Targeted Therapy with a Novel Superantigen-based Fusion Protein Against Interleukin-13 Receptor α2-overexpressing Tumor Cells: An In-silico Study

Abstract

Background & objective: Superantigens are bacterial toxins that induce a massive immune response in the host. Superantigen staphylococcal enterotoxin B (SEB) can form a ternary complex with its receptors, MHC class II (MHCII) and TCR, and can be used in tumor-targeting therapy, particularly when cooperating with a specific vector. In this study, SEB was fused to interleukin-13 (IL13), which forms a complex with IL13 receptor α2 (IL13Rα2) overexpressed in glioblastoma multiforme (GBM) cells for therapeutic goals.

Methods: We designed four fusion proteins based on the arrangement of SEB (N- or C-terminal domain) and provided a flexible inter-domain linker (no or yes), resulting in the formation of SEB-IL13, SEB-L-IL13, IL13-SEB, and IL13-L-SEB, respectively. These fusion proteins were then evaluated for their various physicochemical properties and structural characteristics. Bioinformatics tools were employed to predict, refine, and validate the three-dimensional structure of the fusion proteins. In addition, the fusion proteins were docked with IL13Rα2, MHCII, and TCR receptors through the HADDOCK 2.4 server. The candidate fusion protein was subjected to molecular dynamics simulation.

Results: There were differences among the designed fusion proteins. The model with the N-terminal domain of IL13 and containing an inter-domain linker (IL13-L-SEB) was stable and had a long half-life. The docking analysis revealed that the IL13-L-SEB fusion protein had a higher binding affinity to the IL13Rα2, MHCII, and TCR receptors. Finally, using molecular dynamics simulation through iMODS, acceptable results were obtained for the IL13-L-SEB docked complexes.

Conclusion: The results suggest IL13-L-SEB is a promising novel fusion protein for cancer therapeutic application.

Keywords: Fusion protein; Glioblastoma multiforme; Interleukin-13; Molecular docking; Staphylococcal enterotoxin B..

Fig. 1

Schematic illustration of the designed SEB-IL13, SEB-L-IL13, IL13-SEB, and IL13-L-SEB fusion proteins, L (linker peptide); GGGGSGGGGSGGGGS.

Fig. 2

The 3D structure of the final (after energy minimization) (A) SEB-IL13, (B) SEB-L-IL13, (C) IL13-SEB, and (D) IL13-L-SEB fusion proteins predicted by Phyre2. The red color depicts the α-helix, the blue color depicts the ß-strand, and the gray color depicts a random coil. The conformation of the linker is shown in pink. Side chains (as sticks) are displayed for the last residue of the N-terminal domain and the first residue of the C-terminal domain in the models containing the inter-domain linker.

Fig. 3

The 3D structure of IL13-L-SEB (IL13, linker, and SEB are depicted in purple, red, and dark gray, respectively) in complex with IL13Rα2 (white). 2D interactions of IL13-L-SEB with IL13Rα2; hydrogen bonds and salt bridge bonds are represented by green and red dash lines, respectively, whereas hydrophobic interactions are indicated by spoked arcs.

Fig. 4

The 3D structure of IL13-L-SEB (IL13, linker, and SEB are depicted in purple, red, and dark gray, respectively) in complex with MHC class II α-chain (yellow) and TCR β-chain variable (green). 2D interactions of IL13-L-SEB with MHCII and TCR; hydrogen bonds and salt bridge bonds are represented by green and red dash lines, respectively, whereas hydrophobic interactions are indicated by spoked arcs.

Fig. 5

Molecular dynamics simulation of the IL13-L-SEB/IL13Rα2 docked complex. (A) Main chain deformability graph, (B) B-factor analysis, (C) Eigenvalue plot, (D) Variance plot, (E) Covariance matrix, (F) Elastic network model.

Fig. 6

Molecular dynamics simulation of the MHCII/IL13-L-SEB/TCR docked complex. (A) Main chain deformability graph, (B) B-factor analysis, (C) Eigenvalue plot, (D) Variance plot, (E) Covariance matrix, (F) Elastic network model.