Host defense peptides (HDP) have emerged as versatile therapeutic candidates showing promise not only against pathogens but also against cancer cells. Tilapia piscidin 4 (TP4), a fish-derived HDP, demonstrates potent activity against non-small cell lung cancer, synovial sarcoma, and triple-negative breast cancer, and with notable selectivity against malignant cell over healthy cells. While recent work has explored the metabolic impacts of TP4, detailed biophysical insights into its interaction with cancer cell membrane mimics remain limited. To uncover the molecular basis of TP4’s activity and selectivity, we utilize a suite of biophysical techniques, featuring neutron reflectometry and diffraction in combination with cancer cell assays. Circular dichroism reveals that the TP4 binds strongly to membranes enriched in negatively charged lipids (POPS), while cholesterol impairs binding. X-ray diffraction shows that the presence of negatively charged lipids promotes phase separation and enhances membrane disruption by TP4. Neutron diffraction and reflectometry further demonstrate that TP4 inserts into the acyl chain region of the biolayer and interacts with ∼114 water molecules, highlighting its deep integration within the membrane. Together, these results define the molecular determinants of TP4 selectivity and activity against cancer cells, providing insights to guide the development of future peptide-based cancer therapeutics.
Makambi et al. (Sun,) studied this question.