The α-helix-facilitating amino acid sequence EAAAK dramatically increases the antiviral activity of SARS-CoV-2 fusion-inhibitory peptides via the endosome fusion pathway.

UNLABELLED: The SARS-CoV-2 fusion-inhibitory lipopeptide IPB29 features a unique design incorporating the α-helix-promoting EAAAK sequence as a rigid linker between the peptide backbone and the conjugated lipid, exhibiting greatly improved antiviral activity compared to lipopeptides with conventional flexible linkers. Here, we characterized its mechanism of action and uncovered several critical insights. First, IPB29 remained the most potent inhibitor of emerging Omicron subvariants compared to IPB24 and IPB28, which share the same peptide sequence but employ flexible polyethylene glycol or glycine-serine repeat linker. Notably, this enhanced potency was abolished in Vero E6 cells stably expressing TMPRSS2, where viral entry occurs via the plasma membrane fusion pathway. Visualization using FITC-labeled lipopeptides demonstrated that IPB29 binds to and enters target cells more efficiently. Subsequently, we generated two unconjugated template peptides, P43 (containing EAAAK) and P37 (lacking EAAAK). P43 exhibited superior inhibitory activity compared to P37 in Huh-7 and 293T/ACE2 cells, but not in Vero E6-TMPRSS2 cells. Both peptides showed comparable efficacy in blocking S protein-driven cell-cell fusion. Although P43 displayed enhanced α-helicity relative to P37, both peptides exhibited similar target-binding affinities. Instead, the cellular uptake of P43 was markedly more efficient, further supporting that the improved antiviral effect is attributable to the inhibition of the viral endosomal fusion pathway. Collectively, our studies provide molecular insights into the antiviral mechanism of IPB29 and propose the short EAAAK sequence as a generalizable strategy for enhancing the efficacy of peptide-based therapeutics. IMPORTANCE: The development of effective therapeutics against SARS-CoV-2 remains an urgent priority. IPB29, a potent broad-spectrum lipopeptide fusion inhibitor currently in clinical trials, requires mechanistic elucidation. Here, we demonstrate that the C-terminal EAAAK motif in IPB29-originally designed as a rigid linker connecting the peptide backbone to the lipid moiety-is critical for its antiviral activity and functions via the endosomal fusion pathway. These findings reveal a generalizable strategy for optimizing antiviral drug design.