Discovery and engineering of a novel non-membranolytic peptide, QUB-2069, with enhanced in vitro efficacy against Escherichia coli

Abstract

For many years, bioactive peptides originating from natural sources have been the subject of intensive investigation. These peptides with antimicrobial activity were considered as the antibiotic candidates to solve the drug resistance problem. In this study, different modification strategies were applied to a novel identified random coil peptide to enhance its antibacterial activity, making it a promising clinical antimicrobial agent.

In Chapter 3, QUB-2069 was identified from the green mountain frog, and the antibacterial activity and bactericidal mechanism of the peptide were preliminarily studied. QUB-2069 was found to exhibit antibacterial activity against Escherichia coli (E. coli) and low toxicity and has a different bactericidal mechanism from helical antimicrobial peptides (AMPs). It is considered a valuable AMP for further research.

In Chapter 4, various analogues based on the original peptide QUB-2069 were designed by altering the number of proline residues in the sequence to study the proline function on its biological activity and secondary structure. As the pyrrolidine ring lacks a hydrogen atom to form a hydrogen bond with its following residues, proline residues are considered to impede the helix structure forming. The proline residue in the designed analogue P-A was replaced by the helix promoting residue alanine to form a helix structure, enhancing antibacterial activity and expanding the bactericidal spectrum. Peptide P-A was chosen as the template peptide for subsequent modification.

In Chapter 5, the relationship between peptide function and physicochemical parameters including charge, secondary structure, and amphiphilicity was studied. Firstly, by exchanging the positions of amino acids in the peptide sequence, a perfect amphiphilic peptide was designed, and further analogues were designed by increasing the number of positive charges. In this chapter, 4A-4K was found to have an ideal charge, amphiphilicity, and a clear alpha helical structure, making it the most effective candidate peptide discovered. In addition, 4A-4K also has good stability in salt solutions.

In Chapter 6, peptide modification was studied by conjugating the original peptide QUB-2069 with different kinds of cell penetrating peptides (CPP). TAT-2069 exhibited significant antibacterial activity against E. coli while maintaining the advantage of low toxicity, while KLA-2069 and PLV-2069 have not been further studied due to their high toxicity and negligible antibacterial activity. More effective antibacterial effects were obtained by selecting a combination of peptide antibiotic, colistin, and modified analogues. In summary, through different modification strategies, the antimicrobial activity of QUB-2069 was improved with ideal cytotoxicity and the action mechanism was not changed. The successful modification strategies on QUB-2069 provide further direction for AMPs through random coil secondary structure optimization.

Thesis is embargoed until 31 July 2030.

Date of Award	Jul 2025
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Tianbao Chen (Supervisor), Mei Zhou (Supervisor) & Lei Wang (Supervisor)