Peptides are attracting increasing attention from pharmaceutical companies as promising drug leads. Peptides have high specificity and high-affinity potency towards molecular targets as well as low toxicity, making them viable alternatives to small molecules. However, their lack of membrane permeability, bioavailability and in vivo stability hinders most peptides from becoming actual drugs. Although peptides are generally thought to be poor drugs, disulfide-rich cyclic peptides show great potential as pharmaceutical scaffolds in peptide-based drug design because of their exceptional stability.
Naturally occurring disulfide-rich cyclic peptides have been found in plants and animals. Cyclotides are among the largest naturally occurring disulfide-rich cyclic peptides discovered so far and have been identified in species of Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae and Solanaceae plant families. Cyclotides are distinguished by the cyclic cystine knot (CCK) motif, which comprises a macrocyclic backbone and three conserved disulfide bonds. The CCK motif provides cyclotides with exceptional stability, making them promising scaffolds for peptide drugs. Cyclotides were initially classified into two main subfamilies, Mӧbius (e.g, kalata B1 and kalata B2) and bracelet (e.g, cycloviolacin O2), based on the presence/absence of a cis-Pro peptide bond. A third subfamily, the trypsin inhibitor subfamily, was later introduced following the discovery of two peptides (MCoTI-I and MCoTI-II) obtained from Momordica cochinchinensis seeds. Other trypsin inhibitors in this family have now been identified. This subfamily is homologous to acyclic squash inhibitors and share little sequence similarity to cyclotides from the other two subfamilies. Recently, cyclotides kalata B1 and MCoTI-II have been shown to internalise into cells and were therefore named cyclic cell-penetrating peptides (CCPPs). Another CCPP is SFTI-1, a sunflower trypsin inhibitor that comprises a single disulfide-bond and has been used as a scaffold in drug design.
The aim of my Ph.D. thesis was to understand the mechanism of action and unravel the cell-penetrating properties of disulfide-rich cyclic peptides. Chapter 1 provides an introduction on disulfide-rich cyclic peptides with an emphasis on their structural features and their biological activities, including membrane-binding and cell-penetrating properties, as well as their potential as drug design scaffolds in various therapeutic applications.
Chapter 2 describes the materials and methods used to carry out the experiments in this thesis, which include methods to synthesise peptides that were studied, and biological assays used to evaluate them. In addition biophysical methodologies to evaluate structural features, peptide internalisation and peptide-membrane interactions are also detailed.
Chapter 3 is a research article published in the European Journal of Medicinal Chemistry (2014) that focuses on the structural characteristics affecting the cell-penetrating properties of disulfide-rich cyclic peptides, MCoTI-II and SFTI-1. Sets of MCoTI-II and SFTI-1 analogues were synthesised to investigate the relevance of positively charged, hydrophobic residues and other structural modifications in the cell-penetrating ability of these CCPP scaffolds. This study showed that MCoTI-II cellular uptake can be improved by increasing the overall positive charge of the native sequence. On the other hand, structural mutations to SFTI-1 did not significantly influence its cellular uptake. This study provides information for the development of these disulfide-rich cyclic scaffolds into potential drug candidates towards intracellular therapeutic targets.
In Chapter 4, MCoTI-II is used as a scaffold to target an intracellular protein. In this study, COG1410, an ApoE-derived linear peptide, was engineered onto MCoTI-II with the aim of inhibiting the SET protein, which modulates the NF-κB pathway. The grafted peptides showed high stability in human serum and cytotoxicity towards a cancer cell line. One of the grafted peptides was able to suppress NF-κB dependent gene promoter activity in lipopolysaccharide-stimulated macrophages. This study shows the application of disulfide-rich cyclic peptides as a scaffold for the development of stable antagonists towards an intracellular target.
Chapter 5 discusses the self-association of the cyclotides kalata B1 and kalata B2 in a membrane environment and the significance of its oligomerisation properties to its mechanism of action. Based on the proposed model of cyclotides self-associating in membranes, different mutants of kalata B1 and kalata B2 were synthesised and chemical cross-linking experiments were carried out in presence of liposomes. In this study, a kalata B2 mutant showed oligomer formation in the presence of lipid membranes. This study broadens the knowledge on self-association of cyclotides in membranes, which will help in our understanding on how cyclotides deliver their activities.
In summary, this thesis provides new information on the development of disulfide-rich cyclic peptides as templates in drug design. Cellular uptake studies on disulfide-rich cyclic peptides highlighted the importance of positive charge to the internalisation process. In addition, grafted cyclotides can be directed towards an intracellular therapeutic target to modulate a cellular pathway. Finally, cyclotides can self-associate in a membrane environment, expanding our knowledge on their mechanism of action and membrane binding properties. The findings in this thesis will assist in the development of disulfide-rich cyclic peptides into drugs, further broadening their applications in peptide-based therapeutics.