Synthesis, Characterization, and Antimicrobial Activity of Urea-Containing α/β Hybrid Peptides against Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus.

The insertion of β-amino acids and replacement of the amide bond with a urea bond in antimicrobial peptide sequences are promising approaches to enhance the antibacterial activity and improve proteolytic stability. Herein, we describe the synthesis, characterization, and antibacterial activity of short αβ cationic hybrid peptides LA^U-Orn-β^3,3Ac₆c-PEA, DY-01; LA^U-Lys-β^3,3Ac₆c-PEA, DY-02; and LA^U-Arg-β^3,3Ac₆c-PEA, DY-03 in which a C12 lipid chain is conjugated at the N terminus of peptide through urea bonds. Further, we evaluated all the peptides against both Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) and their multidrug resistant (MDR) clinical isolates. All of the peptides exhibited significant bactericidal efficacy with minimal inhibitory concentration (MIC) values ranging from 2.5 to 6.25 μM (1.4 to 3.9 μg/mL) against P. aeruginosa and its MDR clinical isolates, whereas the MIC values ranging from 0.78 to 6.25 μM (0.45 to 3.9 μg/mL) against MRSA and MDR clinical isolates of S. aureus. To understand the potency and mechanism of action of DY-01 to DY-03, time-kill kinetics, biofilm inhibition and disruption, synergistic interactions with standard antibiotics, swarming motility, scanning electron microscopy (SEM) analyses, and ex vivo infection assay were performed. The SEM images revealed that all of the peptides exert antibacterial activity through a membrane disruption mechanism. Additionally, negligible cytotoxicity was observed against mammalian cell lines RAW 264.7 and J774A.1, with mild hemolysis at higher concentrations. The comprehensive antimicrobial assessments of DY-01 to DY-03 against P. aeruginosa and MRSA highlight their potential for clinical applications in combating resistant microbial infections.