Structure–Activity Study Characterization and Mechanism of Action of an Antimicrobial Peptoid D2 and Its d- and l-Peptide Analogues

摘要

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) constitutes an emerging health problem for companion animals in veterinary medicine. Therefore, discovery of novel antimicrobial agents for treatment of Staphylococcus-associated canine infections is urgently needed to reduce use of human antibiotics in veterinary medicine. In the present work, we characterized the antimicrobial activity of the peptoid >D2 against S. pseudintermedius and Pseudomonas aeruginosa, which is another common integumentary pathogen in dogs. Furthermore, we performed a structure–activity relationship study of >D2, which included 19 peptide/peptoid analogs. Our best compound >D2D, an all d-peptide analogue, showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius (2–4 µg/mL) and P. aeruginosa (4 µg/mL) isolates as well as other selected dog pathogens (2–16 µg/mL). Time–kill assays demonstrated that >D2D was able to inhibit MRSP in 30 min at 1× MIC, significantly faster than >D2. Our results suggest that at high concentrations >D2D is rapidly lysing the bacterial membrane while >D2 is inhibiting macromolecular synthesis. We probed the mechanism of action at sub-MIC concentrations of >D2, >D2D, the l-peptide analog and its retro analog by a macromolecular biosynthesis assay and fluorescence spectroscopy. Our data suggest that at sub-MIC concentrations >D2D is membrane inactive and primarily works by cell wall inhibition, while the other compounds mainly act on the bacterial membrane.