The design of cell-selective tryptophan and arginine-rich antimicrobial peptides by introducing hydrophilic uncharged residues

Antimicrobial peptides (AMPs) are considered to be powerful weapons in the fight against traditional an-tibiotic resistance due to their unique membrane-disruptive mechanism. The combination of traditional and classical hydrophobic tryptophan (W) residues and hydrophilic charged arginine (R) residues is con-sidered as the first choice for the minimalist design of AMPs due to its potent performance in antibacte-rial activity. However, some W-and R-rich AMPs that are not rationally designed and contain excessive repeats of W and R residues may cause severe cytotoxicity and hemolysis. To address this issue, we de-signed the (WRX)n (where X = hydrophilic uncharged amino residues; n = number of repeat units) series engineered peptides with high cell selectivity by introducing hydrophilic uncharged threonine (T), serine (S), glutamine (Q) or asparagine (N) residues into the minimalist design of W-and R-rich AMPs. The re-sults showed that the introduction of these hydrophilic uncharged amino residues, especially T residues, significantly improved the cell selectivity of the W-and R-rich engineered peptides. Among (WRX)n se-ries engineered peptides, T6 presents a mixture structure of beta-turn and alpha-helix. It has broad spectrum and potent antibacterial activity (no activity against probiotics), good biocompatibility, high selectivity index, strong tolerance (physiological salts, serum acid, alkali, and heat conditions), rapid and efficient time-kill kinetics, and no tendency of resistance. Studies on antibacterial mechanism show that T6 exert antibacterial activity mainly by disrupting bacterial cell membrane and inducing the accumulation of re-active oxygen species in bacterial cells. Furthermore, T6 exhibited potent antibacterial and antiinflamma-tory capabilities in vivo in a mouse peritonitis-sepsis model infected with Escherichia coli. In conclusion, our study confirms an effective strategy for the minimalist design of highly cell selective W-and R-rich AMPs by introducing hydrophilic uncharged T residues, which may trigger widespread attention to hy-drophilic uncharged amino acid residues, including T residues, and provide new insights into the design of peptide-based antibacterial biomaterials.