Structure-function relationships of disulfide-rich peptides

Dastpeyman, Mohadeseh (2019) Structure-function relationships of disulfide-rich peptides. PhD thesis, James Cook University.

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This thesis focuses on applying the approach of downsizing disulfide-rich peptides for the development of potential drug leads, and providing insight into important structural features for bioactivity. Disulfide-rich peptides are widely distributed in nature and several hold promise for the development of novel therapeutics and diagnostic agents. This thesis explores the structure-function relationships of two disulfide rich peptides; the scorpion venom peptide chlorotoxin, and the parasitic liver fluke protein Ov-GRN-1.

Chapter 2 focuses on chlorotoxin, a potent tumour-imaging agent that selectively binds to tumour cells. Interestingly, it has been shown that chlorotoxin can have biological effects without disulfide bonds stabilising the native fold. This finding suggests that smaller regions, the inter-cysteine loops, might be responsible for the bioactivity of chlorotoxin. To explore this hypothesis, four small fragments of chlorotoxin were chemically synthesised using Fmoc solid-phase peptide synthesis method. As expected for such small peptides, NMR analysis indicated that the peptides were unstructured in solution. The bioactivity of the fragments was assessed by cell migration and invasion assays, alongside cell surface binding and internalization assays. Our results indicate that a small, unstructured fragment from the C-terminal region plays a critical role in the bioactivity of chlorotoxin. This is an unusual finding as structure is often critical for bioactivity in disulfide-rich peptides.

The remaining experimental chapters focus on the characterization of Ov-GRN-1, a protein isolated from the excretory/secretory (ES) products of the carcinogenic liver fluke Opisthorchis viverrini. Ov-GRN-1 belongs to the granulin family, which are growth factor proteins with a wide range of functions mainly involved in cell modulation. A recombinant version of Ov-GRN-1 causes proliferation of host (human) cells and can accelerate the repair of wounds in animals. However, recombinant expression of Ov-GRN-1 is challenging and leads to a low product yield, impeding its utility as a drug lead.

Chapter 3 focuses on the design, structure and functional analysis of minimized analogues from the N-terminal region of Ov-GRN-1. A series of analogues from the N-terminal region of Ov-GRN-1 were chemically synthesised by solid-phase peptide synthesis, oxidized by air oxidation, purified by HPLC and characterized by mass spectroscopy. The structure of peptides was studied by NMR spectroscopy and the 3D structure was calculated by CYANA and visualized by MolMol. Cell proliferation and wound healing activity were assessed by an in vitro xCELLigence cell proliferation assay and an in vivo mouse-wounding model, respectively. The structural characterization of the Ov-GRN-1 N-terminal truncated peptides indicated that the introduction of a non-native disulfide bond appears to stabilize the fold and allow the peptide to form a β- hairpin structure. This analogue, which is called Ov-GRN₁₂₋₃₅_₃ₛ, induced cell proliferation and in vivo wound healing with similar potency to the full-length Ov-GRN-1.

NMR analysis of Ov-GRN₁₂₋₃₅_₃ₛ indicated the presence of multiple conformations, most likely from proline cis/trans isomerisation. In Chapter 4, a series of analogues involving mutation of the proline residues was synthesised to investigate the role of proline residues in adopting the multiple confirmations by Ov-GRN₁₂₋₃₅_₃ₛ. Utilising the same techniques and methods used in Chapter 3, proline residues were shown to have a significant influence on the structure, activity and folding of Ov-GRN₁₂₋₃₅_₃ₛ. The results obtained for this chapter led to the development of a more potent analogue, GRN(P₄A), with improved folding yield.

Chapter 5 further explores the structure-function relationships of granulin peptides through analysis of the N-terminal region of human granulin A, as well as the C-terminal region of Ov-GRN-1. The former peptide was designed to determine if the non-native disulfide bond present in Ov-GRN₁₂₋₃₅_₃ₛ could also be accommodated in a granulin from another species, whereas the latter represents the first truncation study of the C-terminal region of a granulin peptide. The same techniques and methods as Chapter 4 were used to synthesise and characterise the analogues. Bioactivity of analogues were assessed using an in vitro xCELLigence cell proliferation assay. The results indicated that accommodation of a non-native disuflide bond might be a general phenomenon in the granulin family, as the N-terminal half of the human granulin A protein also folds independently with three disulfide bonds, despite significant sequence differences to the Ov-GRN-1 peptide. We also show for the first time that the equivalent C-terminal half of Ov-GRN-1 does not fold into a well-defined structure, but still displays cell proliferative activity. Our results indicate that well-defined structures are not critical for granulin bioactivity.

In summary, the results highlight the potential of the "downsizing" approach for elucidating bioactive sequences, providing insight into folding processes and the development of novel drug leads. One of the major findings from this thesis is the development of a truncated form of Ov-GRN-1 that is likely to have lower immunogenicity than the full-length protein because of its smaller size, is significantly easier to produce and more potent in a mouse wound healing assay. These features make it a more viable drug lead for wound healing applications, and it is currently being considered for commercial development.

Item ID: 62919
Item Type: Thesis (PhD)
Keywords: chlorotoxin, disulfide-rich peptide, granulin, invasion, metastasis, migration, NMR spectroscopy, nuclear magnetic resonance spectroscopy, oxidative folding, tumor-imaging agent
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Copyright Information: Copyright © 2019 Mohadeseh Dastpeyman.
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Four publications arising from this thesis are stored in ResearchOnline@JCU, at the time of processing. Please see the Related URLs field. The publications are:

Chapter 1: Dastpeyman, Mohadeseh, Smout, Michael J., Wilson, David, Loukas, Alex, and Daly, Norelle L. (2018) Folding of granulin domains. Peptide Science, 110 (3). e24062.

Chapter 2: Dastpeyman, Mohadeseh, Giacomin, Paul, Wilson, David, Nolan, Matthew J., Bansal, Paramjit S., and Daly, Norelle L. (2019) A C-terminal fragment of chlorotoxin retains bioactivity and inhibits cell migration. Frontiers in Pharmacology, 10. 250.

Chapter 3: Bansal, Paramjit S., Smout, Michael J., Wilson, David, Cobos Caceres, Claudia, Dastpeyman, Mohadeseh, Sotillo, Javier, Seifert, Julia, Brindley, Paul J., Loukas, Alex, and Daly, Norelle L. (2017) Development of a potent wound healing agent based on the liver fluke granulin structural fold. Journal of Medicinal Chemistry, 60 (10). pp. 4258-4266.

Chapter 4: Dastpeyman, Mohadeseh, Bansal, Paramjit S., Wilson, David, Sotillo, Javier, Brindley, Paul J., Loukas, Alex, Smout, Michael J., and Daly, Norelle L. (2018) Structural variants of a liver fluke derived granulin peptide potently stimulate wound healing. Journal of Medicinal Chemistry, 61 (19). pp. 8746-8753.

Date Deposited: 27 Apr 2020 04:51
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110106 Medical Biochemistry: Proteins and Peptides (incl Medical Proteomics) @ 35%
11 MEDICAL AND HEALTH SCIENCES > 1115 Pharmacology and Pharmaceutical Sciences > 111502 Clinical Pharmacology and Therapeutics @ 30%
03 CHEMICAL SCIENCES > 0304 Medicinal and Biomolecular Chemistry > 030404 Cheminformatics and Quantitative Structure-Activity Relationships @ 35%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970111 Expanding Knowledge in the Medical and Health Sciences @ 50%
86 MANUFACTURING > 8608 Human Pharmaceutical Products > 860803 Human Pharmaceutical Treatments (e.g. Antibiotics) @ 50%
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