靶向VEGFA结合蛋白的设计与功能表征OA

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.