Modulation of Membrane-Disruptive Activity of Melittin via N- and C-Terminal PEGylation Strategies

Melittin has emerged as a promising therapeutic agent due to its potent antitumor and antimicrobial activities. However, the clinical translation of native Melittin is hindered by substantial challenges, including systemic toxicity and rapid proteolytic degradation, leading to suboptimal pharmacokinetic profiles. Therefore, structure-activity relationship-guided rational design strategies focusing on the molecular determinants of membrane penetration mechanisms are essential for optimizing Melittin's therapeutic index. Herein, we synthesized a series of Melittin derivatives with varying PEG modification lengths and N- or C-terminus. Our evaluation revealed that N-terminal PEGylation substantially mitigated the cytotoxicity and hemolytic activity of Melittin while enhancing its proteolytic stability, where these beneficial properties exhibited progressive enhancement correlating with increasing PEG chain length. Conversely, C-terminal PEGylation demonstrated limited efficacy in modulating Melittin's toxicity profile. Our findings elucidated that the membrane interaction mechanism of Melittin was predominantly mediated by its N-terminal helical domain, rather than the C-terminus, which initiated the cell membrane binding and subsequent pore formation, ultimately culminating in cell demise. This finding underscored the critical role of the N-terminus in the biological activity of Melittin. This study provided insight into the structure-activity relationship of PEGylated Melittin and established guidance for creating the next generation of peptide therapies.