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David J Craik - One of the best experts on this subject based on the ideXlab platform.
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Cyclotides: From Structure to Function
Chemical Reviews, 2019Co-Authors: Simon J. De Veer, David J CraikAbstract:This Review explores the class of plant-derived macrocyclic peptides called cyclotides. We include an account of their discovery, characterization, and distribution in the plant kingdom as well as a detailed analysis of their sequences and structures, biosynthesis and chemical synthesis, biological functions, and applications. These macrocyclic peptides are around 30 amino acids in size and are characterized by their head-to-tail cyclic backbone and Cystine Knot Motif, which render them to be exceptionally stable, with resistance to thermal or enzymatic degradation. Routes to their chemical synthesis have been developed over the past two decades, and this capability has facilitated a wide range of mutagenesis and structure-activity relationship studies. In turn, these studies have both led to an increased understanding of their mechanisms of action as well as facilitated a range of applications in agriculture and medicine, as ecofriendly crop protection agents, and as drug leads or scaffolds for pharmaceutical design. Our overall objective in this Review is to provide readers with a comprehensive overview of cyclotides that we hope will stimulate further work on this fascinating family of peptides.
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Discovery, structure, function, and applications of cyclotides: circular proteins from plants
Journal of Experimental Botany, 2016Co-Authors: Joachim Weidmann, David J CraikAbstract:Cyclotides are plant-derived cyclic peptides that have a head-to-tail cyclic backbone and three conserved disulphide bonds that form a cyclic Cystine Knot Motif. They occur in plants from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families, typically with 10-100 cyclotides in a given plant species, in a wide range of tissues, including flowers, leaves, stems, and roots. Some cyclotides are expressed in large amounts (up to 1g kg wet plant weight) and their natural function appears to be to protect plants from pests or pathogens. This article provides a brief overview of their discovery, distribution in plants, and applications. In particular, their exceptional stability has led to their use as peptide-based scaffolds in drug design applications. They also have potential as natural 'ecofriendly' insecticides, and as protein engineering frameworks.
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Fmoc-Based Synthesis of Disulfide-Rich Cyclic Peptides
Journal of Organic Chemistry, 2014Co-Authors: Olivier Cheneval, Christina I. Schroeder, Thomas Durek, Phillip Walsh, Yen-hua Huang, Spiros Liras, David A. Price, David J CraikAbstract:Disulfide-rich cyclic peptides have exciting potential as leads or frameworks in drug discovery; however, their use is faced with some synthetic challenges, mainly associated with construction of the circular backbone and formation of the correct disulfides. Here we describe a simple and efficient Fmoc solid-phase peptide synthesis (SPPS)-based method for synthesizing disulfide-rich cyclic peptides. This approach involves SPPS on 2-chlorotrityl resin, cyclization of the partially protected peptide in solution, cleavage of the side-chain protecting groups, and oxidization of cysteines to yield the desired product. We illustrate this method with the synthesis of peptides from three different classes of cyclic Cystine Knot Motif-containing cyclotides: Mobius (M), trypsin inhibitor (T), and bracelet (B). We show that the method is broadly applicable to peptide engineering, illustrated by the synthesis of two mutants and three grafted analogues of kalata B1. The method reduces the use of highly caustic and tox...
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Cyclotides as grafting frameworks for protein engineering and drug design applications
Biopolymers, 2013Co-Authors: Aaron G. Poth, Lai Yue Chan, David J CraikAbstract:Cyclotides are a family of naturally occurring backbone-cyclized macrocyclic mini-proteins from plants that have a Knotted trio of intramolecular disulfide bonds. Their structural features imbue cyclotides with extraordinary stability against degradation at elevated temperatures or in the presence of proteolytic enzymes. The plasticity of their intracysteine loop sequences is exemplified by the more than 250 natural cyclotides sequenced to date, and this tolerance to sequence variation, along with their diverse bioactivities, underpins the suitability of the cyclic Cystine Knot Motif as a valuable drug design scaffold and research tool for protein engineering studies. Here, we review the recent literature on applications of cyclotides for the stabilization of peptide epitopes and related protein engineering studies. Possible future directions in this field are also described. (C) 2013 Wiley Periodicals, Inc.
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Cyclotide isolation and characterization.
Methods in Enzymology, 2012Co-Authors: David J Craik, Sónia Troeira Henriques, Joshua S. Mylne, Conan K. WangAbstract:Cyclotides are disulfide-rich cyclic peptides produced by plants with the presumed natural function of defense agents against insect pests. They are present in a wide range of plant tissues, being ribosomally synthesized via precursor proteins that are posttranslationally processed to produce mature peptides with a characteristic cyclic backbone and Cystine Knot Motif associated with their six conserved cysteine residues. Their processing is not fully understood but involves asparaginyl endoproteinase activity. In addition to interest in their defense roles and their unique topologies, cyclotides have attracted attention as potential templates in peptide-based drug design applications. This chapter provides protocols for the isolation of cyclotides from plants, their detection and sequencing by mass spectrometry, and their structural analysis by NMR, as well as describing methods for the isolation of nucleic acid sequences that encode their precursor proteins. Assays to assess their membrane-binding interactions are also described. These protocols provide a “starter kit” for researchers entering the cyclotide field.
Norelle L Daly - One of the best experts on this subject based on the ideXlab platform.
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Structural studies of cyclotides
Advances in Botanical Research, 2015Co-Authors: Norelle L Daly, K. Johan RosengrenAbstract:Over the last two decades, the cyclotides have generated widespread interest both in the plant biology and peptide communities for a number of reasons, including their physiological roles in plants, evolution, and wide range of biological activities with potential therapeutic applications. But perhaps most of all it is their unique structure with a cyclic Cystine Knot Motif that provide remarkable features such as resistance to chemical and biological degradation, which have caught researchers' attention. In this chapter, we summarize the key studies that have contributed to our understanding of the cyclotide fold, the structural features of these peptides, and the role and utilization of these features for native and engineered biological functions.
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Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry
Amino Acids, 2013Co-Authors: Hossein Hashempour, Norelle L Daly, Johannes Koehbach, Alireza Ghassempour, Christian W. GruberAbstract:Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved Cystine-Knot Motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis , whereof ten (vigno 1–10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides.
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Cystine Knot Folding in Cyclotides
ChemInform, 2011Co-Authors: Norelle L Daly, Ulf Goransson, Christian W. Gruber, David J CraikAbstract:Cyclotides are naturally occurring plant-based proteins of approximately 30 amino acids in size that contain a head-to-tail cyclized backbone and a Cystine Knot Motif formed by their three conserved disulfide bonds. Their exceptional stability and unique topology make them valuable frameworks in drug design or protein engineering applications. To facilitate such applications and to explore structure–activity relationships of cyclotides it is useful to be able to chemically synthesize them, a process that is readily achieved via solid phase peptide synthesis followed by oxidative folding. This chapter describes what is known about the oxidative folding of cyclotides, both in chemical folding buffers and assisted by a protein disulfide isomerase enzyme isolated from a cyclotide-producing plant. Formation of the Cystine Knot Motif is readily achieved, despite its apparent topological complexity.
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Isolation and characterization of bioactive cyclotides from Viola labridorica.
Helvetica Chimica Acta, 2010Co-Authors: Jun Tang, Norelle L Daly, Conan K. Wang, Guang-zhi Zeng, Wenyan Xu, Wen-jun He, David J CraikAbstract:Many Violaceae plants contain cyclotides, which are plant cyclopeptides distinguished by a cyclic Cystine Knot Motif with 28-37 amino acid residues. In the current study, four new cyclotides, vila A - D (1-4 resp), together with a known cyclotide, vary D (5), were isolated from Viola labridorica (Violaceae) A chromatography-based method was used to isolate the cyclotides, which were characterized using tandem mass spectrometry and 2D-NMR spectroscopy Several of the cyclotides showed cytotoxic activities against five cancer cell lines, i e, U251 MDA-MB-231, A549 DU145, and BEL-7402, with vila A and B (1 and 2, resp) being the most cytotoxic The isolated cyclotides showed no antibacterial activity against Staphyloccocus aureus and Candida albicans Homology modeling of the cyclotide structures was used to analyze structure-activity relationships.
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Isolation and characterization of cytotoxic cyclotides from Viola tricolor
Peptides, 2010Co-Authors: Jun Tang, Norelle L Daly, Conan K. Wang, Guang-zhi Zeng, Wenyan Xu, Wen-jun He, David J CraikAbstract:Many plants of the Violaceae plant family have been used in traditional remedies, and these plants often contain cyclotides, a particular type of plant cyclopeptide that is distinguished by a cyclic Cystine Knot Motif. In general, bioactive plant cyclopeptides are interesting candidates for drug development. In the current study, a suite of 14 cyclotides, which includes seven novel cyclotides [vitri B, C, D, E, F, vary Hm, and He], together with seven known cyclotides [vary A, D, E, F, H, vitri A, and cycloviolacin O2], was isolated from Viola tricolor, a common flower. A chromatography-based method was used to isolate the cyclotides, which were characterized using tandem mass spectrometry and NMR spectroscopy. Several of the cyclotides showed cytotoxic activities against five cancer cell lines, U251, MDA-MB-231, A549, DU145, and BEL-7402. Three cyclotides, vitri A, vitri F, and cycloviolacin O2, were the most cytotoxic. The cytotoxic activity of the cyclotides did not correlate well with their hemolytic activity, indicating that different interactions, most likely with membranes, are involved for cytotoxic and hemolytic activities. Homology modeling of the structures was used in deriving structure-activity relationships. (C) 2010 Elsevier Inc. All rights reserved.
Ulf Goransson - One of the best experts on this subject based on the ideXlab platform.
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Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense
Planta, 2016Co-Authors: Blazej Slazak, Małgorzata Kapusta, Sohaib Malik, Jerzy Bohdanowicz, Elżbieta Kuta, Przemysław Malec, Ulf GoranssonAbstract:Main conclusion The distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic Cystine Knot Motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique’s resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa , and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense—relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown.
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Cystine Knot Folding in Cyclotides
ChemInform, 2011Co-Authors: Norelle L Daly, Ulf Goransson, Christian W. Gruber, David J CraikAbstract:Cyclotides are naturally occurring plant-based proteins of approximately 30 amino acids in size that contain a head-to-tail cyclized backbone and a Cystine Knot Motif formed by their three conserved disulfide bonds. Their exceptional stability and unique topology make them valuable frameworks in drug design or protein engineering applications. To facilitate such applications and to explore structure–activity relationships of cyclotides it is useful to be able to chemically synthesize them, a process that is readily achieved via solid phase peptide synthesis followed by oxidative folding. This chapter describes what is known about the oxidative folding of cyclotides, both in chemical folding buffers and assisted by a protein disulfide isomerase enzyme isolated from a cyclotide-producing plant. Formation of the Cystine Knot Motif is readily achieved, despite its apparent topological complexity.
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Synthesis and folding of the circular Cystine Knotted cyclotide cycloviolacin O2
Planta Medica, 2008Co-Authors: Teshome Leta Aboye, David J Craik, Richard J Clark, Ulf GoranssonAbstract:The cyclic Cystine Knot Motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering [1]. However, to date the utilization of this scaffold has been limited by the inability to synthesize members of the most diverse and biologically active subfamily, the bracelet cyclotides. Here we describe the synthesis and first direct oxidative folding of a bracelet cyclotide, cycloviolacin O2, and thus provide an efficient method of exploring the most potent cyclic Cystine Knot peptides [2]. The linear chain of cycloviolacin O2 was assembled using Fmoc solid phase peptide synthesis and cyclized by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single step reaction. The folding pathway was characterized and included predominating fully oxidized intermediates that slowly converted to the native peptide structure. Acknowledgements: This study was supported by the Swedish International Development Cooperation Agency (SIDA), Dept for Research Cooperation (SAREC) and The Royal Swedish Academy of Sciences.. References: 1. Craik, D.J. (2006) Science 311:1563–1564. 2. Leta Aboye, T. et al. (2008) Chembiochem 9:103–13.
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The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity
Phytochemistry, 2008Co-Authors: Anders Herrmann, David J Craik, Richard J Clark, Joshua S. Mylne, Robert Burman, Gustav Karlsson, Joachim Gullbo, Ulf GoranssonAbstract:The cyclotides are currently the largest known family of head-to-tail cyclic proteins. The complex structure of these small plant proteins, which consist of approximately 30 amino acid residues, contains both a circular peptide backbone and a Cystine Knot, the combination of which produces the cyclic Cystine Knot Motif. To date, cyclotides have been found in plants from the Rubiaceae, Violaceace and Cucurbitaceae families, and are believed to be part of the host defence system. In addition to their insecticidal effect, cyclotides have also been shown to be cytotoxic, anti-HIV, antimicrobial and haemolytic agents. In this study, we show that the alpine violet Viola biflora (Violaceae) is a rich source of cyclotides. The sequences of 11 cyclotides, vibi A-K, were determined by isolation and MS/MS sequencing of proteins and screening of a cDNA library of V. biflora in parallel. For the cDNA screening, a degenerate primer against a conserved (AAFALPA) Motif in the cyclotide precursor ER signal sequence yielded a series of predicted cyclotide sequences that were correlated to those of the isolated proteins. There was an apparent discrepancy between the results of the two strategies as only one of the isolated proteins could be identified as a cDNA clone. Finally, to correlate amino acid sequence to cytotoxic potency, vibi D, E, G and H were analysed using a fluorometric microculture cytotoxicity assay using a lymphoma cell line. The IC50-values of the bracelet cyclotides vibi E, G and H ranged between 0.96 and 5.0 μM while the Mobius cyclotide vibi D was not cytotoxic at 30 μM.
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Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular Cystine Knotted cyclotide cycloviolacin O2.
ChemBioChem, 2008Co-Authors: Teshome Leta Aboye, David J Craik, Richard J Clark, Ulf GoranssonAbstract:The cyclic Cystine Knot Motif as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisotion of this scaffold has been limited by the inability to synthesise members of the most diverse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide-cycloviolacin O2-and thus provides on efficient method for exploring the most potent cyclic Cystine Knot peptides. The linear chain of cycloviolacin O2 was assembled by solid-phase Fmoc peptide synthesis and cyclised by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single-step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.
Richard J Clark - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of human prolyl oligopeptidase activity by the cyclotide psysol 2 isolated from Psychotria solitudinum
Journal of Natural Products, 2015Co-Authors: Roland Hellinger, Richard J Clark, Johannes Koehbach, Albert Puigpinos, Teresa Tarragó, Ernest Giralt, Christian W. GruberAbstract:Cyclotides are head-to-tail cyclized peptides comprising a stabilizing Cystine-Knot Motif. To date, they are well known for their diverse bioactivities such as anti-HIV and immunosuppressive properties. Yet little is known about specific molecular mechanisms, in particular the interaction of cyclotides with cellular protein targets. Native and synthetic cyclotide-like peptides from Momordica plants are potent and selective inhibitors of different serine-type proteinases such as trypsin, chymotrypsin, matriptase, and tryptase-beta. This study describes the bioactivity-guided isolation of a cyclotide from Psychotria solitudinum as an inhibitor of another serine-type protease, namely, the human prolyl oligopeptidase (POP). Analysis of the inhibitory potency of Psychotria extracts and subsequent fractionation by liquid chromatography yielded the isolated peptide psysol 2 (1), which exhibited an IC50 of 25 μM. In addition the prototypical cyclotide kalata B1 inhibited POP activity with an IC50 of 5.6 μM. The i...
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Lysine-scanning Mutagenesis Reveals an Amendable Face of the Cyclotide Kalata B1 for the Optimization of Nematocidal Activity
Journal of Biological Chemistry, 2010Co-Authors: Yen-hua Huang, Michelle L Colgrave, Richard J Clark, Andrew C. Kotze, David J CraikAbstract:Cyclotides are a family of macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a Cystine Knot Motif, the combination of which imparts them with extraordinary stability. The prototypic cyclotide kalata B1 is toxic against two economically important gastrointestinal nematode parasites of sheep, Haemonchus contortus and Trichostrongylus colubriformis. A lysine scan was conducted to examine the effect of the incorporation of positive charges into the kalata B1 cyclotide framework. Each of the non-cysteine residues in this 29-amino acid peptide was successively substituted with lysine, and the nematocidal and hemolytic activities of the suite of mutants were determined. Substitution of 11 residues within kalata B1 decreased the nematocidal activity dramatically. On the other hand, six other residues that are clustered on the surface of kalata B1 were tolerant to Lys substitution, and indeed the introduction of positively charged residues into this region increased nematocidal activity. This activity was increased further in double and triple lysine mutants, with a maximal increase (relative to the native kalata B1) of 13-fold obtained with a triple lysine mutant (mutated at positions Thr-20, Asn-29, and Gly-1). Hemolytic activity correlated with the nematocidal activity of all lysine mutants. Our data clearly highlight the residues crucial for nematocidal and hemolytic activity in cyclotides, and demonstrate that the nematocidal activity of cyclotides can be increased by incorporation of basic amino acids.
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Dissecting the Oxidative Folding of Circular Cystine Knot Miniproteins
Antioxidants & Redox Signaling, 2009Co-Authors: Sunithi Gunasekera, Richard J Clark, Norelle L Daly, David J CraikAbstract:Abstract Cyclotides are plant proteins with exceptional stability owing to the presence of a cyclic backbone and three disulfide bonds arranged in a Cystine Knot Motif. Accordingly, they have been proposed as templates to stabilize bioactive epitopes in drug-design applications. The two main subfamilies, referred to as the Mobius and bracelet cyclotides, require dramatically different in vitro folding conditions to achieve the native fold. To determine the underlying elements that influence cyclotide folding, we examined the in vitro folding of a suite of hybrid cyclotides based on combination of the Mobius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1. The folding pathways of the two cyclotide subfamilies were found to be different and influenced by specific residues within intercysteine loops 2 and 6. Two changes in these loops, a substitution in loop 2 and an addition in loop 6, enabled the folding of a cycloviolacin O1 analogue under conditions in which folding does not occur in vitr...
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Synthesis and folding of the circular Cystine Knotted cyclotide cycloviolacin O2
Planta Medica, 2008Co-Authors: Teshome Leta Aboye, David J Craik, Richard J Clark, Ulf GoranssonAbstract:The cyclic Cystine Knot Motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering [1]. However, to date the utilization of this scaffold has been limited by the inability to synthesize members of the most diverse and biologically active subfamily, the bracelet cyclotides. Here we describe the synthesis and first direct oxidative folding of a bracelet cyclotide, cycloviolacin O2, and thus provide an efficient method of exploring the most potent cyclic Cystine Knot peptides [2]. The linear chain of cycloviolacin O2 was assembled using Fmoc solid phase peptide synthesis and cyclized by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single step reaction. The folding pathway was characterized and included predominating fully oxidized intermediates that slowly converted to the native peptide structure. Acknowledgements: This study was supported by the Swedish International Development Cooperation Agency (SIDA), Dept for Research Cooperation (SAREC) and The Royal Swedish Academy of Sciences.. References: 1. Craik, D.J. (2006) Science 311:1563–1564. 2. Leta Aboye, T. et al. (2008) Chembiochem 9:103–13.
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The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity
Phytochemistry, 2008Co-Authors: Anders Herrmann, David J Craik, Richard J Clark, Joshua S. Mylne, Robert Burman, Gustav Karlsson, Joachim Gullbo, Ulf GoranssonAbstract:The cyclotides are currently the largest known family of head-to-tail cyclic proteins. The complex structure of these small plant proteins, which consist of approximately 30 amino acid residues, contains both a circular peptide backbone and a Cystine Knot, the combination of which produces the cyclic Cystine Knot Motif. To date, cyclotides have been found in plants from the Rubiaceae, Violaceace and Cucurbitaceae families, and are believed to be part of the host defence system. In addition to their insecticidal effect, cyclotides have also been shown to be cytotoxic, anti-HIV, antimicrobial and haemolytic agents. In this study, we show that the alpine violet Viola biflora (Violaceae) is a rich source of cyclotides. The sequences of 11 cyclotides, vibi A-K, were determined by isolation and MS/MS sequencing of proteins and screening of a cDNA library of V. biflora in parallel. For the cDNA screening, a degenerate primer against a conserved (AAFALPA) Motif in the cyclotide precursor ER signal sequence yielded a series of predicted cyclotide sequences that were correlated to those of the isolated proteins. There was an apparent discrepancy between the results of the two strategies as only one of the isolated proteins could be identified as a cDNA clone. Finally, to correlate amino acid sequence to cytotoxic potency, vibi D, E, G and H were analysed using a fluorometric microculture cytotoxicity assay using a lymphoma cell line. The IC50-values of the bracelet cyclotides vibi E, G and H ranged between 0.96 and 5.0 μM while the Mobius cyclotide vibi D was not cytotoxic at 30 μM.
Glenn F. King - One of the best experts on this subject based on the ideXlab platform.
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Novel venom-derived inhibitors of the human EAG channel, a putative antiepileptic drug target
Biochemical Pharmacology, 2018Co-Authors: Linlin Ma, Volker Herzig, Yanni K-y Chin, Paul F. Alewood, Zoltan Dekan, Chun Yuen Chow, Jacqueline Heighway, Gilles J. Guillemin, Glenn F. KingAbstract:Abstract Recently, we and other groups revealed that gain-of-function mutations in the human ether a go-go voltage-gated potassium channel hEAG1 (Kv10.1) lead to developmental disorders with associated infantile-onset epilepsy. However, the physiological role of hEAG1 in the central nervous system remains elusive. Potent and selective antagonists of hEAG1 are therefore much sought after, both as pharmacological tools for studying the (patho)physiological functions of this enigmatic channel and as potential leads for development of anti-epileptic drugs. Since animal venoms are a rich source of potent ion channel modifiers that have been finely tuned by millions of year of evolution, we screened 108 arachnid venoms for hEAG1 inhibitors using electrophysiology. Two hit peptides (Aa1a and Ap1a) were isolated, sequenced, and chemically synthesised for structure-function studies. Both of these hEAG1 inhibitors are C-terminally amidated peptides containing an inhibitor Cystine Knot Motif, which provides them with exceptional stability in both plasma and cerebrospinal fluid. Aa1a and Ap1a are the most potent peptidic inhibitors of hEAG1 reported to date, and they present a novel mode of action by targeting both the activation and inactivation gating of the channel. These peptides should be useful pharmacological tools for probing hEAG1 function as well as informative leads for the development of novel anti-epileptic drugs.
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Modulatory features of the novel spider toxin μ-TRTX-Df1a isolated from the venom of the spider Davus fasciatus
British Journal of Pharmacology, 2017Co-Authors: Fernanda C. Cardoso, Irina Vetter, Volker Herzig, Glenn F. King, Paul F. Alewood, Jennifer J Smith, Zoltan Dekan, Jennifer R. Deuis, Richard J. LewisAbstract:Background and purpose Naturally occurring dysfunction in NaV channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel NaV modulators, we investigated spider venoms for new inhibitors of NaV channels. Experimental Approach We used high-throughput screens to identify a NaV modulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modeling and a rodent pain model. Key Results We identified a potent NaV inhibitor named μ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by NaV1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited CaV3 currents but had no activity against KV2. The modelled structure of Df1a, which contains an inhibitor Cystine Knot Motif, is reminiscent of the NaV channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all NaV subtypes tested (hNaV1.1–1.7). Df1a also slowed fast inactivation of NaV1.1, NaV1.3 and NaV1.5, and modified the voltage-dependence of activation and inactivation of most NaV subtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which likely explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviors induced by the NaV activator OD1. Conclusion and Implication μ-TRTX-Df1a shows potential as a new molecule for the development of therapies to treat voltage-gated ion channels mediated pain disorders.
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Characterization of Three Venom Peptides from the Spitting Spider Scytodes thoracica
PLOS ONE, 2016Co-Authors: Nathanial K. Ariki, Lisa E. Muñoz, Elizabeth L. Armitage, Francesca R. Goodstein, Kathryn G. George, Vanessa L. Smith, Irina Vetter, Volker Herzig, Glenn F. King, Nikolaus M. LoeningAbstract:We present the solution-state NMR structures and preliminary functional characterizations of three venom peptides identified from the spitting spider Scytodes thoracica. Despite little sequence identity to other venom peptides, structural characterization reveals that these peptides contain an inhibitor Cystine Knot Motif common to many venom peptides. These are the first structures for any peptide or protein from spiders of the Scytodidae family. Many venom peptides target neuronal ion channels or receptors. However, we have not been able to determine the target of these Scytodes peptides so we can only state with certainty the channels and receptors that they do not target.
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The insecticidal neurotoxin Aps III is an atypical Knottin peptide that potently blocks insect voltage-gated sodium channels
Biochemical Pharmacology, 2013Co-Authors: Niraj S. Bende, Volker Herzig, Mehdi Mobli, Graham M. Nicholson, Eunji Kang, Frank Bosmans, Glenn F. KingAbstract:One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical Knottin peptide; three of the disulfide bridges form a classical inhibitor Cystine Knot Motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel.
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The Neurotoxic Mode of Action of Venoms from the Spider Family Theraphosidae
Spider Ecophysiology, 2012Co-Authors: Volker Herzig, Glenn F. KingAbstract:Spiders from the family Theraphosidae are typically generalist predators of both invertebrates and vertebrates. Due to their large size and popularity in the pet trade, obtaining large amounts of a diverse range of theraphosid venoms is easier than for any other spider family. This explains why ~25 % of all spider toxins described to date originate from theraphosids even though they account for only 2 % of the taxonomic diversity of spiders. The main components of theraphosid venoms are neurotoxic peptides (cysteine-rich mini-proteins) that act on ion channels in the central or peripheral nervous system in order to induce rapid paralysis and/or death of prey. Their venoms also contain toxins that inhibit proteolytic degradation and enzymes that aid in the spread of other venom components. Some compounds in theraphosid venoms cause pain in vertebrates, making them useful for predator deterrence. Most theraphosid venom peptides contain three disulfide bonds that form an inhibitor Cystine Knot Motif, which endows these toxins with extreme chemical and thermal stability, as well as resistance to proteolytic degradation. These properties, as well as their high potency and selectivity for particular molecular targets, have made some theraphosid venom peptides valuable as pharmacological tools and as leads for therapeutics and bioinsecticides.
