Functional characterization and optimization of antimicrobial peptides, QUB2061 and Esculentin-1-OR3, from <i>Odorrana</i> species

In vitro, we evaluated the antimicrobial, anti-proliferative, and haemolytic activities of QUB-2061and its analogues. <br><br>Analogues such as QUB2061-20R and QUB2061-4K18K, with superior therapeutic indices and robust efficacy against resistant pathogens, emerged as highly promising candidates for future antimicrobial drug development, addressing the critical global threat posed by antibiotic-resistant infections.<br><br>In Chapter 4, addressing the inherent limitations associated with antimicrobial peptides (AMPs) has increasingly involved sophisticated bioinformatics analyses and peptide engineering techniques.

Utilizing bioinformatics-based screening methodologies, a set of twelve promising AMPs was recently identified, among which Esculentin-1-OR3, a frog-derived peptide consisting of 46 amino acids, was particularly noteworthy due to its exceptional broad-spectrum antimicrobial properties. Esculentin-1-OR3 demonstrated rapid bactericidal activity against both Gram-positive and Gram-negative bacterial species, including highly resistant pathogens such as P. aeruginosa and A. baumannii.

Mechanistically, this peptide efficiently disrupted bacterial integrity by sequentially permeabilizing the outer membrane and inducing depolarization of the cytoplasmic membrane. However, the antimicrobial effectiveness of Esculentin-1-OR3 significantly diminished under physiological conditions, primarily due to competitive inhibition by divalent cations and vulnerability to degradation by serum proteases. Additionally, while Esculentin-1-OR3 exhibited pronounced cytotoxic effects against various cancer cell types, its indiscriminate mode of action led to considerable cytotoxicity toward healthy mammalian cells, underscoring the critical necessity for structural optimization to improve selectivity and therapeutic index.<br><br>In Chapter 5, to address the inherent limitations associated with Esculentin-1-OR3, we utilized rational peptide engineering approaches to develop several truncated derivatives.

These modifications included optimization of the peptide’s active core region, amidation of the C-terminus, and the strategic incorporation of structural modifications, particularly the introduction of α-aminoisobutyric acid (Aib) residues and D-amino acid substitutions.<br><br>Among the seven analogues synthesized, the variant designated as Aib10,18 exhibited outstanding antimicrobial efficacy against multidrug-resistant bacterial strains, characterized by a geometric mean minimal inhibitory concentration (MIC) of 4.92 μM. Moreover, this analogue demonstrated significantly improved pharmacological properties, including diminished haemolytic activity (HC10 = 52.29 μM) and enhanced resilience under physiological salt conditions.

The time-killing kinetic assays highlighted rapid bactericidal action, achieving complete bacterial clearance within 5 minutes at concentrations corresponding to four times the MIC (4×MIC). Mechanistically, Aib10,18 was shown to exert its antimicrobial effects via a dual-mode membrane disruption process, evidenced by substantial outer membrane permeability as indicated by elevated NPN uptake, coupled with inner membrane depolarization.

Importantly, no significant β-galactosidase leakage was detected in ONPG assays, suggesting selective membrane targeting rather than general pore formation.<br><br>Interestingly, Aib10,18 displayed targeted cytotoxicity against carcinoma cell lines, including H460 (IC50 = 9.5 μM) and MCF-7 (IC50 = 9.8 μM), while exerting negligible toxic effects against normal mammalian cells (IC50 values exceeding 100 μM). Structural characterizations performed through circular dichroism spectroscopy confirmed that Aib10,18 maintained a stable amphipathic α-helical structure under aqueous conditions, distinctly contrasting with the predominantly random coil conformations observed in non-modified analogues.<br><br>Collectively, these results substantiate the effectiveness of our rational design approach in converting naturally occurring, elongated peptides into shorter antimicrobial peptide analogues.

Furthermore, this study establishes a conceptual framework for the development of dual-functional peptides possessing both potent antimicrobial and anticancer activities. The strategic incorporation of Aib residues at critical helical positions (specifically positions 10 and 18) effectively achieves a balance between target specificity and minimized mammalian cell toxicity, representing an important advance toward the realization of clinically applicable peptide-based antibiotics.

Future investigations focusing on pharmacokinetic enhancements and in vivo assessments are essential for fully elucidating the therapeutic potential and translational feasibility of this promising lead compound.<br><br><i>Thesis is embargoed until 31 December 2030.</i>