Identification and functional study of peptides from the skin secretion of Phyllomedusa azurea

Abstract

Antimicrobial peptides (AMPs) from Phyllomedusa frog skin secretions represent a rich source of membrane-active anti-infective agents. In this work, three major AMP lineages, dermaseptins (QUB-2480, DRS-01) and phylloseptins (PS-12), were comprehensively-investigated alongside rationally-designed analogue libraries to elucidate the structural determinants governing antimicrobial, cytotoxic, enzymatic, and biophysical properties. All peptides and analogues were chemically-synthesised and structurally-validated by RP-HPLC, ESI-MS, and circular dichroism spectroscopy, confirming amphipathic α-helical conformations under membrane-mimetic conditions.

QUB-2480, a newly identified dermaseptin from Phyllomedusa azurea, displayed potent broad-spectrum antibacterial activity, with MIC values in the low micromolar range against both Gram-positive and Gram-negative bacteria，particularly against Gram-negative bacteria. Analogues with enhanced cationicity apparently improved potency and therapeutic indices, whereas reduced hydrophobicity or loss of C-terminal amidation attenuated activity. PS-12 displayed moderate antibacterial activity, with MIC values ranging from 2–64 µM across tested strains. Rational modification of cationicity and α-helical propensity generated more potent analogues, with PS-12-1 showing improved activity (MIC = 1–4 µM against S. aureus and E. coli). However, excessive hydrophobicity or structural disruption reduced selectivity. Hydrocarbon stapling (PS-12-5) enhanced resistance to proteolytic degradation but resulted in reduced antimicrobial potency (MIC ≥64 µM), highlighting a trade-off between stability and activity. For the dermaseptin DRS-01, activity was robust across bacterial species but markedly-compromised by trypsin digestion, whereas the N-terminal Nal-modified analogue DRS-N preserved both potency and partial protease resistance. Modifications aimed at sequence shortening or charge redistribution generated apparent loss of activity.

Across all peptide families, mechanistic assays demonstrated rapid bactericidal effects mediated by membrane perturbation, including outer- and inner-membrane permeabilisation, depolarisation, and time-dependent disruption consistent with a carpet-like mode of action. Several analogues exhibited reduced haemolytic activity and improved antiproliferative effects against cancer cell lines, underscoring the tunability of bioactivity profiles. Resistance-development assays revealed minimal MIC shifts over serial passaging, indicating a low propensity for resistance induction.

Collectively, these studies have established QUB-2480, PS-12, DRS-01, and their optimised analogues, as promising membrane-active AMPs, and provide detailed structure–activity insights to guide the rational design of next-generation antimicrobial therapeutics with improved stability, potency, and safety.

Thesis is embaragoed until 31 July 2031.

Date of Award	Jul 2026
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Tianbao Chen (Supervisor), Mei Zhou (Supervisor) & Lei Wang (Supervisor)