The identification, bioactive evaluation and exploration of the active sites of antimicrobial peptides from the skin secretion of Odorrana margaretae

Abstract

Antimicrobial resistance continues to escalate globally, underscoring the urgent need for novel therapeutic strategies. Among them, AMPs derived from amphibian skin secretions have attracted significant interest due to their potent and broad-spectrum activity. However, a fundamental challenge persists, the poor understanding of AMP active sites, particularly in non-model species such as Odorrana margaretae. To address this gap, this study employed a multimodal strategy integrating gene cloning, SPPS, RP-HPLC/MALDI-TOF identification, and a series of functional and structural assays to characterise two AMPs: QUB1971 and Palustrin-OM. In Chapter 3, by sequence alignment analysis, Ile13 was identified as a critical residue in QUB1971, essential for antimicrobial activity. Its substitution with valine, analogue 13V, led to a complete loss of function, with MICs exceeding 512 μM across all tested strains. Analogue L, rationally designed based on structural insights, exhibited the best therapeutic index (TI = 8.7) among all derivatives. In Chapter 4, Alanine-scanning mutagenesis further identified Ile6, Lys11, Ile13 and Val14 as functional “hotspot” residues, where substitution led to a marked reduction with MICs exceeding 64 μM across all tested strains. In contrast, modifications at other positions were either neutral or enhanced activity, demonstrating a context-dependent role of individual residues and offering guidance for rational design strategies. In Chapter 5, three truncated analogues of palustrin-OM were designed using the trypsin-cleavage mimicking strategy. Functional screening revealed that truncated peptide 2 not only retained but enhanced antimicrobial efficacy, as indicated by a lower overall GM (4.6 μM vs. 8.5 μM for Palustrin-OM), while markedly improving safety with a higher therapeutic index (TI overall = 6.9 vs. 1.0), thus presenting a promising candidate for further development. Together, these complementary approaches established a robust framework for identifying active sites in antimicrobial peptides, thereby advancing the rational design of next-generation peptide therapeutics with improved potency and selectivity.

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)