MeLysine-Stapling Combined with N-Terminal Acetylation Yields Stable Oncolytic Peptides with Enhanced Antitumor Efficacy

To overcome the proteolytic instability of oncolytic peptides, we developed a novel “MeLysine-stapling” strategy through intramolecular cross-linking of two α-methyl-substituted lysine residues. Applying this strategy to the cobra-derived peptide OH-CM6 yielded a series of stabilized analogues. The dual-modified lead candidate MeLS-5 (MeLysine-stapling and N-terminal acetylation), exhibited an exceptional serum half-life exceeding 48 h─a 32-fold increase over the parent peptide. MeLS-5 demonstrated potent broad-spectrum cytotoxicity (geometric mean IC50 = 3.9 μM across nine cancer cell lines) and low toxicity to human erythrocytes and normal mammalian cells. Mechanistic investigations confirmed that MeLS-5 acts via rapid, irreversible physical disruption of cancer cell membranes. In a syngeneic 4T1 xenograft model, intravenous administration of MeLS-5 (5 mg/kg, every other day) inhibited tumor growth by 63% without systemic toxicity or exacerbated pathologies. Collectively, this study validates MeLysine-stapling as a robust strategy for engineering stable peptide therapeutics and positions MeLS-5 as a promising preclinical candidate.