Antibody Hotspot-Centric de Novo Design of Peptide Inhibitors of Influenza A Virus Membrane Fusion

Abstract Here we report the applicability of a hotspot-centric approach for de novo computational design of l-peptides based on crystal structures of antigen–antibody complexes. Broadly neutralizing antibodies (bnAbs) FI6v3 and CR9114, which target influenza A virus hemagglutinin (HA), guided the peptide design. Disembodied hotspot residues from these bnAbs were selected and anchored onto known peptide scaffolds (≤35 amino acids) from the Protein Data Bank (PDB). The top-scoring designs were subsequently synthesized, folded, tested in vitro, and structurally characterized in complex with HA. The designed peptides demonstrate structural and functional mimicry by recapitulating the binding mode of the bnAbs on HA and act as inhibitors of low-pH-dependent conformational transitions in HA that facilitate membrane fusion. These findings demonstrate that hotspot-guided design strategy, previously applied to proteins and d-peptides, can be effectively extended to l-peptide scaffolds, enabling the development of functional HA inhibitors informed by key residues at antigen–antibody interfaces.