Computational design of newly engineered DARPins as HER2 receptor inhibitors for breast cancer treatment

Abstract

Background and purpose: Human epidermal growth factor receptor 2 (HER2) is overexpressed in approximately 25% of breast cancer patients; therefore, its inhibition is a therapeutic target in cancer treatment.

Experimental approach: In this study, two new variants of designed ankyrin repeat proteins (DARPins), designated EG3-1 and EG3-2, were designed to increase their affinity for HER2 receptors. To this end, DARPin G3 was selected as a template, and six-point mutations comprising Q26E, I32V, T49A, L53H, K101R, and G124V were created on its structure. Furthermore, the 3D structures were formed through homology modeling and evaluated using molecular dynamic simulation. Then, both structures were docked to the HER2 receptor using the HADDOCK web tool, followed by 100 ns of molecular dynamics simulation for both DARPins / HER2 complexes.

Findings/results: The theoretical result confirmed both structures' stability. Molecular dynamics simulations reveal that the applied mutations on DARPin EG3-2 significantly improve the receptor binding affinity of DARPin.

Conclusion and implications: The computationally engineered DARPin EG3-2 in this study could provide a hit compound for the design of promising anticancer agents targeting HER2 receptors.

Keywords: Breast cancer; Designed ankyrin repeat proteins; Docking; Molecular dynamic simulation.

Fig. 1

The Ramachandran plot of the designed (A) DARPin EG3-1 and (B) DARPin EG3-2. DARPin EG, Designed ankyrin repeat proteins engineered G.

Fig. 2

Molecular dynamic simulation results of the DARPin EG3-1 variant compared with the DARPin EG3-2 during 100 ns. Plots illustrate (A) RMSD, (B) RMSF, (C) Rg, and (D) solvent-accessible surface area trends for the DARPin EG3-1 and DARPin EG3-2. DARPin EG, Designed ankyrin repeat proteins engineered G; RMSD, root mean square deviation; RMSF, root mean square fluctuation; Rg, radius of gyration.

Fig. 3

Molecular dynamic simulation results of the DARPin EG3-1 variant compared with the DARPin EG3-2 during 100 ns. Plots illustrate (A) intramolecular (protein-protein) hydrogen bonds of DARPin EG3-1 and DARPin EG3-2 and (B) intermolecular (protein-solvent) hydrogen bonds of DARPin EG3-1 and DARPin EG3-2. DARPin EG, Designed ankyrin repeat proteins engineered G.

Fig. 4

Two-dimensional representation of protein-protein interactions for the complex (A) DARPin EG3-1 and (B) DARPin EG3-2 binding mode and the position of the hydrogen bonds of the extracellular domain of human epidermal growth factor receptor 2. Dashed green lines indicate hydrogen bonds, and the half-moon indicates hydrophobic interactions. DARPin EG, Designed ankyrin repeat proteins engineered G.

Fig. 5

Two-dimensional representation of protein-protein interactions for the complex (A) DARPin EG3-1 and (B) DARPin EG3-2 binding mode and the position of the hydrophobic bonds of the extracellular domain of human epidermal growth factor receptor 2. DARPin EG, Designed ankyrin repeat proteins engineered G.

Fig. 6

Structure of the designed DARPin EG3-2 / human epidermal growth factor receptor 2 complex and its mutation site (magnified). Mutated residues in the DARPin EG3-2 variant are represented by gold spheres mode. The DARPin, linker, BAX-Smac, and receptor domains are depicted in purple, gold, magenta, and light sea green, respectively. Image created using Chimera 1.12. DARPin EG, Designed ankyrin repeat proteins engineered G; BAX, Bcl-2-associated X protein; Smac, second mitochondria-derived activator of caspase.