Toggling Preassembly with Single-Site Mutation Switches the Cytotoxic Mechanism of Cationic Amphipathic Peptides

Precise regulation of membrane-active peptide activity is a frontier of research to facilitate its applicational translation. A clear understanding of how a peptide's physicochemical properties determine its mode of action (MOA) will aid the process. Herein, anionic glutamate residue-based scanning was applied to the hydrophobic surface of a self-assembling lysine-rich cationic amphipathic peptide (CAP) KL1. Single-site mutations from leucine to glutamate dramatically changed the MOA of all mutants from membranolytic to nonlytic. An apoptosis-inducing mutant L2E unable to self-assemble under extracellular anions exhibited a different conformational transformation process in the amphiphilic environment than KL1. Further adjustment of the overall positive charge allowed regulation of cytotoxic potency without affecting the MOA determined by the lack of preassembly formation. Compared with KL1, hemolytic toxicities of nonmembranolytic peptides were greatly reduced, with safety indices increased. This work thus provided novel insights into and integrated rationales on the improvement of CAPs for both anticancer activity and safety profile.