Design and functional validation of protein binders targeting ErbB2
10.3781/j.issn.1000-7431.2025.2503-0167
- VernacularTitle:靶向ErbB2的结合蛋白的设计与功能验证
- Author:
Fan XU
1
;
Yali LIU
;
Yongning HE
Author Information
1. 上海交通大学医学院附属仁济医院,上海市肿瘤研究所,肿瘤系统医学全国重点实验室,上海 200127;上海市肿瘤系统调控与转化重点实验室,上海 200127
- Publication Type:Journal Article
- Keywords:
De novo protein design;
Human epidermal growth factor receptor-2;
ErbB2;
Ovarian cancer;
Pancreatic cancer
- From:
Tumor
2025;45(2):101-110
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To design protein binders of human epidermal growth factor receptor-2(HER2,also known as ErbB2)using de novo protein design and to preliminarily evaluate their biological effects on tumor cells.Methods:Based on de novo protein design strategy,RosettaDesign and ProteinMPNN algorithms were used to design the binding proteins targeting ErbB2.Concurrently,AlphaFold was applied to assess structural accuracy of the protein binders and their interactions with ErbB2.Subsequently,yeast surface display technology combined with dual-fluorescence high-throughput flow cytometry were performed to screen for protein binders which were capable of specifically binding to ErbB2 specifically.Selected binding proteins were expressed and purified in Eschrichia coli system.Bio-layer interferometry(BLI)was used to validate the binding specificity of the purified protein binders to ErbB2.Finally,Western blotting and CCK-8 assay were used to evaluate the effects of the candidate binding protein on the downstream signaling of ErbB2 and cell proliferation capacity in ovarian cancer(SK-OV-3)and pancreatic cancer(BxPC-3)cell lines,respectively.Results:In this study,the protein binders targeting ErbB2 were successfully designed,a corresponding binding protein library was established,and a protein binder that specifically binds to ErbB2 was screened out from the library.After BLI verification,this protein binder could effectively bind to ErbB2.Further investigation revealed that in ErbB2-high-expressing SK-OV-3 and BxPC-3 cells,this protein binder could effectively inhibit cell proliferation and reduce the phosphorylation level of AKT,a signaling molecule downstream of ErbB2.Conclusion:Based on the strategy of de novo protein design,this study successfully constructed a protein binder that can effectively bind to ErbB2.The binder can effectively inhibit the activation of the downstream signaling pathway mediated by ErbB2 and the proliferation of tumor cells.This indicates the potential application value of de novo designed protein binders targeting ErbB2 in cancer therapy,providing a novel research approach for the field of targeted tumor therapy.
