Structures and activities of cystine knot peptides from the Violaceae plant family

Abstract

Cyclotides are defined by the cyclic cystine knot (CCK) motif, formed by three interlinking disulfide bonds and a head-to-tail cyclic backbone. Since the natural role of cyclotides is presumed to be in plant defence, the discovery and characterisation of novel cyclotides plays an important role in finding new biocides. My PhD project focuses on the discovery and applications of cyclotides from the Violaceae plant family, which are abundant producers of diverse cyclotides. My PhD thesis first chapter introduces the state-of-the art in the field and the four subsequent chapters describe the results. In brief, Chapters 2 and 3 focus on the discovery and characterisation of new cyclotides and their linear analogues, called acyclotides. Chapters 4 and 5 focus on the applications of these new cystine knot peptides for agricultural and drug design purposes. Chapter 6 draws together the conclusions deriving from my various experimental studies and makes suggestions for possible future studies. Chapter 2 describes the sequence diversity of novel cyclotides from Vietnamese Viola species including V. arcuata, V. tonkinensis and V. austrosinensis. In total, four novel and six known cyclotides were discovered, sequenced, characterised and screened against a panel of cancer cell lines. An increase in hydrophobicity correlates with higher cytotoxicity of isolated cyclotides, potentially benefitting future drug design studies using these cyclotides as scaffolds. In addition, the sequences discovered in this study expand our understanding of cyclotide diversity in comparison with other cyclotides found in plants from the Asian region. Chapter 3 describes the discovery of cystine knot peptides from Rinorea bengalensis as well as some surprising results surrounding cyclic peptide production in R. bengalensis (understory trees in the violet family (Violaceae)). Unlike other members of the Violaceae family examined thus far, R. bengalensis produces unusually few cyclotides. It represents the first study that examines cystine knot peptides from R. bengalensis through a multifaceted approach to fully understand the transcriptomics, proteomics, functional characterisation and chemical synthesis of these knotted peptides. Key outcomes of the work are the data showing that head-to-tail cyclisation is not essential for the reported cytotoxic activity. Interestingly, 32 acyclotides were found to be produced in this Violaceae plant, whereas only one cyclotide could be identified. All novel cystine knot peptides displayed anticancer activities. This chapter also describes an advanced chemical synthesis approach for cyclotides belonging to the bracelet cyclotide subclass, the members of which have proven recalcitrant to chemical synthesis for decades.Chapter 4 focuses on the insecticidal activity of acyclotides. The biological activities of acyclotides have been less extensively studied than those of cyclotides. In particular, there are no reported studies investigating the insecticidal activities of acyclotides, emphasising this as an under-explored research field. In this chapter, four novel acyclotides and the stem extract from R. bengalensis were tested using in vitro and in vivo assays; their activity against the tropical fruit fly was evaluated in the hope of finding a potential bioinsecticide. Acyclotides were characterised using NMR spectroscopy 2D and 3D analysis. Isolated peptides were tested against a panel of insect cell lines, and it was found that they retained the cytotoxic properties affiliated with cyclotide-like peptides. Acyclotides were highly stable in human serum despite their non-cyclic backbone. Disruption ability of ribe 31 toward insect cell membrane was investigated in this study. In summary, the bracelet acyclotide ribe 31 is a promising bioinsecticide due to its ability to reduce the survival rate of D. melanogaster and its potential in vitro results on other insect cells. Chapter 5 focuses on the synthesis and folding of some cyclotides discovered in this work, as well as some grafted bracelet cyclotides. Cyclotides discovered from the Violaceae family in Chapters 2 and 3 (i.e., Mobius cyO22 and bracelet ribe 33) were used as scaffolds for stabilising bioactive epitopes. This is the first study to introduce bracelet cyclotide scaffolds used to stabilise linear epitopes, which could lead to using bracelet scaffolds in drug design applications. Of interest to those seeking to synthesise bracelet cyclotides, this study opens a new era in grafted peptide drug design through the use of hydrazine methodology.Two main outcomes of this PhD were success in identifying a new potential bio-insecticidal compound for crop protection as well as solving one of the major challenges in the bracelet cyclotide production using chemical synthesis, which has defied previous attempts for nearly a decade. These outcomes could enable the future use of cyclotides/acyclotides for crop protection and provide a method to obtain sufficient quantities of bracelet cyclotides for commercial applications. ​​​​​​​