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Cyclotides: Tuning Parameters Toward Their Use in Drug Design
Uppsala University, Disciplinary Domain of Medicine and Pharmacy.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cyclotides are plant proteins with a unique topology, defined as the cyclic cystine knot motif. The motif endows cyclotides with exceptional chemical and biological stability. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. Hence, cyclotides have become potential candidates in the development of peptide-based drugs; either as scaffolds to stabilize susceptible peptide sequences or as drugs by their own right. In this thesis, important parameters that could be inputs toward this development have been tuned.

An extraction protocol that can be extended to industrial scale production of the cyclotides from natural sources was developed; accordingly, a single maceration with hydroalcoholic solutions of medium polarity represented an optimum extraction method.

Moreover, it was shown that investigating the cyclotide content of cyclotide-bearing plants from diverse environments is a promising approach for extending the knowledge of both structural and biological diversity of these proteins. Five novel cyclotides with new sequence diversity were isolated and characterized from a violet that grows on Ethiopian highlands at an altitude of 3400 m.

One of the areas where the cyclotide framework has attracted interest is the development of stable antimicrobial peptides. A stability study was carried out to determine the stability of the cyclotide framework in a cocktail of bacterial proteases and serum where the native forms of tested cyclotides exhibited high stability profile.

Understanding the modes of cyclotide-cell interaction is certainly an important factor for the potential development of cyclotide-based drugs. Cellular studies were carried out using the comet assay and microautoradiography. A bell-shaped dose response curve was obtained for the DNA damaging effect of the cyclotides in the comet assay, which was the first toxicological assay of its kind on this class of proteins. The microautoradiography study revealed that the cyclotides penetrate into the cells even at cytotoxic concentrations. From previous reports, it was known that the cyclotides interact with membranes; the cellular studies in this thesis added to this knowledge by clearly demonstrating that these proteins have multiple modes of action. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 81 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 156
Keyword [en]
cyclotide, Viola abyssinica, Viola odorata, Extraction, Violaceae, vaby, cytotoxic, genotoxic, cell penetrating, microautoradiography, LC-MS, comet assay, cycloviolacin O2
National Category
Medical and Health Sciences Natural Sciences
Research subject
Pharmacognosy
Identifiers
URN: urn:nbn:se:uu:diva-169712ISBN: 978-91-554-8307-4 (print)OAI: oai:DiVA.org:uu-169712DiVA: diva2:510559
Public defence
2012-04-26, B41, Husargatan 3, Biomedical Center, Uppsala University, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2012-04-04 Created: 2012-03-05 Last updated: 2012-04-19Bibliographically approved
List of papers
1. Optimization of Cyclotide Extraction Parameters
Open this publication in new window or tab >>Optimization of Cyclotide Extraction Parameters
(English)Article in journal (Other academic) Submitted
Abstract [en]

Cyclotides are gene-encoded plant mini-proteins that contain a unique circular and cystine knotted amide backbone. Because of that ultra stable scaffold and the ability to harness a wide variety of sequences and biological activities within the scaffold, cyclotides find interesting potential applications for drug discovery and in agriculture. However, some fundamental knowledge is still missing to exploit these plant compounds including finding the optimal process of their extraction from plant material. In the current work, the extraction parameters solvent type, time of extraction, number of re-macerations and the plant material to solvent ratio have been compared using the sweet violet (Viola odorata L.) as a model plant. That species is a well-characterized and rich source of cyclotides that contains prototypic cyclotides with different chemical and physical properties. We found that hydroalcoholic solutions of medium polarity give good yield of the cyclotide cocktail. In conclusion, single maceration with 50 % MeOH for 6 h at a plant material to solvent ratio of 0.5: 10 (g/mL) represents an optimum extraction method.

Keyword
cyclotide, Extraction, cycloviolacin O2, cycloviolacin O18, cycloviolacin O19, varv A, Viola odorata
National Category
Medical and Health Sciences Natural Sciences
Identifiers
urn:nbn:se:uu:diva-171152 (URN)
Available from: 2012-03-15 Created: 2012-03-15 Last updated: 2012-04-19
2. Cyclotides from an Extreme Habitat: Characterization of Cyclic Peptides from Viola abyssinica of the Ethiopian Highlands
Open this publication in new window or tab >>Cyclotides from an Extreme Habitat: Characterization of Cyclic Peptides from Viola abyssinica of the Ethiopian Highlands
2011 (English)In: Journal of natural products (Print), ISSN 0163-3864, E-ISSN 1520-6025, Vol. 74, no 4, 727-731 p.Article in journal (Refereed) Published
Abstract [en]

As part of ongoing explorations of the structural diversity of cyclotides, the cyclotide content of a native violet of the East African highlands, Viola abyssinica (which grows at altitudes up to 3400 m), was studied. Six new cyclotides, vaby A-E (1-5) and varv E (6), were isolated and characterized by employing HPLC and MS techniques and quantitative amino acid analysis. Cyclotides 1-5 were found to have new sequences, and 1-3 have a further novel feature in their sequences, an alanine moiety in loop 2. Two of the cyclotides (1 and 4) also exhibited cytotoxic properties in a flourometric microculture cytotoxicity assay. The findings corroborate the hypothesis that investigating the cyclotide contents of violets growing in diverse environments is a promising approach for extending our knowledge of both the structural and biological diversity of cyclotides.

Place, publisher, year, edition, pages
American chemical society, 2011
Keyword
cyclotide, Viola abyssinica
National Category
Medical and Health Sciences
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-161105 (URN)10.1021/np100790f (DOI)000289742300029 ()
Available from: 2011-11-07 Created: 2011-11-07 Last updated: 2017-12-08Bibliographically approved
3. Cyclotide stability against Pseudomonas aeruginosa and Staphylococcus aureus secreted proteases
Open this publication in new window or tab >>Cyclotide stability against Pseudomonas aeruginosa and Staphylococcus aureus secreted proteases
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Abstract [en]

Production and release of proteases is among the strategies by which pathogenic bacteria increase their virulence. Moreover, these bacteria are also known to produce a cocktail of enzymes to degrade and inhibit antimicrobial peptides. Cyclotides, on the other hand, are circular plant mini-proteins having a unique cyclic topology (termed as the cyclic cystine knot, CCK) which endows them with exceptional stability. Their ultra-stable framework has attracted interest in drug design as a scaffold to otherwise susceptible drugs including antibacterial peptides. We have determined the stability of three peptides with the CCK motif, i.e., the cyclotides cycloviolacin O2, kalata B1 and MCoTI-II in elastase and aureolysin and also in the supernatant cultures of P. aeruginosa and S. aureus. The cyclotides have resisted enzymatic degradation both from the specific proteases and the bacterial cultures, with MCoTI-II being the least stable of the three. The cyclotides find application in drug engineering as their stable framework can be used as a scaffold or as a template for unstable drugs. The findings of the current study further support this idea; the cyclotide structure can be utilized as a novel strategy in enhancing the stability of antibacterial peptides.

Keyword
cyclotides, cycloviolacin O2, kalata B1, MCoTI-II, Pseudomonas aeruginosa, Staphylococcus aureus, elastase, aureolysin, Viola odorata, Oldeanlandia affinis, Momordica cochinchinensis, protease, serum
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-171141 (URN)
Available from: 2012-03-16 Created: 2012-03-15 Last updated: 2016-10-05
4. Genotoxicity and Cellular Uptake of Cyclotides: Evidence for Multiple Mode of Action
Open this publication in new window or tab >>Genotoxicity and Cellular Uptake of Cyclotides: Evidence for Multiple Mode of Action
2012 (English)In: Mutation research. Genetic toxicology and environmental mutagenesis, ISSN 1383-5718, E-ISSN 1879-3592, Vol. 747, no 2, 176-181 p.Article in journal (Refereed) Published
Abstract [en]

Cyclotides are a family of ultra stable, head-to-tail cyclic plant mini-proteins with each member comprising about 30 amino acid residues. Their stability is associated with the unique structural topology where the cyclic backbone and two disulfide bonds make up an embedded ring which is knotted by a third disulfide bond. The cyclotides find potential applications in drug industry as a drug scaffolds for unstable drugs and also as medicinal agents due to the wide range of inherent pharmacological activities they possess. However, there is a lack of fundamental toxicological studies on these classes of compounds. The current study determined a possible DNA damaging effect of three cyclotides, i.e., cycloviolacin O2, vaby D, and kalata B1 in human lymphoma cells using the alkaline version of the comet assay. The three cyclotides induced massive DNA fragmentation at lethal concentrations. At a sublethal concentration, cycloviolacin O2 and vaby D gave a bell shaped dose-response curve for their DNA-damaging effect. Kalata B1 caused no significant DNA damage at sub cytotoxic concentrations. Single cell microautoradiography was carried out on tritium labeled cycloviolacin O2 in order to understand the mechanism behind the dose-response curve. The results revealed that the peptide is taken up into the cell, at both cytotoxic and at low concentrations. Most biological effects of the cyclotides have been taken to follow from the disruption of cell membranes, but even if the intracellular mechanisms/targets still remain unknown, the current study has unequivocally demonstrated that these compounds also must have other dose-dependent modes of action. 

Keyword
cyclotide, cycloviolacin O2, vaby D, kalata B1, comet assay, DNA damage, microautoradiography, mode of action, viola odorata, viola abyssinica, oldenlandia affinis, cell penetrating, radiolabeling
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-171150 (URN)10.1016/j.mrgentox.2012.05.006 (DOI)000307148600003 ()
Available from: 2012-03-15 Created: 2012-03-15 Last updated: 2017-12-07Bibliographically approved

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