Membrane-Anchored and Sequence-Oriented Antiviral Activity of Fusion-Inhibitory Lipopeptides Derived from the SARS-CoV-2 Spike Glycoprotein S2 Subunit

Background: SARS-CoV-2 fusion inhibitory peptides represent promising antiviral candidates. Recently, a 19-mer peptide (PN19)—designed in our laboratory to mimic the internal fusion peptide of the SARS-CoV-2 spike S2 subunit—demonstrated potent antiviral activity and stable conformational features. Objectives: To investigate how this antiviral activity depends on membrane interactions, we designed synthetic PN19 lipopeptide derivatives and evaluated their efficacy against SARS-CoV-2 replication. Methods: Lipopeptides were synthesized by conjugating cholesterol to either the N- or C-terminus of the PN19 peptide, utilizing a Gly/Ser pentapeptide (GSGSG) and/or various polyethylene glycol (PEG) spacers. Antiviral activity against SARS-CoV-2 variants was evaluated by plaque reduction assays, and cytotoxicity was assessed in Vero E6 cells. Results: The lipopeptides exhibited potent inhibitory activity at sub-micromolar concentrations. Compared to the unmodified PN19 peptide, antiviral efficacy was significantly enhanced by cholesterol conjugation at either terminus. Evaluation of six PN19 lipopeptides bearing the GSGSG sequence and different PEG spacers revealed that C-terminal cholesterol conjugation yielded higher antiviral activity than N-terminal derivatives. Furthermore, thirteen shorter PN19 lipopeptide derivatives (8–13-mers) confirmed this robust efficacy, which was most pronounced with C-terminal cholesterol conjugation and further enhanced by the spacers. Noteworthy, all tested PN19 lipopeptides displayed broad activity against multiple SARS-CoV-2 variants in the absence of cytotoxicity. Conclusions: Collectively, peptides conjugated with cholesterol at the C-terminus emerged as highly potent inhibitors of SARS-CoV-2, likely driven by enhanced peptide–membrane interactions. These findings warrant further investigation to fully elucidate the role of lipidation in the inhibitory mechanism, supporting the development of novel antiviral lipopeptides for SARS-CoV-2 therapy.