Structure-function relationships of disulfide-rich peptides

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

A C-Terminal Fragment of Chlorotoxin Retains Bioactivity and Inhibits Cell Migration

Chlorotoxin was originally isolated from the venom of the Israeli scorpion Leiurus quinquestriatus, and has potential as a tumor imaging agent based on its selective binding to tumor cells. Several targets have been suggested for chlorotoxin including voltage-gated chloride channels, and it has been shown to have anti-angiogenic activity and inhibit cell migration. The structure of chlorotoxin is stabilized by four disulfide bonds and contains β-sheet and helical structure. Interestingly, the reduced form has previously been shown to inhibit cell migration to the same extent as the wild type, but structural analysis indicates that the reduced form of the peptide does not maintain the native secondary structure and appears unstructured in solution. This lack of structure suggests that a short stretch of amino acids might be responsible for the bioactivity. To explore this hypothesis, we have synthesized fragments of chlorotoxin without disulfide bonds. As expected for such small peptides, NMR analysis indicated that the peptides were unstructured in solution. However, the peptide corresponding to the eight C-terminal residues inhibited cell migration, in contrast to the other fragments. Our results suggest that the C-terminal region plays a critical role in the bioactivity of chlorotoxin

Folding of granulin domains

Granulins are a family of protein growth factors that are involved in a range of biological functions, including wound repair, inflammation, and tumor growth. They are often expressed as part of large precursor proteins containing multiple granulin domains. Individual granulin domains are characterized by a conserved arrangement of 12 cysteine residues that form six disulfide bonds. Despite the conservation of the cysteine residues, there is significant sequence variation between granulins from different species. The initial structure determined for this family indicated the presence of a well‐defined structure with a laddered arrangement of the six disulfide bonds and a β‐hairpin stack. However, subsequent studies have shown the structure‐function relationships of granulins are quite complex. Recent studies have indicated some granulins might have potential as wound healing agents, and studies aimed at understanding the structure‐function relationships of this family are likely to enhance this potential in drug design. This review provides an overview of the structure‐based studies of granulins, including the folding of truncated peptides derived from granulins from different species

Structural variants of a liver fluke derived granulin peptide potently stimulate wound healing

Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver fluke Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in recombinant form at high yield, impeding its utility as a drug lead. Previously we reported that a truncated analogue (Ov-GRN12–35_3s) promotes healing of cutaneous wounds in mice. NMR analysis of this analogue indicates the presence of multiple conformations, most likely as a result of proline cis/trans isomerization. To further investigate whether the proline residues are involved in adopting the multiple confirmations, we have synthesized analogues involving mutation of the proline residues. We have shown that the proline residues have a significant influence on the structure, activity, and folding of Ov-GRN12–35_3s. These results provide insight into improving the oxidative folding yield and bioactivity of Ov-GRN12–35_3s and might facilitate the development of a novel wound healing agent

Development of a Potent Wound Healing Agent Based on the Liver Fluke Granulin Structural Fold

Granulins are a family of protein growth factors that are involved in cell proliferation. An orthologue of granulin from the human parasitic liver fluke Opisthorchis viverrini, known as <i>Ov</i>-GRN-1, induces angiogenesis and accelerates wound repair. Recombinant <i>Ov</i>-GRN-1 production is complex and poses an obstacle for clinical development. To identify the bioactive region(s) of <i>Ov</i>-GRN-1, four truncated N-terminal analogues were synthesized and characterized structurally using NMR spectroscopy. Peptides that contained only two native disulfide bonds lack the characteristic granulin β-hairpin structure. Remarkably, the introduction of a non-native disulfide bond was critical for formation of β-hairpin structure. Despite this structural difference, both two and three disulfide-bonded peptides drove proliferation of a human cholangiocyte cell line and demonstrated potent wound healing in mice. Peptides derived from <i>Ov</i>-GRN-1 are leads for novel wound healing therapeutics, as they are likely less immunogenic than the full-length protein and more convenient to produce