Abstract
Membrane-active peptides (MAPs) have garnered significant attention as potential alternatives to conventional cancer therapies, which are frequently limited by severe side effects. Among them, antimicrobial peptides (AMPs) that leverage differences between the plasma membranes of cancer cells and healthy cells are particularly attractive. While several AMPs have demonstrated anticancer potency, structure-function relationship studies are lacking to explain the molecular basis of their selectivity and to help design improved analogs. Here, we contribute to filling this gap by investigating Nile tilapia piscidin 4 (TP4), an AMP with demonstrated activity against several solid organ cancers. First, we discover through biological assays that the anticancer activity of the peptide, which underscores a promising therapeutic window, is associated with increased plasma membrane permeability in cancer cells compared to normal cells and positively (negatively) correlated with enzymes that enrich (deplete) anionic PS in the outer leaflet. Next, we utilize a suite of complementary techniques on model membranes to investigate the interactions of TP4 with membranes, uncovering behaviors not previously observed in related AMPs. Circular dichroism experiments reveal that TP4 preferentially binds to zwitterionic phosphatidylcholine (PC) membranes enriched in anionic PS, while cholesterol markedly impairs binding. X-ray diffraction demonstrates that TP4 disrupts PC-PS membranes by inducing lipid segregation. Covering a range of biologically relevant peptide concentrations with neutron diffraction and reflectometry measurements in fluid bilayers and MD simulations, we unveil how TP4 and associated water gradually insert into the hydrocarbon region and cause convoluted membrane deformations to breach the membrane barriers. These studies highlight the pivotal role of the TP4 polyarginine tail in driving selective membrane binding and disruption on membranes enriched with the anionic lipid PS. Together, our results elucidate the molecular determinants underpinning the selective anticancer effects of TP4, providing a strategic framework for the rational design of advanced membrane-active therapeutics.