Structure-activity relationships and development of disulfide-rich cyclic peptides as pharmaceutical templates

Abstract

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 cellpenetrating 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 iv 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,...