Design and discovery of cyclic peptides with applications in drug development

Muharrem Akcan (2011). Design and discovery of cyclic peptides with applications in drug development PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Muharrem Akcan
Thesis Title Design and discovery of cyclic peptides with applications in drug development
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2011-04
Thesis type PhD Thesis
Total pages 184
Total colour pages 47
Total black and white pages 137
Subjects 11 Medical and Health Sciences
Abstract/Summary In recent years, the use of peptides in drug design and development applications has gained increasing prominence. Cyclotides are plant-derived disulfide-rich peptides with high stability, and their cyclic cystine knot motif makes them very useful scaffolds for protein engineering purposes. Conotoxins are another class of disulfide rich peptides. They are obtained from the venoms of marine cone snails and some of them have similar topologies to cyclotides. Because of their highly selective and potent activities, several conotoxins are being used to develop novel drugs. However, being peptides their susceptibility to proteolysis potentially limits their use as drugs. It is proposed in this thesis that chimeric cyclotide-conotoxin peptides might have enhanced resistance to enzymatic digestions. Thus, the overall aim of this thesis is to develop new cyclic peptides with potential therapeutic applications. The thesis comprises seven chapters that describe a range of studies on different aspects of the challenges of using natural peptides to develop drugs. Chapter 1 introduces the peptides studied in the thesis and Chapter 2 describes the materials and methods used throughout the thesis. The remaining chapters describe the experimental findings. Peptide backbone cyclization is a widely used approach to improve the activity and stability of small peptides, but until recently had not been applied to peptides with multiple disulfide bonds. In Chapter 3, the backbone cyclization of ω-conotoxins MVIIA and CVID and the P-superfamily conotoxins Gm9a and Bru9a was studied by joining the N- and C-termini with short peptide linkers using intramolecular native chemical ligation chemistry. The cyclised derivatives of MVIIA have potent activity at N-type calcium channels, similar to the native peptide, highlighting the potential of this approach in developing active ω-conotoxin analogues. Backbone cyclization of Gm9a and Bru9a resulted in correctly folded cyclic peptides with high serum stability. x In Chapter 4, the aim was to re-engineering of a scorpion venom peptide, chlorotoxin (CTX), by substituting Lys15 and Lys23 with Ala or Arg to produce a mono-labeled peptide for regulatory purposes and to synthesize a cyclic version with high stability. The most remarkable feature of CTX is that it selectively binds to glioma, a type of tumor cells in the brain. An optical imaging contrast agent called “Tumor Paint” is being developed to enable surgeons to distinguish cancer cells from adjacent normal tissue. Tumour paint is a bioconjugated form of CTX with a near infrared fluorescent (NIRF) molecule Cy5.5. The studies described in this chapter were aimed to improve the stability and labeling efficiency of conjugated CTX. Encouragingly, the results showed that the bioconjugates of the engineered peptide are functionally equivalent to the native CTX:Cy.5.5 and backbone cyclization resulted in a more stable peptide. In Chapter 5, an alanine scanning mutagenesis of CTX was carried out to determine which residues are important for binding to tumor cells. All alanine mutants were synthesized by Fmoc solid phase peptide chemistry and tested for binding to tumors in a ND2:SmoA1 medulloplastoma mouse model after tail-vein injection. Biophotonic images of mice brains were obtained with a Xenogen near infrared imaging system. Twenty six alanine mutants were tested, with six showing an increase in tumor targeting. The results of this study will be used in the design and synthesis of more active mutants and cyclization will be used to increase the stability of the active analogues. In a complementary approach to the backbone cyclization of peptides described in Chapters 3-5, Chapter 6 explores a grafting approach to transfer the bioactive residues of a conotoxin onto a more stable cyclotide framework. The second loop of MVIA, containing the most active residue Tyr13, was grafted onto the second loop of kalata B1. The grafted peptide has a native fold and higher serum stability than MVIIA. However, no inhibition of N-type VGCCs was observed. xi In summary, this thesis has provided fundamental new insights into the backbone cyclization of disulfide-rich peptides and grafting of the bioactive epitopes to produce drug leads with enhanced stability. The findings from the structure activity studies have the potential to facilitate the development of stable and more effective molecular imaging agents. Overall, the study has provided new knowledge on the tolerance of disulfide-rich peptide scaffolds to chemical reengineering.
Keyword Cyclotides, conotoxins, chlorotoxin, structure-activity relationships, backbone cyclization
NMR Spectroscopy
peptides, cancer, molecular imaging
Additional Notes 23, 26, 28,29, 31, 34-36, 39, 40, 42, 55, 74, 78, 83, 87, 92-98, 100, 101, 104, 105, 112, 115, 118-120, 123, 132, 135, 137, 141, 143, 145, 152, 156, 157, 159, 165, 166, 168,177

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Created: Thu, 27 Oct 2011, 11:25:43 EST by Mr Muharrem Akcan on behalf of Library - Information Access Service