Identification and modification of antimicrobial peptides from the dark-spotted frog, Pelophylax nigromaculatus, with improved antibacterial potency and haemolytic activity

Abstract

Antimicrobial peptides (AMPs) represent a promising alternative to conventional antibiotics due to their broad-spectrum activity and low resistance potential. This study focused on identifying and modifying AMPs from the skin secretions of Pelophylax nigromaculatus to investigate their structure–activity relationships and optimise therapeutic potential. Four peptides—pelophylaxin-1T, -2, -3, and -4—were characterised. Pelophylaxin-1T exhibited selective activity against E. coli with low cytotoxicity, while pelophylaxin-2 showed broad-spectrum antibacterial activity but high cytotoxicity. Pelophylaxin-3 displayed balanced activity and safety, whereas pelophylaxin-4 was effective mainly against Gram-positive bacteria but demonstrated significant haemolysis.

Pelophylaxin-1T was selected for analogue development to explore the role of the 'Ranabox' domain and to enhance antibacterial efficacy. The removal of this domain significantly reduced activity, highlighting its functional importance. Analogues with increased positive charge and reduced hydrophobic moment, particularly -5K11K, -5K10K, and -5K10K11K, maintained antibacterial activity while reducing cytotoxicity. These peptides selectively disrupted the outer membrane of E. coli. Pelophylaxin-2 was also modified due to its toxicity, and two analogues were designed to reduce cytotoxicity while preserving antimicrobial activity. Modifications involving lysine substitution on the hydrophobic face were effective in lowering haemolysis. In a further attempt to enhance in vivo stability, analogues of -5K11K incorporating D-lysine substitutions were created. However, single D-lysine substitutions did not improve salt or serum resistance and sometimes reduced efficacy under physiological conditions.

Overall, lysine incorporation into the hydrophobic face of AMPs proved an effective strategy to reduce cytotoxicity. -5K11K emerged as a lead candidate with strong anti-E. coli activity and low cytotoxicity, though its efficacy declined under simulated in vivo conditions. These findings underscore the delicate balance between activity, selectivity, and stability in AMP design.

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)