Binding loop of sunflower trypsin inhibitor 1 serves as a design motif for proteolysis-resistant antimicrobial peptides

Abstract

Although antimicrobial peptides (AMPs) have become powerful drug candidates in the post-antibiotic era, but their low protease stability hinders their clinical application. In the present study, the natural sunflower trypsin inhibitor 1 (SFTI-1) binding loop (CTKSIPPIC) was used to design and synthesize a specific anti-proteolytic sequence template ((RX)_n W (RX)_n CTKSIPPIC (n = 2, 3; X represents A, I, L, V, F, and W)). After several antibacterial, bactericidal, and toxicity tests, RV3 stood out from the variants and had the highest average selectivity index (SI _all = 156.03). It is highly stable in serum, varying pH, temperature, and salt ions as well as under high trypsin, pepsin, or papain concentrations. In a mouse skin inflammation model, established by Pseudomonas aeruginosa infection, RV3 could effectively kill the pathogen, promote wound healing, inhibit inflammatory cell infiltration, and inhibit mRNA and protein expression of TNF-α, IL-6, and IL-1β inflammatory factors. The antibacterial mechanisms of RV3 include combining with lipopolysaccharides and increasing cell membrane permeability, leading to cell membrane rupture and death. These findings indicate that RV3 has great potential for the treatment of bacterial infections.

Keywords: Antimicrobial peptides; Protease stability; SFTI-1; Skin inflammation; Structure-function relationship.