De Novo Generation of Short Antimicrobial Peptides with Enhanced Stability and Cell Specificity

Objectives: Though antimicrobial peptides (AMPs) show great potential as novel antibiotics, therapeutic applications are hindered by their low stability, toxicity and high manufacturing cost. Various chemical modification strategies are employed to overcome these problems. However, chemical modifications often significantly increase the manufacturing cost of AMPs with only limited pharmacokinetic advantages. Therefore, we developed AMPs with enhanced stability and cell specificity that can be economically produced.

Methods: Peptides were designed by systematic amino acid arrangement without the incorporation of both non-natural amino acids and peptidomimetics. Antimicrobial activities were measured against Gram-positive bacteria, Gram-negative bacteria and fungi by MIC evaluation under both standard and physiologically relevant conditions. Cytotoxicity towards human cells was evaluated to verify selective antimicrobial activity. The antibacterial mechanism of the peptides was elucidated by β-galactosidase assay and scanning electron microscopy.

Results: Among the designed peptides, GNU6 and GNU7 showed potent antimicrobial activity against bacteria and fungi and maintained their activity in the presence of 150 mM NaCl and 10% serum. These peptides were not digested by exposure to trypsin, chymotrypsin and aureolysin for up to 12 h and showed potent antimicrobial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. Moreover, they did not affect the viability of erythrocytes, keratinocytes and fibroblasts up to 128 mg/L. A membrane permeabilization assay and scanning electron microscopy analysis showed that GNU6 and GNU7 compromised membrane integrity and function in microorganisms.

Conclusions: This study suggests that GNU6 and GNU7 might overcome serious problems that currently prevent the clinical use of AMPs and be developed as novel antimicrobial agents.