TOAC-LABELED CTX(ILE21)-HA PEPTIDE ANALOGUES INTERACT DIFFERENTLY IN POPC LIPOSSOMES AND MONOLAYERS

Antimicrobial peptides (AMP) have demonstrated remarkable properties in permeabilizing bacterial membranes, leading to cell death, making them a key focal point in the pursuit of new antibiotics. Several AMPs have been discovered across various vertebrate and invertebrate species, including magainin, ceratotoxin, and others. Among these promising candidates, the antimicrobial peptide Ctx(Ile21)-Ha, isolated from the Brazilian frog Boana albopunctatus, has garnered particular interest due to its potent biological activities against fungi and bacteria, along with its moderate toxicity towards human cells. This study aims to understand the interaction between Ctx(Ile21)-Ha peptide and its TOAC-labeled analogues in phospholipid liposomes and monolayers, mimicking cell membranes but remains poorly understood at the molecular level. To achieve this, kinetic adsorption and Langmuir monolayer techniques were employed to investigate the peptide surface activity and its affinity to POPC monolayers at physiological pH. In addition, circular dichroism (CD) and electron spin resonance (ESR) assays were conducted using several membrane mimetics, including POPC, POPC/CHOL, POPC/ERG, POPC/POPG, and POPE/POPG, to mimic human, bacterial, and fungal membranes. The results suggest that kinetic adsorption of the peptide is faster in phosphate buffer than in the water subphase. Furthermore, maintaining the peptide concentration the same as the peptide: lipid ratio influences the surface activity, with higher concentrations of Ctx(Ile21)-Ha resulting in greater surface activity. The surface isotherms suggest that the peptide is inserted into the phospholipid monolayer, and the interaction between Ctx(Ile21)-Ha and POPC monolayers does not compromise the peptide surface activity. Lastly, CD and ESR studies showed that TOAC-labeled peptides exhibit higher affinities for the anionic liposomes, with POPE/POPG vesicles showing the highest affinity. Interestingly, cholesterol significantly decreases the peptide affinity to the membrane, providing a protective effect that is important for mammalian cells. Notably, the peptides acquire an α-helical structure within the membranes, yet they significantly aggregate in POPG-containing vesicles, especially in POPE-POPG environments. ESR assays also showed that peptide dynamics are more restricted in the anionic membranes.
This work was supported by São Paulo Research Foundation - FAPESP (process numbers 2021/06706-9 and 2022/09379-1).