rα2)-overexpressed cancer cells. For this purpose, IL13Rα2 was selected as a receptor and IL13 and IL13.E13K were evaluated as native and mutant ligands, respectively. In addition, Pep-1 and A2b11 were chosen as the peptide ligands for targeted cancer therapy.

Experimental approach: Several bioinformatics servers were used for designing constructs and optimization. The structures of the chimeric proteins were predicted and verified by I-TASSER, Q-Mean, ProSA, Ramachandran plot, and Verify3D program. Physicochemical properties, toxicity, and antigenicity were predicted by ProtParam, ToxinPred, and VaxiJen. HawkDock, LigPlot+, and GROMACS software were used for docking and molecular dynamics simulation of the ligand-receptor interaction.

Findings/results: The in silico results showed AraA-A2b11 has higher values of confidence score and Q-mean score was obtained for high-resolution crystal structures. All chimeric proteins were stable, non-toxic, and non-antigenic. AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 retained its natural structure and based on ligand-receptor docking and molecular dynamic analysis, the binding ability of AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 to IL13Rα2 was sufficiently strong.

Conclusion and implications: Based on the bioinformatics result AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 was a stable fusion protein with two separate domains and high affinity with the IL13Rα2 receptor. Therefore, AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 fusion protein could be a new potent candidate for target cancer therapy.

Keywords: Arazyme; Cancer; In silico; Interleukin 13; Peptide ligand; Targeting therapy.

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Conflict of interest statement

The authors declared no conflict of interest in this study.

Figures

Fig. 1
Fig. 1
Schematic model of designed chimeric constructs. (A) AraA + linker + native IL13; (B) AraA + linker + IL13.E13; (C) AraA + linker + Pep1; (D) AraA + linker + A2b11.
Fig. 2
Fig. 2
Model. 1 of 3D designed structures was predicted by I-TASSER.
Fig. 3
Fig. 3
3D structures of the selected fusion protein were predicted by (A) Robetta and (B) RaptorX; (C) Robetta model aligned with I-TASSER model; (D) model RaptorX aligned with I-TASSER model; and (E) the results obtained from TM-align server for aligned AraA-IL13 fusion protein with protein data bank files of AraA and IL 13 using Chimera software 1.10.2. AraA, Arazyme; IL, interleukin.
Fig. 4
Fig. 4
(A) Interaction between IL13 and IL13Rα2; (B) interaction between chimeric protein and IL13Rα2 receptor; (C) the 2D view of the docked complexes were analyzed using LigPlot+ software. The hydrogen bond (green dashed lines) and hydrophobic interactions (spoked arc) between the IL13-IL13Rα2; (D) chimeric IL13-IL13Rα2. IL, Interleukin.
Fig. 5
Fig. 5
(A) The Cα-RMSD graph for the IL13Rα2 and chimeric-IL13Rα2 in the complex state; (B) the Cα-RMSD graph for the IL13 and chimeric-IL13 at the complex state; (C) the Cα-RMSF graph for the IL13 in the complexes state. IL13Rα2, Interleukin 13 receptor alpha 2.
Fig. 6
Fig. 6
The 3D view of the final protein-protein complexes. (A) IL13Rα2-IL13; (B) IL13Rα2-IL13 in chimeric state. Residues referring to protein-protein interface sites (cyan and magenta sticks) are labeled. The IL13Rα2 is represented as the red cartoon and IL13 as the yellow cartoon. IL13Rα2, Interleukin 13 receptor alpha 2.
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