Introduction:

Short cationic peptides are valuable chemical templates for the design of clinically available antibiotics. The discovery and development of these peptides face numerous challenges, including time-consuming screening processes, toxicity, and stability issues. Computational methods have been integrated into this process, with successful outcomes.

Methods:

In this study, we designed eight arginine-rich peptides, which were analysed using in silico tools to predict their toxicity and antimicrobial properties. We then confirmed the in vitro antibacterial effects and toxicity to fibroblasts and red blood cells of these synthetic peptides using absorbance-based assays. The activity of these peptides was also tested in environments that were rich in proteases. Three chemically modified peptides were synthesised based on the most active peptide. The antibiofilm action and bactericidal effects were evaluated using fluorescent markers and microscopy. We also monitored the levels of reactive oxygen species (ROS) when the peptides were incubated with bacteria.

Results:

In summary, we observed discrepancies between the in silico predictions and the in vitro screening results. R4F4 was the most promising peptide candidate, but its function was impaired when incubated with serum or trypsin. The lipidated analogue was toxic and did not exhibit similar antimicrobial effects. However, cyclisation and D-amino acid substitution strategies enhanced the stability and activity of novel analogues. These arginine-rich peptides act through a dual mechanism, integrating damage to the bacterial membrane and an increase in reactive oxygen species levels.

Conclusions:

Overall, this study demonstrates the significant contributions of the cyclisation and D-amino acid substitution approaches in enhancing the stability and activity of arginine-rich peptides. It represents an important step forward in the development of peptide-based candidates, which could form the basis of future antibacterial interventions.