Peptide Double-Stapling and Arginine <i>N</i>-Glycosylation Triggered the Development of Therapeutic Antimicrobial Peptides Capable of Killing Drug-Resistant Bacteria in Mice

Antimicrobial peptides SAAP-148 exhibited excellent antimicrobial activities but suffered from inherent disadvantages, including cytotoxicity and poor proteolytic stability. Herein, we developed a novel strategy combining one unique silver-catalyzed solid-phase glycosylation-enabled arginine <i>N</i>-glycosylation strategy and all-hydrocarbon peptide double-stapling, and five-round peptide libraries were rationally constructed containing over 50 stapled and/or arginine <i>N</i>-glycosylated peptides. SLP-51 consisting of two introduced all-hydrocarbon staples and the C-terminal arginine glycosylation exhibited superior in vitro antimicrobial activities against drug-resistant Gram-positive or -negative clinical isolates. SLP-51 also exhibited improved proteolytic stability than the parent peptide SLP-0, and importantly, significantly weakened hemolysis. Experimental and modeling mechanism research indicated that SLP-51 exerted similar but stronger killing abilities by destroying the integrality of the bacterial membranes. In both skin wound and drug-resistant bacterial pneumonia models, SLP-51 showcased a potent therapeutic effect in treating both <i>MRSA</i> and <i>Klebsiella pneumoniae</i> infection in vivo and dramatical improvement of inflammatory injury.