The Experts below are selected from a list of 1341 Experts worldwide ranked by ideXlab platform
David J. Craik - One of the best experts on this subject based on the ideXlab platform.
-
evaluation of the in vivo aphrodisiac activity of a Cyclotide extract from hybanthus enneaspermus
Journal of Natural Products, 2020Co-Authors: Yen-hua Huang, Abhishek Bajpai, Majbrit Frosig Jorgensen, Guangzu Zhao, David J. CraikAbstract:Hybanthus enneaspermus is an Indian folk medicinal herb that has been widely used as a libido enhancer. This plant belongs to the Violaceae plant family, which ubiquitously contains disulfide-rich cyclic peptides named Cyclotides. Cyclotides are an expanding plant-derived peptide family with numerous interesting bioactivities, and their unusual stability against proteolysis has attracted much attention in drug design applications. Recently, H. enneaspermus has been reported to be a rich source of Cyclotides, and hence, it was of interest to investigate whether Cyclotides contribute to its aphrodisiac activity. In this study, we evaluated the in vivo aphrodisiac activity of the herbal powder, extract, and the most abundant Cyclotide, hyen D, extracted from H. enneaspermus on rats in a single dose regimen. After dosing, the sexual behaviors of male rats were observed, recorded, analyzed, and compared with those of the vehicle group. The results show that the extract and hyen D significantly decreased the intromission latency of sexually naive male rats and the extract improved a range of other measured sexual parameters. The results suggest that the extract could enhance libido as well as facilitate erectile function in male rats and that the Cyclotide hyen D could contribute to the libido-enhancing activity of this ethnomedicinal herb.
-
exploring the sequence diversity of Cyclotides from vietnamese viola species
Journal of Natural Products, 2020Co-Authors: Tien T Dang, Quentin Kaas, Yen-hua Huang, Lai Yue Chan, Linh T T Nguyen, Tien Huynh, David J. CraikAbstract:Viola is the largest genus in the Violaceae plant family and is known for its ubiquitous natural production of Cyclotides. Many Viola species are used as medicinal herbs across Asia and are often consumed by humans in teas for the treatment of diseases, including ulcers and asthma. Previous studies reported the isolation of Cyclotides from Viola species in many countries in the hope of discovering novel compounds with anti-cancer activities; however, Viola species from Vietnam have not been investigated to date. Here, the discovery of Cyclotides from three Viola species (V. arcuata, V. tonkinensis, and V. austrosinensis) collected in the northern mountainous region of Vietnam is reported. Ten Cyclotides were isolated from these three Viola species: four are novel and six were previously reported to be expressed in other plants. The structures of three of the new bracelet Cyclotides are similar to that of cycloviolacin O2. Because cycloviolacin O2 has previously been shown to have potent activity against a wide range of cancer cell lines including HeLa (human cervical cancer cells) and PC-3 (human prostate cancer cells), the cancer cytotoxicity of the Cyclotides isolated from V. arcuata was assessed. All tested Cyclotides were cytotoxic against cancer cells, albeit to varying degrees. The sequences discovered in this study significantly expand the understanding of Cyclotide diversity, especially in comparison with other Cyclotides found in plants from the Asian region.
-
co expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta
Journal of Experimental Botany, 2018Co-Authors: Simon Poon, David J. Craik, Karen S Harris, Thomas Durek, Edward K Gilding, Nicole L Van Der Weerden, Mark A Jackson, Owen C Mccorkelle, Marilyn A AndersonAbstract:Cyclotides are ultra-stable, backbone-cyclized plant defence peptides that have attracted considerable interest in the pharmaceutical industry. This is due to their range of native bioactivities as well as their ability to stabilize other bioactive peptides within their framework. However, a hindrance to their widespread application is the lack of scalable, cost-effective production strategies. Plant-based production is an attractive, benign option since all biosynthetic steps are performed in planta. Nonetheless, cyclization in non-Cyclotide-producing plants is poor. Here, we show that cyclic peptides can be produced efficiently in Nicotiana benthamiana, one of the leading plant-based protein production platforms, by co-expressing Cyclotide precursors with asparaginyl endopeptidases that catalyse peptide backbone cyclization. This approach was successful in a range of other plants (tobacco, bush bean, lettuce, and canola), either transiently or stably expressed, and was applicable to both native and engineered cyclic peptides. We also describe the use of the transgenic system to rapidly identify new asparaginyl endopeptidase cyclases and interrogate their substrate sequence requirements. Our results pave the way for exploiting Cyclotides for pest protection in transgenic crops as well as large-scale production of cyclic peptide pharmaceuticals in plants.
-
Understanding the Diversity and Distribution of Cyclotides from Plants of Varied Genetic Origin
2017Co-Authors: Anjaneya S. Ravipati, Michelle L Colgrave, Aaron G. Poth, Sónia Troeira Henriques, Murari Bhandari, Yen-hua Huang, Jaime Nino, David J. CraikAbstract:Cyclotides are a large family of naturally occurring plant-derived macrocyclic cystine-knot peptides, with more than 400 having been identified in species from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families. Nevertheless, their specialized distribution within the plant kingdom remains poorly understood. In this study, the diversity of Cyclotides was explored through the screening of 197 plants belonging to 43 different families. In total, 28 Cyclotides were sequenced from 15 plant species, one of which belonged to the Rubiaceae and 14 to the Violaceae. Every Violaceae species screened contained Cyclotides, but they were only sparsely represented in Rubiaceae and nonexistent in other families. The study thus supports the hypothesis that Cyclotides are ubiquitous in the Violaceae, and it adds to the list of plants found to express kalata S and cycloviolacin O12. Finally, previous studies suggested the existence of Cyclotide isoforms with either an Asn or an Asp at the C-terminal processing site of the Cyclotide domain within the precursor proteins. Here we found that despite the discovery of a few Cyclotides genuinely containing an Asp in loop 6 as evidenced by gene sequencing, deamidation of Asn during enzymatic digestion resulted in the artifactual presence of Asp isoforms. This result is consistent with studies suggesting that peptides can undergo deamidation after being subjected to external factors, including pH, temperature, and enzymatic digestion
-
isolation and characterization of Cyclotides from brazilian psychotria significance in plant defense and co occurrence with antioxidant alkaloids
Journal of Natural Products, 2016Co-Authors: Helio Nitta Matsuura, Aaron G. Poth, Anna Carolina Alves Yendo, Arthur Germano Fettneto, David J. CraikAbstract:Plants from the genus Psychotria include species bearing Cyclotides and/or alkaloids. The elucidation of factors affecting the metabolism of these molecules as well as their activities may help to understand their ecological function. In the present study, high concentrations of antioxidant indole alkaloids were found to co-occur with Cyclotides in Psychotria leiocarpa and P. brachyceras. The concentrations of the major Cyclotides and alkaloids in P. leiocarpa and P. brachyceras were monitored following herbivore- and pathogen-associated challenges, revealing a constitutive, phytoanticipin-like accumulation pattern. Psyleio A, the most abundant Cyclotide found in the leaves of P. leiocarpa, and also found in P. brachyceras leaves, exhibited insecticidal activity against Helicoverpa armigera larvae. Addition of ethanol in the vehicle for peptide solubilization in larval feeding trials proved deleterious to insecticidal activity and resulted in increased rates of larval survival in treatments containing ind...
Norelle L Daly - One of the best experts on this subject based on the ideXlab platform.
-
Structure and Activity of the Leaf-Specific Cyclotide vhl-2
2016Co-Authors: Norelle L Daly, Bin A Chen, Philip C Nguyencong, David J. CraikaAbstract:Cyclotides are plant-derived macrocyclic peptides with potential applications in the pharmaceutical and agricultural industries. In addition to their presumed natural function as host-defence peptides arising from their insecticidal activity, their other biological activities include antimicrobial, haemolytic, and cytotoxic activities, but at present, only limited information is available on the structural and chemical features that are important for these various activities. In the current study, we determined the three-dimensional structure of vhl-2, a leaf-specific Cyclotide. Although the characteristic cyclic cystine knot fold of other Cyclotides is maintained in vhl-2, it has more potent haemolytic activity than well-characterized Cyclotides such as kalata B1 and kalata B8. Analysis of surface hydrophobicity and haemolytic activity for a range of Cyclotides indicates a correlation between them, with increasing hydrophobicity resulting in increased haemolytic activity.This correlation is consistent with membrane binding being a vital step in mediating the various cytotoxic activities of Cyclotides. The gene sequence for vhl-2 was determined and indicates that vhl-2 is processed from a multidomain precursor protein that also encodes the Cyclotide cycloviolacin H3
-
efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase
Nature Communications, 2015Co-Authors: Karen S Harris, Quentin Kaas, Norelle L Daly, Aaron G. Poth, Ivana Saska, Thomas Durek, Edward K Gilding, Brendon Conlan, Nicole L Van Der Weerden, David J. CraikAbstract:Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of Cyclotide biosynthesis and confers a measure of stability on the resultant Cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the Cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis Cyclotide precursors, implicating additional proteases in Cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to Cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.
-
oxytocic plant Cyclotides as templates for peptide g protein coupled receptor ligand design
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Johannes Koehbach, Norelle L Daly, Margaret Obrien, Markus Muttenthaler, Marion Miazzo, Muharrem Akcan, Alysha G Elliott, Peta J Harvey, Sarah ArrowsmithAbstract:Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, Cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native Cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as “kalata-kalata,” the Cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a receptors, members of the G protein-coupled receptor family, as molecular targets for this Cyclotide. Furthermore, we show that Cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of Cyclotide-based peptide ligands.
-
phosphatidylethanolamine binding is a conserved feature of Cyclotide membrane interactions
Journal of Biological Chemistry, 2012Co-Authors: Sonia Troeira Henriques, Yen-hua Huang, Miguel A R B Castanho, Luis A Bagatolli, Secondo Sonza, Gilda Tachedjian, Norelle L DalyAbstract:Cyclotides are bioactive cyclic peptides isolated from plants that are characterized by a topologically complex structure and exceptional resistance to enzymatic or thermal degradation. With their sequence diversity, ultra-stable core structural motif, and range of bioactivities, Cyclotides are regarded as a combinatorial peptide template with potential applications in drug design. The mode of action of Cyclotides remains elusive, but all reported biological activities are consistent with a mechanism involving membrane interactions. In this study, a diverse set of Cyclotides from the two major subfamilies, Mobius and bracelet, and an all-d mirror image form, were examined to determine their mode of action. Their lipid selectivity and membrane affinity were determined, as were their toxicities against a range of targets (red blood cells, bacteria, and HIV particles). Although they had different membrane-binding affinities, all of the tested Cyclotides targeted membranes through binding to phospholipids containing phosphatidylethanolamine headgroups. Furthermore, the biological potency of the tested Cyclotides broadly correlated with their ability to target and disrupt cell membranes. The finding that a broad range of Cyclotides target a specific lipid suggests their categorization as a new lipid-binding protein family. Knowledge of their membrane specificity has the potential to assist in the design of novel drugs based on the Cyclotide framework, perhaps allowing the targeting of peptide drugs to specific cell types.
-
discovery of an unusual biosynthetic origin for circular proteins in legumes
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Aaron G. Poth, Michelle L Colgrave, Norelle L Daly, Russell E. Lyons, David J. CraikAbstract:Cyclotides are plant-derived proteins that have a unique cyclic cystine knot topology and are remarkably stable. Their natural function is host defense, but they have a diverse range of pharmaceutically important activities, including uterotonic activity and anti-HIV activity, and have also attracted recent interest as templates in drug design. Here we report an unusual biosynthetic origin of a precursor protein of a Cyclotide from the butterfly pea, Clitoria ternatea, a representative member of the Fabaceae plant family. Unlike all previously reported Cyclotides, the domain corresponding to the mature Cyclotide from this Fabaceae plant is embedded within an albumin precursor protein. We confirmed the expression and correct processing of the Cyclotide encoded by the Cter M precursor gene transcript following extraction from C. ternatea leaf and sequencing by tandem mass spectrometry. The sequence was verified by direct chemical synthesis and the peptide was found to adopt a classic knotted Cyclotide fold as determined by NMR spectroscopy. Seven additional Cyclotide sequences were also identified from C. ternatea leaf and flower, five of which were unique. Cter M displayed insecticidal activity against the cotton budworm Helicoverpa armigera and bound to phospholipid membranes, suggesting its activity is modulated by membrane disruption. The Fabaceae is the third largest family of flowering plants and many Fabaceous plants are of huge significance for human nutrition. Knowledge of Fabaceae Cyclotide gene transcripts should enable the production of modified Cyclotides in crop plants for a variety of agricultural or pharmaceutical applications, including plant-produced designer peptide drugs.
Christian W. Gruber - One of the best experts on this subject based on the ideXlab platform.
-
Cyclotides isolated from an ipecac root extract antagonize the corticotropin releasing factor type 1 receptor
Frontiers in Pharmacology, 2017Co-Authors: Christian W. Gruber, Mohsen Fahradpour, Peter Keov, Carlotta Tognola, Estela Perezsantamarina, Peter J Mccormick, Alireza GhassempourAbstract:Cyclotides are plant derived, cystine-knot stabilized peptides characterized by their natural abundance, sequence variability and structural plasticity. They are abundantly expressed in Rubiaceae, Psychotrieae in particular. Previously the Cyclotide kalata B7 was identified to modulate the human oxytocin and vasopressin G protein-coupled receptors (GPCRs), providing molecular validation of the plants’ uterotonic properties and further establishing Cyclotides as valuable sources for novel GPCR ligand design. In this study we screened a Cyclotide extract derived from the root powder of the South American medicinal plant ipecac (Carapichea ipecacuanha) for its GPCR modulating activity of the corticotropin-releasing factor type 1 receptor (CRF1R). We identified and characterized seven novel Cyclotides. One Cyclotide, caripe 8, isolated from the most active fraction, was further analyzed and found to antagonize the CRF1R. A nanomolar concentration of this Cyclotide (260 nM) reduced CRF potency by ~4.5-fold. In contrast, caripe 8 did not inhibit forskolin-, or vasopressin-stimulated cAMP responses at the vasopressin V2 receptor, suggesting a CRF1R-specific mode-of-action. These results in conjunction with our previous findings establish Cyclotides as modulators of both class A and class B GPCRs. Given the diversity of Cyclotides, our data point to other Cyclotide-GPCR interactions as potentially important sources of drug-like molecules.
-
Peptidomics of Circular Cysteine-Rich Plant Peptides: Analysis of the Diversity of Cyclotides from Viola tricolor by Transcriptome and Proteome Mining
2016Co-Authors: Johannes Koehbach, Douglas E Soltis, Eric J Carpenter, Gane Kashu Wong, Christian W. GruberAbstract:*S Supporting Information ABSTRACT: Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology Cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel Cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and Cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of Cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The Cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 Cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to a
-
Inhibition of Human Prolyl Oligopeptidase Activity by the Cyclotide Psysol 2 Isolated from Psychotria solitudinum
2016Co-Authors: Johannes Koehbach, Richard J Clark, Ernest Giralt, Teresa Tarrago, Christian W. GruberAbstract:ABSTRACT: 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 inhibitory activity appeared to be selective for POP, since neither psysol 2 nor kalata B1 were able to inhibit the proteolytic activity of trypsin or chymotrypsin. The enzyme POP is well known for its role in memory and learning processes, and it is currently being considered as a promisin
-
peptidomics of circular cysteine rich plant peptides analysis of the diversity of Cyclotides from viola tricolor by transcriptome and proteome mining
Journal of Proteome Research, 2015Co-Authors: Roland Hellinger, Johannes Koehbach, Douglas E Soltis, Eric J Carpenter, Gane Kashu Wong, Christian W. GruberAbstract:Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology Cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel Cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and Cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of Cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The Cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 Cyclotides were discovered by nucleic acid and peptide analysis in V. t...
-
Peptidomics of Circular Cysteine-Rich Plant Peptides: Analysis of the Diversity of Cyclotides from Viola tricolor by Transcriptome and Proteome Mining
2015Co-Authors: Roland Hellinger, Johannes Koehbach, Douglas E Soltis, Eric J Carpenter, Gane Kashu Wong, Christian W. GruberAbstract:Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology Cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel Cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and Cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of Cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The Cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 Cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to as many as 150 000 individual Cyclotides. Encompassing the diversity of V. tricolor as a combinatorial library of bioactive peptides, this commercially available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies
Johannes Koehbach - One of the best experts on this subject based on the ideXlab platform.
-
Inhibition of Human Prolyl Oligopeptidase Activity by the Cyclotide Psysol 2 Isolated from Psychotria solitudinum
2016Co-Authors: Johannes Koehbach, Richard J Clark, Ernest Giralt, Teresa Tarrago, Christian W. GruberAbstract:ABSTRACT: 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 inhibitory activity appeared to be selective for POP, since neither psysol 2 nor kalata B1 were able to inhibit the proteolytic activity of trypsin or chymotrypsin. The enzyme POP is well known for its role in memory and learning processes, and it is currently being considered as a promisin
-
Peptidomics of Circular Cysteine-Rich Plant Peptides: Analysis of the Diversity of Cyclotides from Viola tricolor by Transcriptome and Proteome Mining
2016Co-Authors: Johannes Koehbach, Douglas E Soltis, Eric J Carpenter, Gane Kashu Wong, Christian W. GruberAbstract:*S Supporting Information ABSTRACT: Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology Cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel Cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and Cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of Cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The Cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 Cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to a
-
peptidomics of circular cysteine rich plant peptides analysis of the diversity of Cyclotides from viola tricolor by transcriptome and proteome mining
Journal of Proteome Research, 2015Co-Authors: Roland Hellinger, Johannes Koehbach, Douglas E Soltis, Eric J Carpenter, Gane Kashu Wong, Christian W. GruberAbstract:Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology Cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel Cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and Cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of Cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The Cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 Cyclotides were discovered by nucleic acid and peptide analysis in V. t...
-
Peptidomics of Circular Cysteine-Rich Plant Peptides: Analysis of the Diversity of Cyclotides from Viola tricolor by Transcriptome and Proteome Mining
2015Co-Authors: Roland Hellinger, Johannes Koehbach, Douglas E Soltis, Eric J Carpenter, Gane Kashu Wong, Christian W. GruberAbstract:Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology Cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel Cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and Cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of Cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The Cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 Cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to as many as 150 000 individual Cyclotides. Encompassing the diversity of V. tricolor as a combinatorial library of bioactive peptides, this commercially available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies
-
Inhibition of Human Prolyl Oligopeptidase Activity by the Cyclotide Psysol 2 Isolated from Psychotria solitudinum
2015Co-Authors: Roland Hellinger, Richard J Clark, Johannes Koehbach, Albert Puigpinós, 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 inhibitory activity appeared to be selective for POP, since neither psysol 2 nor kalata B1 were able to inhibit the proteolytic activity of trypsin or chymotrypsin. The enzyme POP is well known for its role in memory and learning processes, and it is currently being considered as a promising therapeutic target for the cognitive deficits associated with several psychiatric and neurodegenerative diseases, such as schizophrenia and Parkinson’s disease. In the context of discovery and development of POP inhibitors with beneficial ADME properties, Cyclotides may be suitable starting points considering their stability in biological fluids and possible oral bioavailability
Craik, David J. - One of the best experts on this subject based on the ideXlab platform.
-
Discovery and mechanistic studies of cytotoxic Cyclotides from the medicinal herb Hybanthus enneaspermus
'American Society for Biochemistry & Molecular Biology (ASBMB)', 2020Co-Authors: Du Qingdan, Kaas Quentin, Chan, Lai Y., Gilding, Edward K., Henriques, Sónia Troeira, Condon, Nicholas D., Ravipati, Anjaneya S., Huang, Yen Hua, Craik, David J.Abstract:Cyclotides are plant-derived peptides characterized by an ∼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain Cyclotides. In the current study, we isolated 11 novel Cyclotides and 1 known Cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these Cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known Cyclotides. The most abundant Cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known Cyclotides. We also provide mechanistic insights into how these novel Cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these Cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin Cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered Cyclotides with potential anticancer activity
-
Discovery and mechanistic studies of cytotoxic Cyclotides from the medicinal herb Hybanthus enneaspermus
'American Society for Biochemistry & Molecular Biology (ASBMB)', 2020Co-Authors: Du Qingdan, Huang Yen-hua, Kaas Quentin, Chan, Lai Y., Gilding, Edward K., Henriques, Sónia Troeira, Condon, Nicholas D., Ravipati, Anjaneya S., Craik, David J.Abstract:Cyclotides are plant-derived peptides characterized by a ~30-amino-acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain Cyclotides. In the current study, we isolated 11 novel Cyclotides and one known Cyclotide (cycloviolacin O2) from and used tandem MS to determine their amino acid sequences. We found that among these Cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known Cyclotides. The most abundant Cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known Cyclotides. We also provide mechanistic insights into how these novel Cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, M, and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these Cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin Cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb and have uncovered Cyclotides with potential anticancer activity
-
Circular permutation of the native enzyme-mediated cyclization position in Cyclotides
'American Chemical Society (ACS)', 2020Co-Authors: Smithies, Bronwyn J., Huang Yen-hua, Gilding, Edward K., Jackson, Mark A., Yap Kuok, Harris, Karen S., Anderson, Marilyn A., Craik, David J.Abstract:Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate Cyclotide maturation. One of these ligases, OaAEP1b, originates from the Cyclotide-producing plant, , and can be produced recombinantly for use as an alternative to chemical cyclization of recombinant substrates. However, not all engineered Cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular Cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular Cyclotide for pharmaceutical applications, will be useful in designing bioactive Cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic Cyclotides alike
-
Harnessing Cyclotides to design and develop novel peptide GPCR ligands
'Royal Society of Chemistry (RSC)', 2020Co-Authors: Muratspahić Edin, Koehbach Johannes, Gruber, Christian W., Craik, David J.Abstract:Cyclotides are plant-derived cyclic, disulfide-rich peptides with a unique cyclic cystine knot topology that confers them with remarkable structural stability and resistance to proteolytic degradation. Recently, Cyclotides have emerged as promising scaffold molecules for designing peptide-based therapeutics. Here, we provide examples of how engineering Cyclotides using molecular grafting may lead to the development of novel peptide ligands of G protein-coupled receptors (GPCRs), today's most exploited drug targets. Integrating bioactive epitopes into stable Cyclotide scaffolds can lead to improved pharmacokinetics and oral activity as well as selectivity and high enzymatic stability. We also discuss and highlight the importance of engineered Cyclotides as novel tools to study GPCR signaling
-
Exploring the sequence diversity of Cyclotides from Vietnamese Viola species
'American Chemical Society (ACS)', 2020Co-Authors: Dang, Tien T., Huang Yen-hua, Kaas Quentin, Chan, Lai Y., Nguyen, Linh T. T., Huynh Tien, Craik, David J.Abstract:is the largest genus in the Violaceae plant family and is known for its ubiquitous natural production of Cyclotides. Many species are used as medicinal herbs across Asia and are often consumed by humans in teas for the treatment of diseases, including ulcers and asthma. Previous studies reported the isolation of Cyclotides from species in many countries in the hope of discovering novel compounds with anti-cancer activities; however, species from Vietnam have not been investigated to date. Here, the discovery of Cyclotides from three species (, , and ) collected in the northern mountainous region of Vietnam is reported. Ten Cyclotides were isolated from these three species: four are novel and six were previously reported to be expressed in other plants. The structures of three of the new bracelet Cyclotides are similar to that of cycloviolacin O2. Because cycloviolacin O2 has previously been shown to have potent activity against a wide range of cancer cell lines including HeLa (human cervical cancer cells) and PC-3 (human prostate cancer cells), the cancer cytotoxicity of the Cyclotides isolated from was assessed. All tested Cyclotides were cytotoxic against cancer cells, albeit to varying degrees. The sequences discovered in this study significantly expand the understanding of Cyclotide diversity, especially in comparison with other Cyclotides found in plants from the Asian region
