| 1. |
- Burman, Robert, 1979-, et al.
(författare)
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Evaluation of toxicity and anti-tumour activity of cycloviolacin O2 in mice.
- 2010
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 94:5, s. 626-634
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Tidskriftsartikel (refereegranskat)abstract
- Cycloviolacin O2 is a small cyclic cysteine-rich protein belonging to the group of plant proteins called cyclotides. This cyclotide has been previously shown to exert cytotoxic activity against a variety of human tumor cell lines as well as primary cultures of human tumor cells in vitro. This study is the first evaluation of its tolerability and antitumor activity in vivo. Maximal-tolerated doses were estimated to 1.5 mg/kg for single intravenous (i.v.) dosing and 0.5 mg/kg for daily repeated dosing, respectively. Two different in vivo methods were used: the hollow fiber method with single dosing (i.v. 1.0 mg/kg) and traditional xenografts with repeated dosing over 2 weeks (i.v. 0.5 mg/kg daily, 5 days a week). The human tumor cell lines used displayed dose-dependent in vitro sensitivity (including growth in hollow fibers to confirm passage of cycloviolacin O2 through the polyvinylidene fluoride fibers), with IC50 values in the micromolar range. Despite this sensitivity in vitro, no significant antitumor effects were detected in vivo, neither with single dosing in the hollow fiber method nor with repeated dosing in xenografts. In summary, the results indicate that antitumor effects are minor or absent at tolerable (sublethal) doses, and cycloviolacin O2 has a very abrupt in vivo toxicity profile, with lethality after single injection at 2 mg/kg, but no signs of discomfort to the animals at 1.5 mg/kg. Repeated dosing of 1 mg/kg gave a local-inflammatory reaction at the site of injection after 2–3 days; lower doses were without complications.
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| 2. |
- Burman, Robert, 1979-, et al.
(författare)
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Cytotoxic potency of small macrocyclic knot proteins : Structure-activity and mechanistic studies of native and chemically modified cyclotides
- 2011
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 9:11, s. 4306-4314
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Tidskriftsartikel (refereegranskat)abstract
- The cyclotides are a family of circular and knotted proteins of natural origin with extreme enzymatic and thermal stability and active in a wide range of biological activities make them promising tools for pharmaceutical and crop-protection applications. The cyclotides are divided into two subfamilies depending on the presence (Möbius) or absence (bracelet) of a cis-Pro peptide bond. In the current work we report a series of experiments to give further insight into the structure activity relationship of cyclotides in general, and the differences between subfamilies and the role of their hydrophobic surface in particular. Selective chemical modifications of Glu, Arg, Lys and Trp residues was tested for cytotoxic activity and derivatives in which the Trp residue was modified showed low effect, suggesting the existence of a connection between hydrophobicity and activity. However, over the full set of cyclotides examined, there was no strong correlation between the cytotoxic activity and their hydrophobicity. Instead, it seems more like that the distribution of charged and hydrophobic residues determines the ultimate degree of potency. Furthermore, we found that while the Glu residue is very important in maintaining the activity of the bracelet cyclotide cycloviolacin O2, it is much less important in the Möbius cyclotides. However, despite these differences, a systematic test of mixtures of cyclotides, even from both subfamilies revealed that they act in an additive way.
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| 3. |
- Burman, Robert, 1979-
(författare)
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Distribution and Chemical Diversity of Cyclotides from Violaceae : Impact of Structure on Cytotoxic Activity and Membrane Interactions
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During the last decade there has been increased interest in the cyclotide protein family, which consist of a circular chain of approximately 30 amino acids, including six cysteines that form three disulfide bonds, arranged in a cyclic cystine knot motif. This thesis gives new insights in cyclotide distribution and occurrence in the plant family Violaceae, structure-activity relationships for cytotoxic effects, membrane disruption and adsorption on lipid membranes, and evaluates toxicity and anti-tumor activity in vivo. A large-scale analysis was done on over 200 samples covering 17 of the 23 genera in Violaceae, and cyclotides were positively identified in almost 150 of approximately 900 known species. Conclusions are that the Violaceae is an extremely rich source of cyclotides, and that they are ubiquitous among all species in that plant family. After investigating the cyclotides' cytotoxicity it was evident that the effects were immediate and occurred at low micromolar concentrations. To understand the relationships between structure and activity, approximately 30 cyclotides and cyclotide derivates were assayed for cytotoxicity. Results showed that the overall charge is of minor influence on activity and revealed a strong correlation between an intact hydrophobic molecular surface and cytotoxic effect. The cytotoxic activity is mainly due to interactions between peptides and target membranes, illustrated by prototypic cyclotides' ability to induce liposome leakage and adsorb to lipid membranes. Cyclotides were strongly lytic against zwitterionic liposomes, less when cholesterol was included, while for anionic liposomes, activity depend on the net charge of cyclotide. A similar pattern was observed for the adsorption of the cyclotides to anionic bilayers, in which strong lytic activity was coupled with high adsorption. To further evaluate cyclotides cytotoxic effects, in vivo studies were conducted, both for acute toxicity and anti-tumor efficacy in mice. Two different methods were used: hollow fiber method and traditional xenografts, but no significant anti-tumor effects were detected. The results indicate that anti-tumor effects are minor or absent at tolerable doses and that cyclotides have a very abrupt in vivo toxicity profile, with lethality after single injection at 2.0 mg/kg.
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| 4. |
- Burman, Robert, 1979-, et al.
(författare)
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Membrane integrity as a target for cyclotide cytotoxic activity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The cyclotides are a family of plant-derived proteins that occur in plants from the Violaceae (violet) and Rubiaceae (coffee) families and have a diverse range of biological activities, including cytotoxic, hemolytic, antimicrobial, and insecticidal activities; the latter suggests their natural function lies in plant defense. In the current study we have investigated the membrane-disrupting and adsorption ability of prototypic cyclotides and correlated these findings to their cytotoxic properties. We also included modifications of selected charged amino acids in cycloviolacin O2, previously shown to be of importance for its cytotoxic activity. The cyclotides’ cytotoxic activity, ability to adsorb and disrupt model lipid membranes of different charge densities was investigated, e.g. by fluorescence spectroscopy, ellipsometry, and circular dichroism. Cytotoxicity of the native cyclotides was demonstrated to correlate to membrane adsorption and lytic activity. Hence, the activity of native cyclotides is mainly due to interactions between the proteins and the phospholipids in the target membrane. Striking effects of single amino acid variations in cycloviolacin O2 on its membrane interaction were also demonstrated.
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| 5. |
- Jacobsson, Erik, 1986-
(författare)
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Studies on cysteine-rich peptides from Nemertea and Violaceae : Proteomic and transcriptomic discovery and characterization
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The overall aims of the projects included in this thesis were to discover, synthesize and characterize disulphide-stabilized peptides from marine worms (Nemertea sp.) and plants (Viola sp.). One of the main outcomes of this thesis is the discovery of a new family of highly active cysteine-rich toxins, alpha nemertides, from nemertean worms (paper II). Functional characterization and production routes of nemertides were further explored (papers II-III). In addition, 12 new cyclotides from the bog violet were discovered (paper I). Finally, transcriptomes and mucus of the Antarctic nemertean Parborlasia corrugatus were investigated for toxin content (paper IV). In paper I wild-type leaf and callus tissue of the endangered bog violet, V. uliginosa, were analyzed using transcriptomics and LC-MS, resulting in the discovery of 12 new cyclotides (i.e. cysteine-rich cyclic peptides). In addition, cyclotide expression under different cell-growth conditions was monitored.In paper II the discovery and initial characterization of a new family of highly active peptides, the alpha nemertides, from the epidermal mucus of the world’s longest animal; Lineus longissimus is described. The most abundant alpha nemertide, alpha-1, was extracted in minute amounts, prompting the use solid phase peptide synthesis (SPPS) for further characterization. The tertiary structure of alpha-1 was elucidated and revealed an inhibitory cystine knot (ICK) framework. The knotted core-structure is similar to the cyclic cystine knot (CCK) motif, found in the cyclotides described in paper I.In manuscript III, the production route established in paper II was used to produce nemertides alpha 1-7. These were tested in vivo in an Artemia microwell assay as well as on an extended panel of voltage-gated sodium channels (NaV1.1 – 1.8 and BgNaV1). All seven alpha nemertides were highly active in the in vivo Artemia assay with EC50 values in the sub to low µM range. The alpha nemertides were also active in the NaVs tested. However, differences in the activity profiles were observed, indicating an opportunity for future optimization of alpha nemertides to reach higher specificity to certain NaV subtypes.In manuscript IV, the exploration of nemertide toxins was extended to include the Antarctic P. corrugatus. Resulting findings include a set of cysteine-rich peptides, some similar to the nemertides previously discovered in paper II. Two purified peptides and one fraction were evaluated for their membranolytic activity.
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| 6. |
- Mohotti, Supun, et al.
(författare)
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Screening for bioactive secondary metabolites in Sri Lankan medicinal plants by microfractionation and targeted isolation of antimicrobial flavonoids from Derris scandens
- 2020
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Ingår i: Journal of Ethnopharmacology. - : Elsevier BV. - 0378-8741 .- 1872-7573. ; 246
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Tidskriftsartikel (refereegranskat)abstract
- Ethnopharmacological relevance: Sri Lanka is known to have very diverse flora. Many of these species are used for plant-based remedies, which form the integral part of two Sri Lankan systems of traditional medicine, Ayurveda and Deshiya Chikitsa. Despite their widespread use, only a limited number of studies have probed into the scientific evidence for bioactivity of these medicinal plants. Such studies rarely progress to the identification of bioactive natural products. Aim of the study: The primary aim was to develop a bioactivity screening method and apply it to 50 Sri Lankan medicinal plants where antimicrobial properties could be relevant for its traditional use. The subsequent aim was the progression into defining and characterising potent isolates within targeted compound classes from such plants, i.e. Derris scandens and its antimicrobial flavonoids. Material and methods: The plant collection comprised 24 species of Fabaceae, 15 Rubiaceae, 7 Solanaceae and 4 Cucurbitaceae plants. These 50 species were collected based on their ethnopharmacological importance and use in Sri Lankan traditional medicine. Crude extracts from each species were initially subjected to radial disc diffusion and microdilution assays. Subsequently, aqueous extracts of all plants were microfractionated in deep well plates using reversed-phase HPLC. Fractions were tested for antibacterial and cytotoxic activities and masses of target bioactive compounds were identified using mass spectrometry. Bioactive compounds with the masses identified through microfractions were isolated from Derris scandens using reversed-phase HPLC. The isolated pure compounds were characterised using LC-MS and NMR. Results: Crude aqueous extracts from 19 species showed activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) in the radial disc diffusion assay. Crude aqueous extracts from 34 plant species and organic extracts from 46 plant species were active against S. aureus (<= 4 mg mL(-1)) in the microdilution assay. Microfractionation demonstrated antibacterial activity for 19 plants and cytotoxicity for 6 plants. Furthermore, target bioactive compounds and their molecular ions were identified during microfractionation. Dalpanitin and vicenin-3, two of the flavonoids isolated from Derris scandens gave MICs of 23 mu g mL(-1) against S. aureus. Dalpanitin also exhibited relevant MICs on Gram-negative bacteria (94 mu g mL(-1)) against Escherichia coli and Pseudomonas aeruginosa). Conclusion: The microfractionation protocol developed in this study enabled time-efficient screening of many plants species, using a small quantity of sample material. In addition, microfractionation served as a guiding tool for identifying individual antimicrobial compounds. Through this process, flavonoids were isolated from Derris scandens, out of which dalpanitin and vicenin-3 showed activity in the low micromolar range. The high hit rate for in vitro antibacterial properties from this ethnopharmacologically guided sample collection gives credence to Sri Lankan traditional herbal medicine as a source for drug discovery.
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