Rational two-round modification for developing isopeptide bond-containing antimicrobial peptide with enhanced...

The peptide CPF-7, developed by our group previously, is a modified analog of the natural antimicrobial peptide CPF-C1, which demonstrated increased antibacterial activity. However, its perfect α-helical structure contributes to high hemolysis, thereby limiting its applications. Herein, to improve the druggability of CPF-7, we first introduced isopeptide bonds to modulate helicity, generating analogs with a favorable therapeutic index. But these modifications resulted in poor serum stability. Based on the cleavage sites analysis, a second round of modification was performed by substituting amino acids. The lead peptide IP2-D4 was screened and identified potent antibacterial activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii (MIC = 4-8 μg/mL), reduced hemolysis (MHC₂₀ = 256 μg/mL), and high stability (t_1/2 = 2.932 h). It also showed a low tendency to induce resistance and potent eradication of persisters and biofilms. Importantly, IP2-D4 demonstrated significant antimicrobial efficacy in vivo, effectively treating skin wound infections and catheter-associated biofilm infections. RNA sequencing revealed that it up-regulated the expression of membrane genes in bacteria, and we further confirmed its antibacterial mechanism of membrane destruction.