Design and functional characterization of VEGFA-targeted binding proteins

doi:10.3969/j.issn.1674-8115.2026.09.006

Abstract

Abstract:

Objective ·To construct vascular endothelial growth factor A (VEGFA)-targeted binding proteins by using a de novo protein design strategy, and verify the effects of these proteins on VEGFA-induced biological activities in human umbilical vein endothelial cells (HUVECs). Methods ·A multi-algorithm workflow integrating Rosetta RifDock, FastDesign, and ProteinMPNN was used to design VEGFA-targeted binding proteins. The structural plausibility of the binding proteins and their interactions with VEGFA were assessed using the AlphaFold prediction model. Yeast surface display combined with dual-fluorescence-activated cell sorting was then used to screen specific VEGFA-targeted binding proteins, which were subsequently expressed and purified using the E. coli expression system. The binding affinities between the binding proteins and VEGFA were measured using bio-layer interferometry (BLI). Finally, cell counting kit-8 (CCK-8) assays, Annexin Ⅴ/PI double-staining, Western blotting, scratch wound healing assays, and in vitro tube formation assays were performed to evaluate the effects of the binding protein on VEGFA-induced biological activities in HUVECs. Results ·VEGFA-targeted binding proteins were successfully designed and a candidate binding protein library was constructed. One protein, minibinder-3 (MB3), with moderate affinity for VEGFA was obtained after yeast surface display screening and BLI measurement. Subsequent in vitro experiments demonstrated that MB3 inhibited VEGFA-induced survival, resistance to apoptosis, migration, and angiogenesis, and also reduced the VEGFA-induced phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK), which are downstream signaling molecules of vascular endothelial growth factor receptor (VEGFR) (all P<0.05). Conclusion ·Based on the de novo protein design workflow, MB3, a VEGFA-binding protein with moderate affinity, was obtained. In vitro experiments confirmed that MB3 inhibited VEGFA-mediated HUVEC functions and related signaling pathways, thereby demonstrating the feasibility of the design workflow for developing anti-VEGFA artificial proteins and providing new insights into tumor vascular-targeted therapy.

Key words: de novo protein design, vascular endothelial growth factor A (VEGFA), binding protein, anti-angiogenic therapy

CLC Number:

Lu Yue, Yan Weikang, Liu Yingbin, He Yongning. Design and functional characterization of VEGFA-targeted binding proteins[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026,(9): 1212-1221.

Figures/Tables 5

Fig 1 Ribbon diagram of the VEGFA/VEGFR2 complex structure (A) and the design sites (B) of VEGFA-targeted binding proteins

Fig 2 Experimental screening of VEGFA-binding proteins and schematic diagram of MB3/VEGFA complex structure prediction

Fig 3 Effects of MB3 on VEGFA-induced survival in HUVECs

Fig 4 Effects of MB3 on VEGFA-induced anti-apoptotic effects in HUVECs

Fig 5 Effects of MB3 on VEGFA-induced migration, angiogenesis, and downstream phosphorylation signaling pathways in HUVECs

TrendMD

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