Structure-Guided Design of Proteomimetics Targeting the SARS-CoV-2 S-RBD/hACE2 Interface

The SARS-CoV-2 Spike receptor-binding domain (S-RBD)/hACE2 interaction represents a challenging protein-protein interaction (PPI) target due to its large, shallow binding interface. Here, in silico alanine mutagenesis guided the structure-based design of constrained peptidomimetics that reproduce key hACE2 recognition elements. α1-helix-derived mimetics (Glu23-Ser44) were stabilized using peptide stapling strategies, while antiparallel β-sheet mimetics (Thr347-Leu359) were generated through head-to-tail macrocyclization incorporating a d-Pro/l-Pro motif. Covalent linkage of these two secondary structure mimetics yielded proteomimetic 28, designed to preserve the spatial organization of the hACE2 binding interface. Compound 28 selectively binds SARS-CoV-2 S-RBD and disrupts the S-RBD/hACE2 interaction in biophysical and cellular assays, inhibiting pseudovirus entry (IC₅₀ of 6.6 μM). Importantly, 28 displays high stability in lung epithelial models (t_1/2 > 24 h) and low epithelial permeability (P_app = 2.03 × 10^-8 cm·s^-1), supporting its potential for intranasal antiviral delivery. These findings establish a proof-of-concept for proteomimetics as promising inhibitors of challenging PPIs.