Background/Objectives: Multidrug-resistant Gram-negative ESKAPE pathogens, including
E. coli,
K. pneumoniae,
P. aeruginosa, and
A. baumannii, pose a significant global health threat. Gramicidin S, a potent cyclic antimicrobial peptide, is largely ineffective against these bacteria, and its high haemolytic toxicity
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Background/Objectives: Multidrug-resistant Gram-negative ESKAPE pathogens, including
E. coli,
K. pneumoniae,
P. aeruginosa, and
A. baumannii, pose a significant global health threat. Gramicidin S, a potent cyclic antimicrobial peptide, is largely ineffective against these bacteria, and its high haemolytic toxicity limits its clinical usage. This study reports on several novel gramicidin S analogues with improved efficacy and safety profiles against multidrug-resistant Gram-negative bacteria. Methods: A total of 19 gramicidin S derivatives were synthesised using Fmoc-based solid-phase peptide synthesis with targeted substitutions to enhance cationicity and modulate hydrophobicity. Minimum inhibitory concentrations (MICs) were determined against standard Gram-negative and Gram-positive strains. Haemolytic toxicity and in vitro nephrotoxicity were evaluated using human red blood cells and HEK-293 cells, respectively. All peptides were characterised by RP-HPLC and HRMS. Results: The selective incorporation of
DArg and Trp significantly enhanced activity against Gram-negative bacteria while reducing cytotoxicity. Peptide
8 improved the therapeutic index (TI) against
E. coli by 10-fold (MIC: 8 µg/mL; TI: 4.10) compared to gramicidin S (MIC: 32 µg/mL; TI: 0.38). Peptide
9 exhibited an 8-fold potency increase against
K. pneumoniae and a 25-fold TI improvement. Peptide
19 enhanced activity against
P. aeruginosa 8-fold over gramicidin S, while peptide
7 showed a 27-fold TI enhancement. All active peptides retained broad-spectrum activity against
S. aureus, including MRSA. Conclusions: The findings highlight the critical role of balancing hydrophobicity and cationicity to overcome species-specific resistance mechanisms. Our gramicidin S analogues demonstrate potent broad-spectrum activity with significantly reduced toxicity compared to the parent peptide, providing a robust platform for the development of new antibiotics against ESKAPE bacterial pathogens.
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