Engineering and discovery of bioactive disulfide-rich peptides

Chan, Lai Yue (2012). Engineering and discovery of bioactive disulfide-rich peptides PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Chan, Lai Yue
Thesis Title Engineering and discovery of bioactive disulfide-rich peptides
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2012-03
Thesis type PhD Thesis
Supervisor Norelle L. Daly
David J. Craik
Total pages 237
Total colour pages 51
Total black and white pages 186
Language eng
Subjects 060101 Analytical Biochemistry
060112 Structural Biology (incl. Macromolecular Modelling)
060199 Biochemistry and Cell Biology not elsewhere classified
Formatted abstract
Peptides are an exciting class of compounds for the development of drug leads that can potentially target a range of human diseases. The advantages of peptides as drug leads include high specificity towards targets, low molecular weight and limited toxicity. However, naturally-occurring peptides have encountered difficulties in pre-clinical trials as a result of issues with poor oral absorption, membrane permeability, rapid enzymatic degradation, and instability. Therefore to increase the potency and stability of peptides, chemical modifications are required and several approaches have been applied including the use of cyclization, N-methylation and the addition of lipophilic particles. In addition, disulfide-rich peptides are currently being explored for drug leads because of the stability that can be conferred by the tightly folded conformations often present in these peptides.

One of the largest classes of disulfide-rich peptides is the cyclotides, naturally occurring plant peptides from the Violaceae (Violet), Rubiaceae (coffee), Cucurbitaceae (cucumber), and Fabaceae (pea) plant families. There are more than 210 cyclotides characterized. Although these plant families have been studied extensively, limited study has focused on the Cucurbitaceae plant family. In particular, only three cyclic peptides were isolated from Momordica cochinchinensis prior to the current study.

The cyclotides have approximately 30 residues, including six cysteine residues that form three disulfide bonds. The three disulfide bonds form a cystine knot motif that is coupled with a cyclic backbone to form the cyclic cystine knot motif (CCK). The CCK motif results in the cyclotides being extremely stable and has led to the hypothesis they will be useful as scaffolds for stabilizing bioactive sequences. Other naturally occurring cyclic peptides have also been recently discovered, including SFTI-1, a sunflower trypsin inhibitor with a single disulfide bond. SFTI-1, although smaller than the cyclotides, also has potential as a scaffold in drug design. The ultra stable structural motif of the cyclotides has also inspired the use of backbone cyclization of bioactive peptides to confer stability.

The major aim of this thesis was to explore the diversity and potential applications of disulfide-rich peptides. In particular, the diversity of peptides present in the seeds of M. cochinchinensis was examined in addition to the use of cyclic, disulfide-rich peptide scaffolds as templates for “grafting” bioactive sequences. The scaffolds were based on the naturally occurring cyclotides and a sunflower trypsin inhibitor. In addition to stabilizing small bioactive sequences via grafting onto cyclic peptide scaffolds, an alternative approach to stabilizing bioactive peptides via backbone cyclization of a naturally occurring peptide was explored.

Chapter 1 describes the significance of using nature as a source for the discovery of new disulfide-rich peptides and how they can be used as tools in drug design for targeting a wide range of therapeutic applications.

Chapter 2 is a comprehensive guide to the materials and methods used for conducting all experiments in this thesis. This includes extraction and isolation of peptides from the M. cochinchinensis seeds, proteomics, peptide synthesis, a range of in-house biological assays and structure determination using NMR.

Chapter 3 describes the discovery of novel linear disulfide-rich peptides and new trypsin inhibitors from M. cochinchinensis seeds. M. cochinchinensis is a plant from the Cucurbitaceae family that is commonly found in Vietnam. Vietnamese people have used the flesh of the fruit for flavoring rice and previous studies have found that this genus contains anti-cancer compounds. In the current study analysis of the seed extract has resulted in six new linear disulfide-rich and five new trypsin inhibitor peptides. These studies have provided insight in the understanding of the different classes of peptides that occur in the seeds, and the newly identified trypsin inhibitors have aided in the discovery of the genes for the precursor proteins encoding trypsin inhibitors in M. cochinchinensis.

Chapter 4 describes the discovery of eight new cyclotides (Viphi A-H) along with eight known cyclotides from Viola philippica, an ornamental flower that is native to China, North Korea and Japan. This plant is also widely used as a traditional medicine. Novel cyclotides were found in this plant with only a few amino acids differences from previously known sequences. This has resulted in an increased knowledge on the sequence variation of cyclotides in the Violaceae plant family and also expanded the number of species known to contain cyclotides. In addition, these peptides are cytotoxic against several cancer cell lines. With the sequence variation and cytotoxic activity data gained from this study, this study provides additional information on the structure–activity relationships of the newly characterized cyclotides.

Chapter 5 reports the design and engineering of cyclic peptide frameworks as scaffolds for incorporating bioactive epitopes. MCoTI-II (M. cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1) are the cyclic peptide scaffolds used in this study and three angiogenic epitopes were incorporated into the loop 6 of MCoTI-II and the trypsin inhibitor loop of SFTI-1. SFTI-OPN was identified as the most potent grafted peptide and was shown to have nanomolar angiogenic activity with a greater stability than its original linear peptide. This study provides compelling evidence that disulfide-rich cyclic peptides are useful as templates for drug design.

Chapter 6 describes the cyclization and structure determination of gomesin, a potent antimicrobial peptide isolated from hemocytes of the spider Acanthoscurria gomesiana. Modification of the native linear form of gomesin peptide was done via cyclization. Interestingly, the cyclic form of gomesin was more stable than the native form and has improved potency for a range of biological activities. Therefore, this study suggests that cyclization can be utilized on a diverse range of linear peptides for improving stability and with possibility of the enhancement of bioactivity.

In summary, the results from this thesis have expanded the knowledge of the diversity of disulfide-rich peptides from plants and helped provide insight into their biosynthetic origin. In addition, the use of backbone cyclization for stabilizing bioactive peptides was explored highlighted by the development of an angiogenic compound with nanomolar activity. The findings in this thesis thus provide support for the potential of disulfide-rich peptides as templates for drug design and targeting a wide range of therapeutic applications.
Keyword Cyclotides
Linear cystine knot peptides
Momordica cochinchinensis
Trypsin inhibitors
Grafting studies
Pro-angiogenesis
Gomesin peptide
Sequencing
Structures
Bioassays
Additional Notes 22,28,31,33,34,36,40,49,51,52,56,58,61,63-65,69,73,78,80-82,93,98,129,131-134,138,140-144,146-149,155,156,158,162,172,176,181,186,187,190,194,232

 
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Created: Tue, 03 Jul 2012, 04:43:03 EST by Ms Lai Chan on behalf of Scholarly Communication and Digitisation Service