The development of the cyclotide MCoTI-II as a molecular engineering framework in drug design

Kathryn Greenwood (2008). The development of the cyclotide MCoTI-II as a molecular engineering framework in drug design PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Kathryn Greenwood
Thesis Title The development of the cyclotide MCoTI-II as a molecular engineering framework in drug design
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2008-10
Thesis type PhD Thesis
Supervisor Professor David Craik
Dr Norelle Daly
Total pages 261
Total colour pages 26
Total black and white pages 235
Subjects 06 Biological Sciences
Abstract/Summary This thesis focuses on a member of the cyclotide family, MCoTI-II, and its development as a peptide drug delivery vehicle. The cyclotides are a large family of mini-proteins isolated from common plants and feature a cyclic peptide backbone and a disulfide rich core. More than 100 cyclotide sequences are currently known. They fall into three subfamilies and recent predictions suggest that the family may comprise >50,000 members. The existence of these circular peptides challenges the conventional understanding that proteins have distinct N and C termini and suggests that a cyclic backbone may provide these mini-proteins with an advantage over linear proteins. The serendipitous discovery of the prototypic cyclotide kalata B1 as the active ingredient in tea made from the leaves of the Kalata-Kalata plant in the African Congo provided the first evidence of the exceptional stability of the cyclotides. Kalata B1 endured boiling and then resisted degradation from biological challenges in vivo before eliciting a uterotonic effect. The stability and proposed biocompatibility of kalata B1 are promising features for the development of cyclotides in peptide based drug design. The structural and biological characterisation of cyclotides gained momentum in the mid 1990s and led to a prediction that they would make suitable drug scaffolds. The early discoveries of cyclotides were made from studies screening for a range of biological activities, which highlighted the fact that cyclotides could deliver potent activities that potentially could be harnessed in drug development. These compact, stable peptides were found to have a well defined structure in which several loops of amino acids extend from a tightly bound core, providing sites for incorporation of pharmacophoric sequences. Alone, bioactive peptides may not meet the requirements of stability and bioavailability but their incorporation into a cyclotide scaffold may stabilise their interactions with the environment. The most recently discovered cyclotide subfamily, the trypsin inhibitor cyclotides, has two members MCoTI-I and MCoTI-II, which are the only cyclotides to share a high sequence homology with a family of acyclic peptides. This thesis focuses on the development of the most highly expressed trypsin inhibitor cyclotide, MCoTI-II, as a scaffold to deliver short bioactive peptide sequences to biological targets. MCoTI-II was favoured amongst the cyclotides as a drug scaffold firstly because seed extracts from Momordica cochinchinensis, from which MCoTI-II is derived, have a history of use in traditional Chinese medicine, suggesting that MCoTI-II is biocompatible. Secondly MCoTI-II shares almost no sequence homology with the other cyclotide subfamilies, indicating a propensity for sequence change in achieving the global fold common to the cyclotide family. This thesis reports on investigations made into properties of MCoTI-II that could support its use as a molecular scaffold and on the proof of principle studies conducted for peptide drug delivery in vitro. Chapter 1 introduces the attributes of cyclotides, focussing specifically on MCoTI-II. Chapter 2 describes the materials and methods used throughout this project. Chapter 3 reports on the passage of labelled MCoTI-II into cells, followed by an investigation of the mechanism by which MCoTI-II was internalized. Chapters 4-6 describe the extent to which the native MCoTI-II sequence could be modified, by grafting in a range of sequences encoding different biological activities into different peptide loops. Specifically, in Chapter 4 the grafting studies commenced with manipulating the innate trypsin inhibitory activity of MCoTI-II. Loops 1 and 5 of MCoTI-II were replaced with the inhibitory loop from SFTI-1 and the grafting success was measured by the potency of trypsin inhibition. In Chapters 5 and 6, the grafted sequences were derived from proteins which interact with intracellular pathways of therapeutic significance. A 10 residue sequence from β-arrestin2 protein and an 11 residue sequence from the NEMO binding domain of the IKKα/β complex respectively were grafted into loop 6 of MCoTI-II and their intracellular activity assessed. The folded peptides were then structurally characterised to determine which secondary structural features the grafted regions had acquired that may explain either their activity or inactivity. The grafting studies were concluded by investigating the toxicity and stability of the grafted MCoTI-II peptides that have the potential to limit their effectiveness as peptide drugs in vivo. In the final chapter, Chapter 7, crystals of MCoTI-II bound to its target trypsin were grown with the aim to elucidate the structure and define the geometry of MCoTI-II in its active conformation. Overall, in this thesis project, substantial progress was made into the development of MCoTI-II as a peptide drug delivery vehicle. MCoTI-II proved a suitable scaffold for accommodating novel peptide sequences and was able to deliver one graft to the intracellular environment of cells of the immune system to produce significant pro-inflammatory responses. This work provides a basis for further investigations into the relationship between structural features and biological function for the design and eventual adoption of MCoTI-II as a peptide drug scaffold.
Keyword Cyclotide
Drug Design
cell penetrating peptide
Additional Notes 27, 30-31, 35, 40, 45, 55, 68, 92, 97-100, 112, 114, 118-119, 121, 123, 139, 142, 145, 164, 166, 171, 186

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Created: Wed, 16 Sep 2009, 19:24:09 EST by Ms Kathryn Greenwood on behalf of Library - Information Access Service