De novo Design Protein Binders Targeting the Hydrophobic Surface of DENV4 and ZIKV NS1 Dimer

Abstract

The hydrophobic surface of the flavivirus NS1 dimer—comprised primarily of the β-roll domain and the greasy finger loop—is essential for cell surface association and NS1 oligomerization. However, no antibodies targeting this epitope region have been reported to date. In this study, we employed a pipeline including RFdiffusion, ProteinMPNN, and AlphaFold to design protein binders that target the hydrophobic surface of dengue virus serotype 4 (DENV4) and Zika virus (ZIKV) NS1 dimers. Experimental validation identified five binders for each virus that could be expressed and exhibited specific binding, with equilibrium dissociation constants (K_D) ranging from 45.2 nM to 2.2 μM. Cryo-EM structures of DENV4 NS1 in complex with D4NB4 and D4NB11 and of ZIKV NS1 in complex with ZNB6 confirmed the predicted binding modes. Functional assays demonstrated that these binders inhibit NS1 binding to cell surfaces, highlighting the therapeutic potential of these computationally designed protein binders.