Proline-Modified (RW)<i>n</i> Peptides: Enhancing the Antimicrobial Efficacy and Selectivity against Multidrug-Resistant Pathogens

The growing threat of multidrug-resistant (MDR) bacteria necessitates the development of novel antimicrobial agents. This study investigates the potential of proline-modified (RW)<i>n</i> peptides as a platform for combating MDR pathogens with minimal toxicity. We synthesized and evaluated (RW)<i>n</i> peptides (<i>n</i> = 4, 6, and 8) with and without central proline residues against five clinically relevant bacterial strains, including ESKAPE pathogens. Antimicrobial activity, cytotoxicity, and synergistic effects with conventional antibiotics were assessed. Proline incorporation significantly enhanced the peptide selectivity and broadened the spectrum of activity, particularly against Gram-negative bacteria. RW6P and RW8P demonstrated exceptional efficacy (MICs ≤ 0.25 μg/mL) against methicillin-resistant <i>Staphylococcus aureus</i> and <i>Escherichia coli</i> with minimal toxicity to human cells. Notably, RW8P restored ampicillin susceptibility in <i>Pseudomonas aeruginosa</i> (MIC < 0.25 μg/mL) without dose-dependent toxicity. Exceptionally, RW6-2P demonstrated efficacy against all Gram-positive and Gram-negative bacteria, except <i>Klebsiella pneumoniae</i> (Kp), without toxicity. Furthermore, several Gram-negative isolates were rendered susceptible to vancomycin when combined with these peptides, addressing a key limitation of glycopeptide antibiotics. Gram-negative <i>Klebsiella pneumoniae</i> (Kp) was rendered susceptible to vancomycin when combined with RW4P and RW6-2P. RW4P and RW6-2P, with and without antibiotics, have shown selectivity. This study presents proline-modified (RW)<i>n</i> peptides as promising candidates for developing broad-spectrum antimicrobials with enhanced selectivity and the potential to revitalize existing antibiotics against MDR pathogens.