Discovery, structural characterisation and targeted engineering of bioactive peptides from amphibian skin secretion

QUB-2531 produced a low degree of cytotoxicity and haemolysis without decreasing the antimicrobial effect dramatically. However, QUB-1376 demonstrated lower antimicrobial potency. Interestingly, QUB-3307 and QUB-2531 exhibited an inhibitory effect on trypsin, which may be due to the presence of a typical Kunitz inhibitor motif, -KCK-, at their C-terminals.

However, QUB-1376 failed to retain the inhibitory effect, which indicated that an intact Rana Box domain could contribute to the trypsin inhibition.<br><br>In chapter 4, a novel brevinin-1 peptide was identified from the skin secretion of the Indian cricket Frog, Fejervarya limnocharis, and was named QUB-2605. It demonstrated varying degrees of antimicrobial activities with high haemolysis. The modifications of the peptide were conducted to explain the structure-activity relationship from the N-terminus.

QUB-2605 and its analogues shared the same family feature - the ‘Rana Box’- and the subfamily fragment feature motif, FLP-. Meanwhile, the introduction of Lys and Trp in the analogue peptide sequences revealed that antimicrobial activity of these designed analogues remained unchanged once the hydrophobicity and charge reached a threshold. Meanwhile, the correlation between hydrophobicity and haemolysis explained the structure-activity relationship.

Hence, a new design idea that the hydrophobicity saturations in different situations related to antimicrobial activity could be the starting point for the generation of peptides with specific antimicrobial activity.<br>In chapter 5, a novel temporin peptide was identified from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis), and was named QUB-1368. It demonstrated a broad-spectrum antimicrobial activity against several microorganisms except for Gram-negative bacteria.

Target-modifications were carried out to further enhance the antimicrobial activity against Gram-negative bacteria via coupling QUB-1343 as the common functional domain of Onc112. Synergy checkerboard assay indicated that the hybrid peptide coupling of Onc112 and QUB-1368 contributed to functional improvement instead of cooperation of each fragment. Results indicated that hybrid peptide QUB-2696 exhibited its antimicrobial activity through multiple mechanisms, more than just membrane permeability.<br><br>In conclusion, the structure-activity relationships of AMPs were explained from modifications in both the N-terminus and C-terminus.

Moreover, QUB-1952 and QUB-2696 represented excellent antimicrobial agents with high therapeutic indices.