Development of a Novel Antibacterial Peptide, PAM-5, via Combination of Phage Display Selection and Computer-Assisted Modification

Abstract

Antibacterial peptides (ABPs) have been proposed as potential candidates for alternative antibacterial agents due to the extensive dissemination of antibiotic resistance. However, ABP isolation from natural resources can be tedious without consistent yield. Moreover, many natural ABPs are not developed for clinical application due to potential toxicity to mammalian cells. Therefore, the objective of this study was to develop a potent ABP with minimal toxicity via phage display selection followed by computer-assisted modification. Briefly, a 12-mer phage-displayed peptide library was used to isolate peptides that bound to the cell surface of Pseudomonas aeruginosa with high affinity. The affinity-selected peptide with the highest selection frequency was modified to PAM-5 (KWKWRPLKRKLVLRM) with enhanced antibacterial features by using an online peptide database. Using in vitro microbroth dilution assay, PAM-5 was shown to be active against a panel of Gram-negative bacteria and selected Gram-positive bacteria. Interestingly, the peptide was stable in human plasma by exhibiting a similar bactericidal effect via ex vivo assay. Scanning electron microscopy and SYTOX Green uptake assay revealed that PAM-5 was able to cause membrane disruption and permeabilization of the bacteria. Additionally, the peptide was also able to bind to bacterial DNA as demonstrated by gel retardation assay. In the time-kill assay, PAM-5 was shown to kill the bacteria rapidly in 10 min. More importantly, PAM-5 was non-cytotoxic to Vero cells and non-haemolytic to human erythrocytes at all concentrations tested for the antibacterial assays. Thus, this study showed that the combination of phage display screening and computer-assisted modification could be used to develop potent novel ABPs, and PAM-5 derived from these approaches is worth to be further elucidated for its potential clinical use.

Advances in screening, synthesis, modification, and biomedical applications of peptides and peptide aptamers

“…The results showed that the MIC ranged from 0.98 to 500 μM, while the MBC ranged from 4 to 650 μM, indicating a bactericidal effect. Yuen et al 237 combined phage display technology with computer‐assisted modifications and developed a novel antimicrobial peptide called PAM‐5. This peptide exerts antimicrobial activity by disrupting bacterial membranes and interacting with bacterial DNA.…”

3-Substituted Coumarins Inhibit NorA and MepA Efflux Pumps of Staphylococcus aureus

“…The dye SYTOX Green (a DNA intercalator) was used for this test, as performed by Yuen et al [65]. Initially, the inocula of S. aureus 1199B and K2068 were distributed in the 96-well black plate, and then, we added C1 coumarin until final concentrations of 200 µg/mL, 100 µg/mL, 50 µg/mL, and 25 µg/mL were obtained.…”