Peptides are considered as drugs of the future because of their advantageous features of high specificity and low toxicity. However, the complete therapeutic potential of peptides has not yet been realized because of the in vivo instability displayed by most potential peptides. In this thesis, two naturally derived cyclic peptides, cyclotides and sunflower trypsin inhibitor 1 (SFTI-1), were utilized to impart stability to linear bioactive epitopes and enhance their therapeutic potential in a biological environment. Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclised backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Sunflower typsin inhibitor 1 (SFTI-1) is a stable cyclic peptide containing a single disulfide bond. Taking advantage of these stable cyclic peptide frameworks, novel drug leads to inhibit/stimulate angiogenesis were developed by using the approach of ‘epitope grafting’ in which linear epitopes were grafted onto the cyclic peptide frameworks. Angiogenesis is a physiological condition that is unregulated in the progression of many diseases, including cancers and cardiovascular diseases. Thus the drug leads designed in the current project have potential therapeutic applications to combat cancers and cardiovascular diseases. To fully exploit cyclotides as drug scaffolds, it is imperative to understand their folding. Two main subfamilies, referred to as the Möbius and bracelet cyclotides have been identified and interestingly, they require dramatically different in vitro folding conditions to achieve formation of the conserved cyclic cystine knot motif. To determine the underlying structural elements that influence cyclotide folding, the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1 was examined in this thesis. The pathways of folding of the two cyclotide subfamilies were found to be different and primarily dictated by specific residues harboured within inter-cysteine loops 2 and 6. Two changes in these loops, an amino acid substitution in loop 2 and an amino acid addition in loop 6 enabled the folding of cycloviolacin O1 under conditions where folding does not occur in vitro for the native peptide. Thus, the study identified key residues that are not in close proximity in the primary sequence or three-dimensional structure which assist folding in cyclotides. A key intermediate species in the folding pathway was isolated and characterised, and found to contain a native-like hairpin structure that appears to be a nucleation locus early in the folding process. The intermediate does not have native disulfide connectivities, but disulfide shuffling processes ultimately lead to a rearrangement to the native form. Overall these mechanistic findings on the folding of cyclotides are potentially valuable for protein engineering applications that utilize cystine-rich peptides as scaffolds in the design of new drug leads. The current study has also enabled the extention of the grafting studies to the bracelet cyclotide subfamily, which was intractable to grafting prior to this work. Cyclotides are gene encoded macrocyclic proteins and another way to exploit their potential as drug scaffolds, would be to develop combinatorial cyclotide libraries. The most efficient way to generate engineered cyclotides would be via recombinant expression, which currently remains unsuccessful, partly due to lack of understanding of the mechanism of cyclotide backbone cyclization. Understanding how the cyclotide precursor folds may provide clues to how cyliclization occurs. A conserved region known as the N-terminal repeat (NTR) region in the cyclotide precursor has been speculated to play an important role in precursor folding. In this thesis, the function of the NTR in the folding of the cyclotide precursor in vitro was examined via the design of a series of constructs for the precursor protein for the prototypic kalata B1 cyclotide, with incremental additions of the NTR region. Analysis of the constructs by NMR spectroscopy for evidence of secondary structure revealed that the NTR does not assist folding of the cyclotide precursor in vitro. Using diffusion NMR, the unstructured nature of the constructs was localized to the NTR region. In a complementary study, structural analysis of the full length cyclotide precursor was carried out by expressing the precursor gene for kalata B1 in a bacterial expression system. The full-length precursor was found to be unstructured in solution despite approximately half of the precursor comprising the mature domain and NTR, both of which are structured in isolation. The unstructured nature of the cyclotide precursor suggested that a different environment, or indeed interaction of the NTR with a particular enzyme involved in processing, is necessary for it to adopt a well-defined conformation and allow processing to produce the mature circular protein. The information that NTR alone may not assist folding of the cyclotide precursors has provided new impetus to examine the role of other potential folding auxiliaries such as protein disulfide isomerase in cyclotide folding and has indirectly advanced the production of cyclotides via transgenic means. In summary, this thesis has provided a fundamental insight into the folding of cyclotides, both when expressed as part of a precursor protein and in isolation via solid phase chemical synthesis, and has exploited the potential of cyclic peptide scaffolds in drug design applications.
| Identifer | oai:union.ndltd.org:ADTP/286650 |
| Creators | Sunithi Gunasekera |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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