Discovery, modification, and functional study of a natural Kunitz-type peptide kunitzin-PE from <i>Pelophylax esculentus</i> skin secretion

In this project, the lyophilised skin secretion of the green frog, Pelophylax esculentus, was subject to molecular cloning for identifying a naturally occurring kunitz-type inhibitor, namely kunitzin-PE. In addition to studying the potential applications through functional screening, four analogues (C7, C8, C9 and C19) were designed, synthesised, and tested to investigate the effect of the length of the disulphide region on their biological activities.

In Chapter 4, C8 was used as a template to design a set of variants to explore the effect of the cationicity within the disulphide domain on the bioactivity of C8. Additionally, the relationship between the hydrophobicity inside the convex loop and the bioactivity of C9 was also investigated and discussed in Chapter 5.<br><br>Results: The results showed that the anticancer cell proliferation, trypsin inhibition and chymotrypsin inhibition of the kunitzin peptide were enhanced by changing the length of its disulphide domain.

The cationicity within the disulphide domain of the kunitzin peptide is important for the interaction between the peptide and the anionic cell membrane, but the hydrophobicity within the disulphide domain is less important. Two peptide variants, C7 and K2, were found to have improved anticancer and antibacterial activities while maintaining their protease inhibitory effects. Moreover, these peptides exhibited low haemolysis/cytotoxicity at their minimum concentration for the antibacterial/anticancer action.<br>Conclusions: In summary, the length of the disulphide domain and the cationicity within the convex loop play an important role for protease inhibitory, anticancer and antibacterial activity of kunitizin peptide.

In addition, these findings provide an effective alternative to novel antibiotic drugs or anticancer drugs.<br><br><i>Thesis is embargoed until 31 July 2028</i>.<br>