Discovery and functional study of peptides from natural products and rational engineering of bioactive peptides

In Chapter 3, a novel temporin peptide, named temporin-RC, was identified from bullfrog, Rana catesbeiana. This 14-amino-acid peptide had the typical hydrophobic N-terminus, C-terminal amidation and α-helical structure, characteristic of the temporin peptide family. Temporin-RC displayed potent anti-bacterial activity against Gram-positive bacteria, while it was of limited use against Gram-negative bacteria.

It also showed anti-proliferation activity against various cancer cells and normal cells and relatively low haemolytic ability. Three amino acids on the relative hydrophobic face were substituted with lysine and this analogue was named, temporin-RC-K. The modified peptide had three additional net charges but retained its α-helical secondary structure. This promoted its antimicrobial activity against both Gram-positive and Gram-negative bacteria.

The efficiency of bactericidal ability and membrane disruption of temporin-RC-K was enhanced over its parent peptide. Also, the haemolytic activity of this cationicity-enhanced analogue, was dramatically decreased. In Chapter 4, the temporin peptides were conjugated to a serine protease inhibitor, sunflower trypsin inhibitor (SFTI).

To explore the structure-activity relationships, the SFTI was conjugated at the C-terminus and the N-terminus of temporin-RC, and the conjugated peptides lost anti-microbial ability, but the N-terminal modified analogue showed a prominent anti-proliferation ability. The relationship between linking type between the two different moieties and biofunctionality, was studied. This suggested that the temporin peptide directly conjugated to the single Cys-loop of SFTI, enhanced membrane permeabilisation activity against bacteria and cancer cells.

Using a linker to join the two moieties, could distinctly increase respective activities and the characteristics of the linker influenced the physicochemical properties of peptides, which effected their lysis of erythrocytes.In Chapter 5, the modification of specific temporin-RC positions, was studied. There were two temporin peptides from an amphibian skin and a wasp venom, which have similar amino acid sequences. These were used as templates for analogue design.

The replacement of the proline at the third position illustrated that this helix breaker limited the antimicrobial ability to some extent, and the hinge amino acid was important at the third position for decreasing the cytotoxicity to erythrocytes. In addition, the enhancement of net positive charge was not a determining factor in increasing selectivity. Furthermore, the truncation of the C-terminus to build a much shorter peptide, failed in retaining the biological activities of the parent template peptide.In summary, a novel peptide from Rana catesbeiana was discovered and its bio-activities and mechanisms of action were reported in this thesis.

Also, several modified methods were designed and applied to enhance its anti-microbial and anti-proliferation activities along with decrease its cytotoxicity to normal cells. The study on the relationship between peptide structures and bio-functions produce a profound implication on the development of peptides to clinical drug. Although there are few peptides used in medical practice recently, they still are considered as effective drug leads in medical treatment of diseases in future.

Through the optimisation using peptide rational design, the peptide will achieve more advances in improved bioactivities and less side effects that would make them more ideal to be used in clinic.<br><br><i>Thesis embargoed until 31 July 2027.</i>