RESUMO
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 IC5o 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.
Assuntos
Antineoplásicos/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Ciclotídeos/farmacologia , Proteínas de Plantas/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/administração & dosagem , Ciclotídeos/administração & dosagem , Ciclotídeos/genética , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas de Plantas/administração & dosagem , Proteínas de Plantas/genética , Estrutura Terciária de Proteína , Transplante HeterólogoRESUMO
Cyclotides are a family of plant defense proteins that are highly resistant to adverse chemical, thermal, and enzymatic treatment. Here, we present the first crystal structure of a cyclotide, varv F, from the European field pansy, Viola arvensis, determined at a resolution of 1.8 A. The solution state NMR structure was also determined and, combined with measurements of biophysical parameters for several cyclotides, provided an insight into the structural features that account for the remarkable stability of the cyclotide family. The x-ray data confirm the cystine knot topology and the circular backbone, and delineate a conserved network of hydrogen bonds that contribute to the stability of the cyclotide fold. The structural role of a highly conserved Glu residue that has been shown to regulate cyclotide function was also determined, verifying its involvement in a stabilizing hydrogen bond network. We also demonstrate that varv F binds to dodecylphosphocholine micelles, defining the binding orientation and showing that its structure remains unchanged upon binding, further demonstrating that the cyclotide fold is rigid. This study provides a biological insight into the mechanism by which cyclotides maintain their native activity in the unfavorable environment of predator insect guts. It also provides a structural basis for explaining how a cluster of residues important for bioactivity may be involved in self-association interactions in membranes. As well as being important for their bioactivity, the structural rigidity of cyclotides makes them very suitable as a stable template for peptide-based drug design.
Assuntos
Ciclotídeos , Motivos Nó de Cisteína , Desenho de Fármacos , Inseticidas , Conformação Proteica , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Ciclotídeos/química , Ciclotídeos/genética , Ciclotídeos/metabolismo , Humanos , Ligação de Hidrogênio , Inseticidas/química , Inseticidas/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Alinhamento de SequênciaRESUMO
This review focuses on the discovery of cyclotides in the Violaceae, their isolation and their anti-cancer effects. These macrocyclic plant peptides consist of about 30 amino acids, including three conserved disulfide bonds in a cystine knotted arrangement, which renders them a remarkable stability. Their unique structure, combined with a wide array of biological activities, makes them of great interest as possible leads in drug development or as carriers of grafted peptide sequences. Here we describe the work conducted in our laboratory, which started with the overall aim of identifying peptides and small proteins of the size 10-50 amino acid residues in plants with novel chemical structures and biological profiles with a potential for drug development or for use as pharmacological tools. Thus we developed a fractionation protocol to directly address major challenges encountered when dealing with plant material, such as removal of chlorophyll, polyphenols, and low molecular compounds omnipresent in plants. Using this protocol, we then discovered a suite of cyclotides, the varv peptides, from the plant Viola arvensis (Violaceae). Following this, separation methods directly targeting cyclotides were developed, e.g. by adsorption, ion exchange chromatography and solvent-solvent partitioning, which then were used in the isolation of additional cyclotides. To structurally examine cyclotides we have also developed methods based on mass spectrometry for cyclotide sequencing and mapping of disulfide bonds. Finally, to assess structure-activity relationships, regarding their anti-cancer and cytotoxic effects that we focus upon, we have also characterized the three dimensional structure of cyclotides by homology modeling techniques.
Assuntos
Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Violaceae/química , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Cromatografia Líquida , Ciclotídeos/metabolismo , Ciclotídeos/farmacologia , Dissulfetos/metabolismo , Humanos , Relação Estrutura-AtividadeRESUMO
Cyclotides, a family of approximately 50 mini-proteins isolated from various Violaceae and Rubiaceae plants, are characterized by their circular peptide backbone and six conserved cysteine residues arranged in a cystine knot motif. Cyclotides show a wide range of biological activities, making them interesting targets for both pharmaceutical and agrochemical research, but little is known about their natural function and the events that trigger their expression. An investigation of the geographical and seasonal variations of cyclotide profiles has been performed, using the native Australian violet, Viola hederacea, and the Swedish sweet violet, Viola odorata, as model plants. The results showed that in the Australian violet the relative peptide levels of some cyclotides remained almost constant throughout the year, while other cyclotides were present only at certain times of the year. Therefore, it appears that V. hederacea expresses a basic armory of cyclotides as well as special "add-ons" whose levels are influenced by external factors. In the Swedish violet, cyclotide levels were increased up to 14 times during the warmest period of the year. The larger variation in expression levels of the Swedish plants may be a reflection of a greater climatic variation.
Assuntos
Ciclotídeos , Variação Genética , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Viola/química , Sequência de Aminoácidos , Clima , Bases de Dados de Proteínas , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , SuéciaRESUMO
A crude fraction of Viola tricolor rich in small lipophilic proteins was prepared and subjected to fractionation guided by bioactivity, using RP-HPLC and a fluorometric cytotoxicity assay. Two human cancer cell lines, U-937 GTB (lymphoma) and RPMI-8226/s (myeloma), were used in this study. The most potent compounds isolated, that is, the compounds showing the lowest IC(50) values, were shown to be three small proteins: vitri A (IC(50) = 0.6 microM and IC(50) = 1 microM, respectively), varv A (IC(50) = 6 microM and IC(50) = 3 microM, respectively), and varv E (IC(50) = 4 microM in both cell lines). Their sequences, determined by automated Edman degradation, quantitative amino acid analysis, and mass spectrometry, were cyclo-GESCVWIPCITSAIGCSCKSKVCYRNGIPC (vitri A), cyclo-GETCVGGTCNTPGCSCSWPVCTRNGLPVC (varv A), and cyclo-GETCVGGTCNTPGCSCSWPVCTRNGLPIC (varv E), of which vitri A is described for the first time. Each forms a head-to-tail cyclic backbone, with six cysteine residues being involved in three disulfide bonds, characteristic of the family of small proteins called the cyclotides. This is the first report on cyclotides from the species V. tricolor and the first report on the sequence of the cytotoxic cyclotide vitri A.
Assuntos
Peptídeos Cíclicos/química , Proteínas de Plantas/química , Plantas Medicinais/química , Viola/química , Sequência de Aminoácidos , Aminoácidos/análise , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Alemanha , Humanos , Concentração Inibidora 50 , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Two polypeptides named vodo M and vodo N, both of 29 amino acids, have been isolated from Viola odorata L. (Violaceae) using ion exchange chromatography and reversed phase HPLC. The sequences were determined by automated Edman degradation, quantitative amino acid analysis, and mass spectrometry (MS). Using MS, it was established that vodo M (cyclo-SWPVCTRNGAPICGESCFTGKCYTVQCSC) and vodo N (cyclo-SWPVCYRNGLPVCGETCTLGKCYTAGCSC) form a head-to-tail cyclic backbone and that six cysteine residues are involved in three disulphide bonds. Their origin, sequences, and cyclic nature suggest that these peptides belong to the family of cyclic plant peptides, called cyclotides. The three-dimensional structures of vodo M and vodo N were modelled by homology, using the experimentally determined structure of the cyclotide kalata B1 as the template. The images of vodo M and vodo N show amphipathic structures with considerable surface hydrophobicity for a protein modelled in a polar environment.
Assuntos
Ciclotídeos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Viola/química , Sequência de Aminoácidos , Simulação por Computador , Bases de Dados de Proteínas , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos Cíclicos/isolamento & purificação , Conformação Proteica , Rubiaceae/química , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
The expression of cyclotides-macrocyclic plant peptides-was profiled in six violets, Viola cotyledon, V. biflora, V. arvensis, V. tricolor, V. riviniana, and V. odorata, by LC-MS. All were found to express notably complex mixtures, with single species containing >50 cyclotides. To facilitate their sequencing by MS-MS, an analytical strategy is presented involving aminoethylation of cysteines. This overcomes a number of problems intimately associated with the cyclotide core structure-that is, their joined N and C termini, disulfide knot, and low or clustered content of positively charged amino acids and enzymatic cleavage sites. As a result, charges as well as cleavage sites are introduced at the most conserved part of their sequence, the cysteines. Combined with tryptic digestion, all intercysteine loops are then of suitable size and charge for MS-MS sequencing. The utility of this strategy is shown by the sequencing of two novel cyclotides isolated from V. cotyledon; vico A (cyclo-(AESCVYIPCFTGIAGCSCKNKVCYYNGSIPC)) and vico B (cyclo-(AESCVYIPCITGIAGCSCKNKVCYYNGSIPC)); their complete sequence could be determined by nanospray MS-MS. The strategy for converting conserved cysteines to enzymatic cleavage sites might also benefit the study of other peptides and proteins displaying similar structural problems for MS analysis.
Assuntos
Cisteína/análogos & derivados , Cisteína/química , Peptídeos Cíclicos/metabolismo , Proteínas de Plantas/metabolismo , Viola/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Cromatografia Líquida , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Peso Molecular , Peptídeos Cíclicos/análise , Peptídeos Cíclicos/química , Proteínas de Plantas/análise , Proteínas de Plantas/química , Viola/químicaRESUMO
Through a reliable and repeatable procedure based on solid-phase extraction techniques, a protein fraction (P fraction) rich in Cys/Gly residues was extracted and captured from oat (Avena sativa L.) seeds. Quantitative amino acid analysis and MS of the P fraction indicated that it contains a series of heterogeneous Cys/Gly-rich proteins with molecular masses of 3.6-4.0 kDa. Preliminary results from bioassays showed that these proteins possess weak to moderate antifungal properties to some fungal strains. From this fraction, a new polypeptide, designated avesin A, was purified and sequenced by Edman degradation. Avesin A consists of 37 amino-acid residues, with 10 glycine residues and eight cysteine residues forming disulfide bridges, and contains a single chitin-binding domain, which indicates that avesin A is a new member of the putative chitin-binding proteins. Avesin A is the first identified hevein-like small protein from cereal grains.
Assuntos
Avena/química , Quitina/metabolismo , Proteínas de Plantas/química , Sementes/química , Sequência de Aminoácidos , Aminoácidos/análise , Antifúngicos/química , Cisteína/química , Dissulfetos/análise , Dissulfetos/química , Glicina/química , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cytotoxic activities of three naturally occurring macrocyclic peptides (cyclotides) isolated from the two violets, Viola arvensis Murr. and Viola odorata L., were investigated. A nonclonogenic fluorometric microculture assay was used to examine cytotoxicity in a panel of 10 human tumor cell lines representing defined types of cytotoxic drug resistance. Additionally, primary cultures of tumor cells from patients, and for comparison normal lymphocytes, were used to quantify cytotoxic activity. All three cyclotides, varv A, varv F, and cycloviolacin 02, exhibited strong cytotoxic activities, which varied in a dose-dependent manner. Cycloviolacin 02 was the most potent in all cell lines (IC50 0.1-0.3 microM), followed by varv A (IC50 2.7-6.35 microM) and varv F (IC50 2.6-7.4 microM), respectively. Activity profiles of the cyclotides differed significantly from those of antitumor drugs in clinical use, which may indicate a new mode of action. This, together with the exceptional chemical and biological stability of cyclotides, makes them interesting in particular for their potential as pharmacological tools and possibly as leads to antitumor agents.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ciclotídeos , Neoplasias/tratamento farmacológico , Peptídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Dados de Sequência Molecular , Peptídeos Cíclicos/isolamento & purificação , Células Tumorais Cultivadas , Viola/químicaRESUMO
Chosen to reflect biodiversity in a phylogenetic sense, 100 fractionated plant extracts were screened in vitro for cytotoxicity following extraction and fractionation (polypeptide isolation). Of these 100 extracts, 30 were selected and then characterized preliminarily for antitumor potency and mode of action by testing them on two cell lines and primary cultures of human tumor cells. On the basis of cytotoxicity potency, 10 of the extracts were further characterized for anticancer activity in 10 human tumor cell lines. This final testing resulted in seven potential lead plants with superior evidence of antitumor potential: Colchicum autumnale L. (Colchicaceae), Digitalis lanata Ehrh. and Digitalis purpurea L. (Plantaginaceae), Helleborus cyclophyllus Boiss. (Ranunculaceae), Menyanthes trifoliata L. (Menyanthaceae), and Viola arvensis Murr. and Viola patrinii Ging. (Violaceae). Within a database of antitumor compounds, the activity profiles of the extracts from these seven plants were compared, by correlation analysis, with those of more than 100 other compounds, including 39 standard drugs from different classes of cytotoxic mechanisms. The activity profiles of six of these candidates were uncorrelated with those of the standard drugs, possibly indicating new pathways of drug-mediated cell death.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Plantas Medicinais/química , Automação , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estudos de Avaliação como Assunto , Humanos , Extratos Vegetais/farmacologia , Controle de Qualidade , Células Tumorais CultivadasRESUMO
A new basic protein, designated ligatoxin B, containing 46 amino acid residues has been isolated from the mistletoe Phoradendron liga (Gill.) Eichl. (Viscaceae). The protein's primary structure, determined unambiguously using a combination of automated Edman degradation, trypsin enzymic digestion, and tandem MS analysis, was 1-KSCCPSTTAR-NIYNTCRLTG-ASRSVCASLS-GCKIISGSTC-DSGWNH-46. Ligatoxin B exhibited in vitro cytotoxic activities on the human lymphoma cell line U-937-GTB and the primary multidrug-resistant renal adenocarcinoma cell line ACHN, with IC50 values of 1.8 microM and 3.2 microM respectively. Sequence alignment with other thionins identified a new member of the class 3 thionins, ligatoxin B, which is similar to the earlier described ligatoxin A. As predicted by the method of homology modelling, ligatoxin B shares a three-dimensional structure with the viscotoxins and purothionins and so may have the same mode of cytotoxic action. The novel similarities observed by structural comparison of the helix-turn-helix (HTH) motifs of the thionins, including ligatoxin B, and the HTH DNA-binding proteins, led us to propose the working hypothesis that thionins represent a new group of DNA-binding proteins. This working hypothesis could be useful in further dissecting the molecular mechanisms of thionin cytotoxicity and of thionin opposition to multidrug resistance, and useful in clarifying the physiological function of thionins in plants.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/isolamento & purificação , Erva-de-Passarinho/química , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Citotoxinas/química , Citotoxinas/isolamento & purificação , Citotoxinas/toxicidade , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/toxicidade , Sequências Hélice-Volta-Hélice , Humanos , Linfoma , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas de Plantas/química , Proteínas de Plantas/toxicidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
A multitarget functional bioassay was optimized as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes, mainly to release elastase detected by p-nitroanilide (pNA) formation. Using this bioassay, 100 fractionated extracts of 96 plants were screened, with results presented in a manner that links recorded biological activity to phylogenetic information. The plants were selected to represent a major part of the angiosperms, with emphasis on medicinal plants, Swedish anti-inflammatory plants, and plants known to contain peptides. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. The extract of Digitalis purpurea enhanced pNA formation, and digitoxin, the active compound, was isolated and identified. Plant extracts that exhibited potent nonselective inhibition (>80% inhibition) were evaluated further for direct inhibition of isolated elastase and trypsin enzyme. The inhibitory effect of most tested extracts on the isolated enzyme elastase was similar to that of PAF- and fMLP-induced pNA formation. Compared to trypsin, inhibition of elastase by extracts of Rubus idaeus and Tabernaemontana dichotoma was significantly higher (80% and 99%, respectively). Inhibition of trypsin by the extract of Reseda luteola was high (97%). Orders such as Lamiales and Brassicales were shown to include a comparably high proportion of plants with inhibitory extracts.