Assessing the Effect of Substitution of Arginine with Homoarginine in Two Antimicrobial Peptides

Purpose: Antimicrobial peptides show promise as broad-spectrum agents and are less likely to induce rapid resistance. However, protease sensitivity limits their potential as antibiotic alternatives. Non-canonical amino acid incorporation may enhance stability but could impact other properties. Methods: The activities of four peptides (oligoarginine (PLR10), oligohomoarginine (PLHR10), Mel4, and Mel4HArg) were tested against strains of S. aureus, P. aeruginosa and E. coli. Circular dichroism (CD) was used to determine their secondary structure. Their MICs against E. coli and P. aeruginosa were re-examined after protease treatment. Cytotoxicities against L929 cell line were determined. Results: MICs for the four AMPs ranged from 31.25 to > 500 µM. Total substitution of arginine with homoarginine had minimal effect on MICs of PLR10, but increased the MICs of Mel4, e.g., against S. aureus ATCC 6538 from 31.25 to > 250 µM. In 30 mM SDS, substitution with homoarginine resulted in the loss of α-helical structures. Trypsin reduced PLR10 activity against E. coli and P. aeruginosa but had less effect on PLHR10. At 62.5 µM, PLR10 reduced E. coli growth by 20% compared to 90% for PLHR10. At 125 µM, PLR10 reduced P. aeruginosa growth by 22% compared to 70% for PLHR10. Mel4HArg retained activity against E. coli post-trypsin treatment but not against P. aeruginosa. The IC₅₀ for PLR10 and PLHR10 (15.625 µM) against L929 cell line was below their MIC (31.25 µM), indicating low therapeutic potential. In contrast, Mel4 and Mel4HArg had IC50 > 500 µM, exceeding Mel4’s MIC for S. aureus (31.25–62.5 µM) and E. coli (125 µM), though not for Mel4HArg’s MIC against P. aeruginosa. Conclusion: The total arginine substitution with homoarginine in Mel4 and PLR10 resulted in improved stability but reduced activity in Mel4 and no improvement in cytotoxicity of PLR10, highlighting the need to find balance in optimizing AMP properties.