Aromatic π-stacking stabilizes an α-helical SARS-CoV-2 MPER peptide that mimics the post-fusion spike and enables potent antiviral activity.

Membrane fusion between SARS-CoV-2 and host cells is mediated by the spike protein and involves the membrane-proximal external region (MPER), a tryptophan-rich sequence implicated in viral entry. Here, we investigated the feasibility of MPER-derived peptides as potential antiviral agents and examined the structural determinants underlying their activity. A series of truncated MPER peptides was synthesized and evaluated for antiviral activity against SARS-CoV-2 in cellular assays, revealing potent inhibitory potency against different variants, with nanomolar IC50 values associated with sequences rich in aromatic residues. Notably, this activity was time-dependent, decreasing in effectiveness when the peptide was added 1 h post-infection. Using NMR spectroscopy, we demonstrate that these peptides adopt a stable α-helical conformation in solution and membrane-mimetic environments that is stabilized by intramolecular aromatic π-π stacking interactions among tryptophan and tyrosine side chains. Structure-activity analysis indicates that this aromatic network promotes α-helix stabilization mimicking MPER structure of the spike post-fusion and correlates with enhanced antiviral potency. Our findings reveal a structural mechanism by which aromatic stacking stabilizes MPER helicity and drives antiviral activity, providing insights into peptide-based inhibition of viral membrane fusion and offering a framework for the rational design of SARS-CoV-2 entry inhibitors.