De Novo Design and Characterization of Novel Antimicrobial Peptides, Lk3 and Lk4, Containing Repeating Unit

Short cationic antimicrobial peptides (AMPs) have an excellent potential for treating various infections.

This study focused on the de novo design of two peptides, LK3 (W(LK)₃W-NH₂) and LK4 (W(LK)₄W-NH₂), based on the most frequent and important amino acids in AMPs using in silico tools. The antibacterial activity of the designed peptides was evaluated under standard conditions, physiological NaCl concentration, and in the presence of serum (10% v/v). The antibiofilm and hemolytic activities of the peptides were assayed.

CAMP_R4 algorithms predicted LK3 and LK4 as AMPs. Compared with LK3, the cationicity of LK4 was increased slightly, while its hydrophobicity was decreased a little. Both peptides had extended coil structures. The antibacterial activity of LK3 was better than that of LK4. The best minimum inhibitory concentration of LK3 (MIC: 64 µg/ml) was found against methicillin-resistant Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa under salt and no salt conditions. However, serum inhibited the antibacterial effect of the peptide. LK3 also exhibited 44.45% antibiofilm activity against P. aeruginosa at 4 × MIC concentration. The hemolytic activity of LK3 and LK4 peptides was lower than 10% up to 128 µg/ml. Among the two designed peptides, LK3 represented better selectivity against Gram-negative bacteria.

In conclusion, LK3 with selective activity against Gram-negative bacteria, good salt stability, and low toxicity appears to be a good candidate for further design investigations to enhance its antibacterial activity. This study contributes to the development of AMPs active against antibiotic-resistant Gram-negative bacteria, a major global health concern.