Development and Biological Evaluation of FL18-Based Novel Antimicrobial Peptide Constructs Against Bacterial Pathogens

Abstract

Antimicrobial resistance has become a serious global health challenge, increasing the need for new therapeutic strategies beyond conventional antibiotics. In this study, FL18 and its chimeric derivatives, FL18–TAT8 and FL18–Ahx–TAT8, were designed to combine antimicrobial activity with the cell-associated advantages of a cell-penetrating peptide motif and were synthesized by Fmoc-based solid-phase peptide synthesis. Their molecular masses were confirmed by MALDI-MS analysis. The antibacterial activities of the peptides were evaluated against representative Gram-negative and Gram-positive bacterial strains, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecium. The tested peptides exhibited minimum inhibitory concentration values in the range of 4–64 μM depending on the peptide sequence and bacterial strain, while TAT8 alone showed no significant antibacterial activity under the tested conditions. Among the constructs, FL18–Ahx–TAT8 displayed the most favorable overall antibacterial profile, particularly against P. aeruginosa and E. faecium. Biocompatibility studies further showed that FL18 and its chimeric derivatives maintained approximately 75–90% HaCaT cell viability across the tested concentration range and exhibited generally low hemolytic activity. In addition, confocal microscopy and flow cytometry revealed peptide-associated fluorescence in HaCaT cells, with the TAT8-containing chimeras showing a broader and more pronounced fluorescence distribution than FL18 alone. Overall, these findings demonstrate that FL18-based chimerization provides a modular and effective strategy for tuning antibacterial activity, biocompatibility, and peptide–cell interaction behavior, underscoring their promise as building blocks for the design of next-generation antimicrobial peptide platforms.