D-amino acid substitution and cyclisation enhance the stability and antimicrobial activity of arginine-rich peptides
Cationic peptides, particularly those rich in arginine and/or lysine residues, are potent antimicrobial agents effective at very low concentrations in laboratory settings. However, their applicability in pharmaceutics and biotechnology is currently limited due to their susceptibility to biological enzymatic processes and (in some cases) toxicity to host cells. To address this, we screened eight linear arginine-rich peptides for their haemolytic properties and antimicrobial activity using a set of computational and experimental assays. Inspired by our previous results on R4F4, we then designed three modified peptides based on a R4F4 backbone: R4F4-C16; D-R4F4; Cyclic R4F4 and one based on R4 (R4-C16). Among the tested linear peptides containing only natural amino acids, R4F4 exhibited the strongest antibacterial activity; however, this effect was reduced in the presence of human serum and trypsin. Conversely, our study demonstrated that cyclisation and substitution to its D-amino acid enantiomer significantly enhanced stability and activity of R4F4, and whilst in the presence of proteases. As revealed by fluorescence imaging, microscopy RNA sequencing analysis, the mode of action involves complex and dynamic events. This multifaceted antimicrobial mechanism integrates alterations in membrane permeability, modulation of intracellular reactive oxygen species levels, and changes in transcriptomic signature profiles. At the molecular level, notable changes were observed in the bacterial expression of genes associated with metabolic pathways and biological processes, Furthermore, R4F4-derived peptides showed substantial antibiofilm activity in preventing the formation and disruption of mature biofilms, together with good cytocompatibility, highlighting the potential for clinical applicability. In conclusion, this study emphasises the importance of optimising the stability of peptide-based antimicrobials, particularly those rich in arginine, and highlights the advantages of incorporating D-amino acids and cyclisation for enhanced performance. This information will prove useful in the future design of antimicrobial peptides. In addition, the molecular perspective on peptide-induced gene expression changes, as identified by RNAseq, broadens our understanding of antimicrobial peptides' activities and provides a clearer picture of their versatile mechanisms