Design and Evaluation of Tyrosine‐Rich Antimicrobial Peptides With Quadruple Repeating Units

Multidrug‐resistant (MDR) bacteria pose a significant threat to global public health by undermining conventional antibiotics. Antimicrobial peptides (AMPs), with their membrane‐targeting mechanisms, offer a promising alternative; however, the clinical translation of natural AMPs is hampered by systemic toxicity and high synthesis costs. This study introduces a novel strategy based on a quadruple repeating unit (A 1 A 2 B 1 B 2 ) n , employing tyrosine (Y) as a key hydrophobic residue to reduce overall hydrophobicity and hemolytic activity while maintaining antimicrobial efficacy. The candidate peptide (RRLY) 4 exhibited broad‐spectrum antimicrobial activity against reference strains (minimum inhibitory concentrations (MICs): 15.63–125 μg/mL), combined with low hemolytic activity (HC 50 > 1500 μg/mL) and a therapeutic index exceeding 31.66. Furthermore, (RRLY) 4 possessed potent antimicrobial effects against five clinically isolated MDR bacteria, with MICs ranging from 15.63 to 31.25 μg/mL, and also showed low salt sensitivity. Additionally, mechanism of action studies revealed that (RRLY) 4 exerted rapid bactericidal activity by disrupting the membrane integrity of Staphylococcus aureus ATCC 25 923 and Escherichia coli ATCC 25 922. This study demonstrates that tyrosine can serve as a hydrophobic residue in the rational design of AMPs, offers a strategy for developing amphipathic AMPs with potent antimicrobial activity and low hemolytic activity.