Optimized proteolytic resistance motif (DabW)-based U1-2WD: A membrane-induced self-aggregating peptide to trigger...

The biggest application bottleneck of antimicrobial peptides (AMPs) is the low oral bioavailability caused by the poor stability of digestive enzymes in the gastrointestinal tract. However, the research methods and evaluation criteria of available studies about anti-proteolytic strategies are not uniform and far from the actual environment in vivo. Here, we developed a research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving the protease stability of AMPs on the same platform for the first time. After a comprehensive analysis, Dab modification is identified as the most effective strategy to improve the trypsin stability of AMPs. By further modulating the proteolytic resistance optimization motif (DabW)_n, U1-2WD is obtained with ideal stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which forms amorphous aggregates in the bacteria environment to trigger the agglutination of bacterial cells to prevent bacterial escape. It then kills bacteria by disrupting bacterial membranes and inhibiting bacterial energy metabolism. Overall, our work has led to a new understanding of the effectiveness of proteolytic resistance strategies and accelerated the development of anti-proteolytic AMPs to combat multidrug-resistant bacterial infections. STATEMENT OF SIGNIFICANCE: We developed research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving protease stability of AMPs on the same platform for the first time. we found effective strategies to resist trypsin hydrolysis: modification with backbone (β-Arg), D-enantiomer (D-Arg) and L-2,4-diaminobutanoic acid (Dab). Further, the proteolytic resistance optimization motif (DabW)_n was designed. When n=3, derivative U1-2WD was obtained with desirable stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which can self-aggregate into amorphous aggregates in the bacteria environment to mediate the agglutination and sedimentation of bacterial cells to prevent bacterial escape, and then kill bacteria by destroying bacterial membranes and inhibiting bacterial energy metabolism.