Antimicrobial peptides RI8 and RW8 target bacterial membranes and genomic DNA to overcome drug resistance.

The global rise of multidrug-resistant (MDR) pathogens invalidates conventional antibiotics, while natural antimicrobial peptides (AMPs) lack proteolytic stability and precise targeting. This study reports a protease-resistant symmetric (RRYY)2P(YYRR)2 scaffold (Y = isoleucine/tryptophan) with alternating cationic-hydrophobic repeats, a central proline hinge, and palindromic topology for dual bacterial membrane-genomic DNA targeting. Two derived peptides, RI8 and RW8, were characterized: RI8 showed broad-spectrum activity against MDR pathogens (MIC: 2-16 μg/mL), high therapeutic index (39.4), negligible hemolysis (<1% at 128 μg/mL), and retained efficacy in physiological salts/serum. RW8 exhibited robust protease resistance (1-2× MIC vs trypsin/chymotrypsin, 0.5× MIC vs 8 mg/mL pepsin for 8 h) and Staphylococcus aureus biofilm inhibition (71.4% at 2× MIC) via downregulating icaD/icaB/sarA. Mechanistically, both peptides disrupted bacterial membranes and bound genomic DNA; molecular dynamics simulations confirmed membrane insertion via helix-to-coil transition. In murine models, RI8/RW8 accelerated MDR-infected wound healing and reduced bacterial burdens by 2-5 logs; for S. aureus pneumonia, they outperformed levofloxacin (3-5 vs 2 logs bacterial load reduction) and restored immune function (improved thymus index, reduced pro-inflammatory cytokines). This work establishes a symmetric peptide scaffold as a versatile AMP platform, with RI8 showing promise for systemic MDR infections and RW8 for localized biofilm-associated infections. Overall, these findings highlight a rational design strategy that may help address key translational limitations of conventional antibiotics.