Comprehensive Mapping of Cyclotides from Viola philippica by Using Mass Spectrometry-Based Strategy.

Cyclotides are plant cyclic peptides with exceptional stability and diverse bioactivity, making them promising candidates for biomedical applications. Therefore, the study of cyclotides has attracted increasing attention in recent years. However, the existing cyclotide detection methods face limitations in sensitivity, accuracy, and reliability. To address these challenges, we developed an integrated strategy using a combination of strong cation exchange chromatography techniques for removing interfering small molecules, Orbitrap Exploris 480 mass spectrometry (OEMS); this is a detection and database searching-based method for cyclotide verification, which greatly improved the sensitivity, accuracy, and reliability of cyclotide identification. This strategy was subsequently employed for cyclotide mapping in Viola with a minute amount of starting tissue, resulting the identification of 65 known and 18 potentially novel cyclotides, which is the largest dataset of cyclotides for Viola philippica. This strategy provided valuable insights into the cyclotide diversity and distribution in V. philippica, with potential applications in drug discovery and other biomedical fields.

Characterization of cyclotides Mra30 and cycloviolacin O17 derived from Viola dalatensis Gadnep.

Bacteria threaten human and animal health, and standard antibiotics no longer effective. Antibiotic-resistant microorganisms can make infection treatment challenging and perhaps fail. Investigating the attributes of cyclotide, a peptide with promising antibacterial properties that holds great potential in the field of antibiotic research. The structure of these cyclic peptides involves six conserved cysteine residues that form three disulfide bonds, resulting in a cyclic cystine knot (CCK). This feature guarantees their durability when exposed to changes in temperature, chemicals, and enzymatic degradation. The two cyclotides, cycloviolacin O17 and mra30, were obtained from Viola dalatensis Gadnep through a series of techniques including the use of a 50% acetonitrile/49% miliQ water/1% formic acid solution for extraction, ammonium salt precipitation, RP-HPLC purification and sequence identification by LC-MS/MS. These cyclotides exhibit antibacterial effects on specific strains of bacteria like Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa at a concentration of 0.2 mg/mL, leading to inhibition zones ranging from 10 to 14 mm. In addition, the disulfide bonds play a crucial role in the antibacterial function of cyclotides. Disrupting the disulfide bonds through ankylation reaction results in the loss of antibacterial properties in the cyclotides (cyO17 and mra30). The minimum inhibitory concentration (MIC) values of mra30 and cyO17 are significantly low, ranging from 0.1 to 0.6 µM. These values are approximately three times lower than the MIC values observed in salt precipitation samples.

New Insights into Involvement of Low Molecular Weight Proteins in Complex Defense Mechanisms in Higher Plants.

Dynamic climate changes pose a significant challenge for plants to cope with numerous abiotic and biotic stressors of increasing intensity. Plants have evolved a variety of biochemical and molecular defense mechanisms involved in overcoming stressful conditions. Under environmental stress, plants generate elevated amounts of reactive oxygen species (ROS) and, subsequently, modulate the activity of the antioxidative enzymes. In addition, an increase in the biosynthesis of important plant compounds such as anthocyanins, lignin, isoflavonoids, as well as a wide range of low molecular weight stress-related proteins (e.g., dehydrins, cyclotides, heat shock proteins and pathogenesis-related proteins), was evidenced. The induced expression of these proteins improves the survival rate of plants under unfavorable environmental stimuli and enhances their adaptation to sequentially interacting stressors. Importantly, the plant defense proteins may also have potential for use in medical applications and agriculture (e.g., biopesticides). Therefore, it is important to gain a more thorough understanding of the complex biological functions of the plant defense proteins. It will help to devise new cultivation strategies, including the development of genotypes characterized by better adaptations to adverse environmental conditions. The review presents the latest research findings on selected plant defense proteins.

The involvement of cyclotides in the heavy metal tolerance of Viola spp.

The Violaceae family is rich in metal-tolerant species and species producing cyclic peptides (cyclotides) that are linked to the resistance to biotic factors. Plants that inhabit areas polluted with heavy metals have developed various mechanisms of tolerance. To test the role of cyclotides in protection against abiotic factors, including heavy metals, cell suspension cultures of Viola species/genotypes (V. lutea ssp. westfalica, V. tricolor, V. arvensis, and V. uliginosa), representing different levels of tolerance to heavy metals (from the most tolerant-MET to the least tolerant populations/species-NMET), were used. The relative abundances of the cyclotides in the control, untreated cell suspensions of all the selected species/genotypes, and cells treated with Zn or Pb (200 µM or 2000 µM) for 24 h or 72 h were determined via MALDI-MS. Transmission electron microscopy with X-ray microanalysis was used to detect putative co-localization of the cyclotides with Zn or Pb in the cells of V. tricolor treated with the highest concentration of heavy metals for 72 h. Cyclotide biosynthesis was dependent on the type of heavy metal and its concentration, time of treatment, plant species, and population type (MET vs. NMET). It was positively correlated with the level of tolerance of particular Viola species. The increased production of cyclotides was observed in the cells of metallophyte species, mostly in Zn-treated cells. The nonmetallophyte-V. uliginosa presented a decrease in the production of cyclotides independent of the dose and duration of the metal treatment. Cyclotides co-localized with Pb more evidently than with Zn, suggesting that cyclotides have heavy metal affinity. V. lutea ssp. westfalica transcriptome mining yielded 100 cyclotide sequences, 16 known and 84 novel named viwe 1-84. These findings support the hypothesis that cyclotides are involved in certain mechanisms of plant tolerance to heavy metals.

Structure and Activity of Reconstructed Pseudo-Ancestral Cyclotides.

Cyclotides are cyclic peptides that are promising scaffolds for the design of drug candidates and chemical tools. However, despite there being hundreds of reported cyclotides, drug design studies have commonly focussed on a select few prototypic examples. Here, we explored whether ancestral sequence reconstruction could be used to generate new cyclotides for further optimization. We show that the reconstructed 'pseudo-ancestral' sequences, named Ancy-m (for the ancestral cyclotide of the Möbius sub-family) and Ancy-b (for the bracelet sub-family), have well-defined structures like their extant members, comprising the core structural feature of a cyclic cystine knot. This motif underpins efforts to re-engineer cyclotides for agrochemical and therapeutic applications. We further show that the reconstructed sequences are resistant to temperatures approaching boiling, bind to phosphatidyl-ethanolamine lipid bilayers at micromolar affinity, and inhibit the growth of insect cells at inhibitory concentrations in the micromolar range. Interestingly, the Ancy-b cyclotide had a higher oxidative folding yield than its comparator cyclotide cyO2, which belongs to the bracelet cyclotide subfamily known to be notoriously difficult to fold. Overall, this study provides new cyclotide sequences not yet found naturally that could be valuable starting points for the understanding of cyclotide evolution and for further optimization as drug leads.

Immunosuppressive Cyclotides: A Promising Approach for Treating Autoimmune Diseases.

The immune system maintains constant surveillance to prevent the infiltration of both endogenous and exogenous threats into host organisms. The process is regulated by effector immune cells that combat external pathogens and regulatory immune cells that inhibit excessive internal body inflammation, ultimately establishing a state of homeostasis within the body. Disruption to this process could lead to autoimmunity, which is often associated with the malfunction of both T cells and B cells with T cells playing a more major role. A number of therapeutic mediators for autoimmune diseases are available, from conventional disease-modifying drugs to biologic agents and small molecule inhibitors. Recently, ribosomally synthesized peptides, specifically cyclotides from plants are currently attracting more attention as potential autoimmune disease therapeutics due to their decreased toxicity compared to small molecules inhibitors as well as their remarkable stability against a number of factors. This review provides a concise overview of various cyclotides exhibiting immunomodulatory properties and their potential as therapeutic interventions for autoimmune diseases.

Nucleation of a key beta-turn promotes cyclotide oxidative folding.

Cyclotides are plant-derived peptides characterized by a head-to-tail cyclic backbone and a cystine knot motif comprised of three disulfide bonds. Formation of this motif via in vitro oxidative folding can be challenging and can result in misfolded isomers with nonnative disulfide connectivities. Here, we investigated the effect of ß-turn nucleation on cyclotide oxidative folding. Two types of ß-turn mimics were grafted into kalata B1, individually replacing each of the four ß-turns in the folded cyclotide. Insertion of d-Pro-Gly into loop 5 was beneficial to the folding of both cyclic kB1 and a linear form of the peptide. The linear grafted analog folded four-times faster in aqueous conditions than cyclic kB1 in optimized conditions. Additionally, the cyclic analogue folded without the need for redox agents by transitioning through a native-like intermediate that was on-pathway to product formation. Kalata B1 is from the Möbius subfamily of cyclotides. Grafting d-Pro-Gly into loop 5 of cyclotides from two other subfamilies also had a beneficial effect on folding. Our findings demonstrate the importance of a ß-turn nucleation site for cyclotide oxidative folding, which could be adopted as a chemical strategy to improve the in vitro folding of diverse cystine-rich peptides.

In Silico Analysis of Natural Plant-Derived Cyclotides with Antifungal Activity against Pathogenic Fungi.

BACKGROUND: Fungal infections in plants, animals, and humans are widespread across the world. Limited classes of antifungal drugs to treat fungal infections and loss of drug efficacy due to rapidly evolving fungal strains pose a challenge in the agriculture and health sectors. Hence, the search for a new class of antifungal agents is imperative. Cyclotides are cyclic plant peptides with multiple bioactivities, including antifungal activity. They have six conserved cysteine residues forming three disulfide linkages (CI-CIV, CII-CV, CIII-CVI) that establish a Cyclic Cystine Knot (CCK) structure, making them extremely resistant to chemical, enzymatic, and thermal attacks. AIM: This in silico analysis of natural, plant-derived cyclotides aimed to assess the parameters that can assist and hasten the process of selecting the cyclotides with potent antifungal activity and prioritize them for in vivo/ in vitro experiments. OBJECTIVE: The objective of this study was to conduct in silico studies to compare the physicochemical parameters, sequence diversity, surface structures, and membrane-cyclotide interactions of experimentally screened (from literature survey) potent (MIC ≤ 20 µM) and non-potent (MIC > 20 µM) cyclotides for antifungal activity. METHODOLOGY: Cyclotide sequences assessed for antifungal activity were retrieved from the database (Cybase). Various online and offline tools were used for sequence-based studies, such as physicochemical parameters, sequence diversity, and neighbor-joining trees. Structure-based studies involving surface structure analysis and membrane-cyclotide interaction were also carried out. All investigations were conducted in silico. RESULTS: Physicochemical parameter values, viz. isoelectric point, net charge, and the number of basic amino acids, were significantly higher in potent cyclotides compared to non-potent cyclotides. The surface structure of potent cyclotides showed a larger hydrophobic patch with a higher number of hydrophobic amino acids. Furthermore, the membrane-cyclotide interaction studies of potent cyclotides revealed lower transfer free energy (ΔG transfer) and higher penetration depth into fungal membranes, indicating higher binding stability and membrane-disruption ability. CONCLUSION: These in silico studies can be applied for rapidly identifying putatively potent antifungal cyclotides for in vivo and in vitro experiments, which will ultimately be relevant in the agriculture and pharmaceutical sectors.

Cyclotides derived from Viola dalatensis Gagnep: A novel approach for enrichment and evaluation of antimicrobial activity.

Cyclotides, plant-derived cysteine-rich peptides, exhibit a wide range of beneficial biological activities and possess exceptional structural stability. Cyclotides are commonly distributed throughout the Violaceae family. Viola dalatensis Gagnep, a Vietnamese species, has not been well studied, especially for cyclotides. This pioneering research explores cyclotides from V. dalatensis as antimicrobials. This study used a novel approach to enhance cyclotides after extraction. The approach combined 30% ammonium sulfate salt precipitation and RP-HPLC. A comprehensive analysis was performed to ascertain the overall protein content, flavonoids content, polyphenol content, and free radical scavenging capacity of compounds derived from V. dalatensis. Six known cyclotides were sequenced utilizing MS tandem. Semi-purified cyclotide mixtures (M1, M2, and M3) exhibited antibacterial efficacy against Bacillus subtilis (inhibitory diameters: 19.67-23.50 mm), Pseudomonas aeruginosa (22.17-23.50 mm), and Aspergillus flavus (14.67-21.33 mm). The enriched cyclotide precipitate from the stem extract demonstrated a minimum inhibitory concentration (MIC) of 0.08 mg/mL against P. aeruginosa, showcasing significant antibacterial effectiveness compared to the stem extract (MIC: 12.50 mg/mL). Considerable advancements have been achieved in the realm of cyclotides, specifically in their application as antimicrobial agents.

Methods for extraction, isolation and sequencing of cyclotides and others cyclic peptides with anti-helminthic activities: An overview.

The mortality rate caused by parasitic worms on their hosts is of great concern and studies have been carried out to find molecules to reduce the prevalence, host-parasite interaction, and resistance of parasites to treatments. Existing drugs on the market are very often toxic and have many side effects, hence the need to find new, more active molecules. It has been demonstrated in several works that medicinal plants constitute a wide range of new molecules that can solve this problem. Several works have already been able to demonstrate that cyclic peptides of plant origin have shown good activity in the fight against different types of helminths. Therefore, this review aims to provide a general overview of the methods and techniques of extraction, isolation, activities and mechanisms of action of cyclotides and other cyclic peptides for application in the treatment of helminthic infections.

High-throughput microfluidic chip with silica gel-C18 channels for cyclotide separation.

Over the past two decades, microfluidic-based separations have been used for the purification, isolation, and separation of biomolecules to overcome difficulties encountered by conventional chromatography-based methods including high cost, long processing times, sample volumes, and low separation efficiency. Cyclotides, or cyclic peptides used by some plant families as defense agents, have attracted the interest of scientists because of their biological activities varying from antimicrobial to anticancer properties. The separation process has a critical impact in terms of obtaining pure cyclotides for drug development strategies. Here, for the first time, a mimic of the high-performance liquid chromatography (HPLC) on microfluidic chip strategy was used to separate the cyclotides. In this regard, silica gel-C18 was synthesized and characterized by Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) and then filled inside the microchannel to prepare an HPLC C18 column-like structure inside the microchannel. Cyclotide extract was obtained from Viola ignobilis by a low voltage electric field extraction method and characterized by HPLC and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). The extract that contained vigno 1, 2, 3, 4, 5, and varv A cyclotides was added to the microchannel where distilled water was used as a mobile phase with 1 µL/min flow rate and then samples were collected in 2-min intervals until 10 min. Results show that cyclotides can be successfully separated from each other and collected from the microchannel at different periods of time. These findings demonstrate that the use of microfluidic channels has a high impact on the separation of cyclotides as a rapid, cost-effective, and simple method and the device can find widespread applications in drug discovery research.

Cyclotide nanotubes as a novel potential Drug-Delivery System: Characterization and biocompatibility.

Cyclotides are a class of cyclic peptides that can be self-assembled. This study aimed to discover the properties of cyclotide nanotubes. We performed differential scanning calorimetric (DSC) to characterize their properties. Then, we incorporated the coumarin as a probe and identified the morphology of nanostructures. The stability of cyclotide nanotubes after 3 months of keeping at -20 °C was determined by field emission scanning electron microscopy (FESEM). The cytocompatibility of cyclotide nanotubes was evaluated on peripheral blood mononuclear cells. In vivo, studies were also conducted on female C57BL/6 mice by intraperitoneally administration of nanotubes at 5, 50, and 100 mg/kg doses. Blood sampling was done before and 24 h after nanotube administration and complete blood count tests were conducted. DSC thermogram showed that the cyclotide nanotubes were stable after heating until 200 °C. Fluorescence microscopy images proved that the self-assembled structures of cyclotide can encapsulate the coumarin. FESEM proved that these nanotubes were stable even after 3 months. The results of the cytotoxicity assay and in-vivo study confirmed that these novel prepared nanotubes were biocompatible. These results suggested that the cyclotide nanotubes could be considered as a new carrier in biological fields while they are biocompatible.

Insights into the synthesis strategies of plant-derived cyclotides.

Cyclotides are plant peptides characterized with a head-to-tail cyclized backbone and three interlocking disulfide bonds, known as a cyclic cysteine knot. Despite the variations in cyclotides peptide sequences, this core structure is conserved, underlying their most useful feature: stability against thermal and chemical breakdown. Cyclotides are the only natural peptides known to date that are orally bioavailable and able to cross cell membranes. Cyclotides also display bioactivities that have been exploited and expanded to develop as potential therapeutic reagents for a wide range of conditions (e.g., HIV, inflammatory conditions, multiple sclerosis, etc.). As such, in vitro production of cyclotides is of the utmost importance since it could assist further research on this peptide class, specifically the structure-activity relationship and its mechanism of action. The information obtained could be utilized to assist drug development and optimization. Here, we discuss several strategies for the synthesis of cyclotides using both chemical and biological routes.

Cysteine-rich peptides: From bioactivity to bioinsecticide applications.

Greater levels of insect resistance and constraints on the use of current pesticides have recently led to increased crop losses in agricultural production. Further, the health and environmental impacts of pesticides now restrict their application. Biologics based on peptides are gaining popularity as efficient crop protection agents with low environmental toxicity. Cysteine-rich peptides (whether originated from venoms or plant defense substances) are chemically stable and effective as insecticides in agricultural applications. Cysteine-rich peptides fulfill the stability and efficacy requirements for commercial uses and provide an environmentally benign alternative to small-molecule insecticides. In this article, cysteine-rich insecticidal peptide classes identified from plants and venoms will be highlighted, focusing on their structural stability, bioactivity and production.

Native and Engineered Cyclic Disulfide-Rich Peptides as Drug Leads.

Bioactive peptides are a highly abundant and diverse group of molecules that exhibit a wide range of structural and functional variation. Despite their immense therapeutic potential, bioactive peptides have been traditionally perceived as poor drug candidates, largely due to intrinsic shortcomings that reflect their endogenous heritage, i.e., short biological half-lives and poor cell permeability. In this review, we examine the utility of molecular engineering to insert bioactive sequences into constrained scaffolds with desired pharmaceutical properties. Applying lessons learnt from nature, we focus on molecular grafting of cyclic disulfide-rich scaffolds (naturally derived or engineered), shown to be intrinsically stable and amenable to sequence modifications, and their utility as privileged frameworks in drug design.

Molecular Peptide Grafting as a Tool to Create Novel Protein Therapeutics.

The study of peptides (synthetic or corresponding to discrete regions of proteins) has facilitated the understanding of protein structure-activity relationships. Short peptides can also be used as powerful therapeutic agents. However, the functional activity of many short peptides is usually substantially lower than that of their parental proteins. This is (as a rule) due to their diminished structural organization, stability, and solubility often leading to an enhanced propensity for aggregation. Several approaches have emerged to overcome these limitations, which are aimed at imposing structural constraints into the backbone and/or sidechains of the therapeutic peptides (such as molecular stapling, peptide backbone circularization and molecular grafting), therefore enforcing their biologically active conformation and thus improving their solubility, stability, and functional activity. This review provides a short summary of approaches aimed at enhancing the biological activity of short functional peptides with a particular focus on the peptide grafting approach, whereby a functional peptide is inserted into a scaffold molecule. Intra-backbone insertions of short therapeutic peptides into scaffold proteins have been shown to enhance their activity and render them a more stable and biologically active conformation.

Effects of a plant cyclotide on conformational dynamics and destabilization of ß-amyloid fibrils through molecular dynamics simulations.

Aggregation of ß-amyloid (Aß) peptide is one of the hallmarks of Alzheimer's disease (AD) which results in chronic and progressive neurodegeneration of the brain. A recent study by our group have shown the ability of cyclic disulfide-rich peptides ("cyclotides") isolated from a medicinal plant, Clitoria ternatea, to inhibit the aggregation of Aß peptides and reduce oxidative stress caused by reactive oxygen species using in vivo models of transgenic Caenorhabditis elegans. In the present study, through extensive computational docking and multi-ns molecular dynamics (MD) simulation, we evaluated if cyclotides can stably bind to Aß molecules and/or destabilize the Aß fibril by preventing conformational changes from α-helical to ß-sheet rich structures. We demonstrate that cyclotides bind effectively and stably to different forms of Aß structures via hydrogen bonding and hydrophobic interactions. One of the conserved hydrophobic interface residues, Tyr10 was mutated to Ala and the impact of this virtual mutation was estimated by additional MD simulations for the wild-type (WT) and mutant protein-peptide complexes. A detailed MD simulation analyses revealed that cyclotides form hydrogen bonds with the toxic amyloid assemblies thereby weakening the inter-strand hydrogen bonds between the Aß peptide. The φ-ѱ distribution map of residues in the cyclotide binding pocket that ideally adopt ß-sheet conformation show deviation towards right-handed ɑ-helical (ɑR) conformation. This effect was similar to that observed for the Tyr10Ala mutant and doubly so, for the cyclotide bound form. It is therefore possible to hypothesise that the opening up of amyloid ß-sheet is due to an unfolding process occurring in the Aß caused by cyclotide binding and inhibition. Our current findings provide novel structural insights on the mode of interaction between cyclotides and Aß fibrils and describe their anti-amyloid aggregation potential. This sheds light on the future of cyclotide-based drug design against protein aggregation, a hallmark event in many neurodegenerative diseases.

Using the Cyclotide Scaffold for Targeting Biomolecular Interactions in Drug Development.

This review provides an overview of the properties of cyclotides and their potential for developing novel peptide-based therapeutics. The selective disruption of protein-protein interactions remains challenging, as the interacting surfaces are relatively large and flat. However, highly constrained polypeptide-based molecular frameworks with cell-permeability properties, such as the cyclotide scaffold, have shown great promise for targeting those biomolecular interactions. The use of molecular techniques, such as epitope grafting and molecular evolution employing the cyclotide scaffold, has shown to be highly effective for selecting bioactive cyclotides.

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila.

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

In vitro Inhibition of HIV-1 by Cyclotide-Enriched Extracts of Viola tricolor.

Since viral infectious diseases continue to be a global health threat, new antiviral drugs are urgently needed. A unique class of therapeutic compounds are antimicrobial peptides (AMPs). They can be found in humans, bacteria and plants. Plants express a wide variety of such defense peptides as part of their innate immune system to protect from invading pathogens. Cyclotides are non-classical AMPs that share a similar structure. Their unique topology consists of a circular peptide backbone and disulfide bonds. In previous studies they have been attributed to a wide range of biological activities. To identify novel cyclotides with antiviral activity, we established a library of plant extracts largely consisting of cyclotide-rich species and screened them as inhibitors of HIV-1 infection. Subsequent extraction and fractionation revealed four cyclotide-containing subfractions from Viola tricolor with antiviral activity. These subfractions inhibited HIV-1 infection with IC50 values between 0.6 and 11.2 µg/ml, and selectivity indices of up to 8.1. The identification and characterization of antiviral cyclotides and the determination of the antiviral mechanisms may allow to develop novel agents to combat viral infections. Therefore, cyclotides represent a natural source of bioactive molecules with prospects for development as therapeutics.