D-amino acid substitution and cyclisation enhance the stability and antimicrobial activity of arginine-rich peptides

Cationic peptides rich in arginine are potent antimicrobials but face challenges due to enzymatic degradation and potential host toxicity. We screened eight linear arginine-rich peptides for antimicrobial and haemolytic activity, identifying R4F4 as the most active. However, its efficacy decreased in the presence of human serum and trypsin. To enhance stability, we developed three R4F4 derivatives—D-R4F4, cyclic R4F4, and R4F4-C16—plus one R4-based peptide (R4-C16). Cyclisation and D-amino acid substitution significantly improved protease resistance and antimicrobial activity. Using fluorescence imaging, microscopy, and RNA sequencing, we revealed a complex mechanism involving membrane disruption, oxidative stress modulation, and transcriptomic changes in bacterial metabolic pathways. The R4F4-based peptides also displayed strong antibiofilm activity and good cytocompatibility. Our findings highlight the benefits of peptide optimisation—particularly D-amino acid incorporation and cyclisation—in improving stability and function. This work informs future design of antimicrobial peptides and deepens understanding of their multifaceted biological effects.