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1.
Sci Rep ; 14(1): 9733, 2024 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-38679643

RESUMO

Cyclotides are a type of defense peptide most commonly found in the Violaceae family of plants, exhibiting various biological activities. In this study, we focused on the Viola japonica as our research subject and conducted transcriptome sequencing and analysis using high-throughput transcriptomics techniques. During this process, we identified 61 cyclotides, among which 25 were previously documented, while the remaining 36 were designated as vija 1 to vija 36. Mass spectrometry detection showed that 21 putative cyclotides were found in the extract of V. japonica. Through isolation, purification and tandem mass spectrometry, we characterized and investigated the activities of five cyclotides. Our results demonstrated inhibitory effects of these cyclotides on the growth of Acinetobacter baumannii and Bacillus subtilis, with minimum inhibitory concentrations (MICs) of 4.2 µM and 2.1 µM, respectively. Furthermore, time killing kinetic assays revealed that cyclotides at concentration of 4 MICs achieved completely bactericidal effects within 2 h. Additionally, fluorescence staining experiments confirmed that cyclotides disrupt microbial membranes. Moreover, cytotoxicity studies showed that cyclotides possess cytotoxic effects, with IC50 values ranging from 0.1 to 3.5 µM. In summary, the discovery of new cyclotide sequences enhances our understanding of peptide diversity and the exploration of their activity lays the foundation for a deeper investigation into the mechanisms of action of cyclotides.


Assuntos
Acinetobacter baumannii , Bacillus subtilis , Ciclotídeos , Testes de Sensibilidade Microbiana , Viola , Ciclotídeos/farmacologia , Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Viola/química , Acinetobacter baumannii/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química
2.
J Biol Chem ; 300(6): 107330, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38679329

RESUMO

The cannabinoid type 2 receptor (CB2R), a G protein-coupled receptor, is an important regulator of immune cell function and a promising target to treat chronic inflammation and fibrosis. While CB2R is typically targeted by small molecules, including endo-, phyto-, and synthetic cannabinoids, peptides-owing to their size-may offer a different interaction space to facilitate differential interactions with the receptor. Here, we explore plant-derived cyclic cystine-knot peptides as ligands of the CB2R. Cyclotides are known for their exceptional biochemical stability. Recently, they gained attention as G protein-coupled receptor modulators and as templates for designing peptide ligands with improved pharmacokinetic properties over linear peptides. Cyclotide-based ligands for CB2R were profiled based on a peptide-enriched extract library comprising nine plants. Employing pharmacology-guided fractionation and peptidomics, we identified the cyclotide vodo-C1 from sweet violet (Viola odorata) as a full agonist of CB2R with an affinity (Ki) of 1 µM and a potency (EC50) of 8 µM. Leveraging deep learning networks, we verified the structural topology of vodo-C1 and modeled its molecular volume in comparison to the CB2R ligand binding pocket. In a fragment-based approach, we designed and characterized vodo-C1-based bicyclic peptides (vBCL1-4), aiming to reduce size and improve potency. Opposite to vodo-C1, the vBCL peptides lacked the ability to activate the receptor but acted as negative allosteric modulators or neutral antagonists of CB2R. This study introduces a macrocyclic peptide phytocannabinoid, which served as a template for the development of synthetic CB2R peptide modulators. These findings offer opportunities for future peptide-based probe and drug development at cannabinoid receptors.


Assuntos
Receptor CB2 de Canabinoide , Receptor CB2 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/química , Humanos , Ligantes , Ciclotídeos/química , Ciclotídeos/farmacologia , Células HEK293 , Descoberta de Drogas
3.
Protein J ; 43(2): 159-170, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38485875

RESUMO

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.


Assuntos
Doenças Autoimunes , Ciclotídeos , Humanos , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Ciclotídeos/uso terapêutico , Ciclotídeos/química , Ciclotídeos/farmacologia , Imunossupressores/uso terapêutico , Imunossupressores/farmacologia , Animais
4.
Protein Pept Lett ; 31(3): 247-260, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38445693

RESUMO

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.


Assuntos
Antifúngicos , Ciclotídeos , Fungos , Ciclotídeos/química , Ciclotídeos/farmacologia , Antifúngicos/farmacologia , Antifúngicos/química , Fungos/efeitos dos fármacos , Simulação por Computador , Testes de Sensibilidade Microbiana , Sequência de Aminoácidos , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia
5.
J Med Chem ; 67(2): 1197-1208, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38174919

RESUMO

Peptides are promising drug modalities that can modulate protein-protein interactions, but their application is hampered by their limited ability to reach intracellular targets. Here, we improved the cytosolic delivery of a peptide blocking p53:MDM2/X interactions using a cyclotide as a stabilizing scaffold. We applied several design strategies to improve intracellular delivery and found that the conjugation of the lead cyclotide to the cyclic cell-penetrating peptide cR10 was the most effective. Conjugation allowed cell internalization at micromolar concentration and led to elevated intracellular p53 levels in A549, MCF7, and MCF10A cells, as well as inducing apoptosis in A549 cells without causing membrane disruption. The lead peptide had >35-fold improvement in inhibitory activity and increased cellular uptake compared to a previously reported cyclotide p53 activator. In summary, we demonstrated the delivery of a large polar cyclic peptide in the cytosol and confirmed its ability to modulate intracellular protein-protein interactions involved in cancer.


Assuntos
Peptídeos Penetradores de Células , Ciclotídeos , Neoplasias , Humanos , Ciclotídeos/farmacologia , Ciclotídeos/metabolismo , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/metabolismo
6.
Toxicon ; 239: 107606, 2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38181837

RESUMO

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.


Assuntos
Ciclotídeos , Viola , Ciclotídeos/farmacologia , Ciclotídeos/química , Viola/química , Viola/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Antibacterianos/química , Vietnã
7.
J Biol Chem ; 300(3): 105682, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38272233

RESUMO

Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of ∼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.


Assuntos
Ciclotídeos , Inseticidas , Oldenlandia , Ciclotídeos/genética , Ciclotídeos/farmacologia , Ciclotídeos/química , Inseticidas/química , Inseticidas/farmacologia , Leucina , Lisina/genética , Mutagênese , Proteínas de Plantas/metabolismo , Oldenlandia/química , Estabilidade Proteica , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos
8.
Parasitol Int ; 98: 102808, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37717651

RESUMO

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.


Assuntos
Ciclotídeos , Parasitos , Plantas Medicinais , Animais , Ciclotídeos/farmacologia , Ciclotídeos/química , Peptídeos Cíclicos/farmacologia , Plantas Medicinais/química
9.
J Control Release ; 359: 26-32, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37236320

RESUMO

The CXCR4 chemokine is a key molecular regulator of many biological functions controlling leukocyte functions during inflammation and immunity, and during embryonic development. Overexpression of CXCR4 is also associated with many types of cancer where its activation promotes angiogenesis, tumor growth/survival, and metastasis. In addition, CXCR4 is involved in HIV replication, working as a co-receptor for viral entry, making CXCR4 a very attractive target for developing novel therapeutic agents. Here we report the pharmacokinetic profile in rats of a potent CXCR4 antagonist cyclotide, MCo-CVX-5c, previously developed in our group that displayed a remarkable in vivo resistance to biological degradation in serum. This bioactive cyclotide, however, was rapidly eliminated through renal clearance. Several lipidated versions of cyclotide MCo-CVX-5c showed a significant increase in the half-life when compared to the unlipidated form. The palmitoylated version of cyclotide MCo-CVX-5c displayed similar CXCR4 antagonistic activity as the unlipidated cyclotide, while the cyclotide modified with octadecanedioic (18-oxo-octadecanoic) acid exhibited a remarkable decrease in its ability to antagonize CXCR4. Similar results were also obtained when tested for its ability to inhibit growth in two cancer cell lines and HIV infection in cells. These results show that the half-life of cyclotides can be improved by lipidation although it can also affect their biological activity depending on the lipid employed.


Assuntos
Ciclotídeos , Infecções por HIV , Neoplasias , Ratos , Animais , Ciclotídeos/farmacologia , Linhagem Celular , Receptores CXCR4
10.
Amino Acids ; 55(6): 713-729, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37142771

RESUMO

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.


Assuntos
Ciclotídeos , Ciclotídeos/farmacologia , Ciclotídeos/uso terapêutico , Ciclotídeos/química , Sequência de Aminoácidos , Plantas/metabolismo , Cisteína , Relação Estrutura-Atividade
11.
J Nat Prod ; 86(5): 1222-1229, 2023 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-37099442

RESUMO

Cyclotides are a unique family of stable and cyclic mini-proteins found in plants that have nematicidal and anthelmintic activities. They are distributed across the Rubiaceae, Violaceae, Fabaceae, Cucurbitaceae, and Solanaceae plant families, where they are posited to act as protective agents against pests. In this study, we tested the nematicidal properties of extracts from four major cyclotide-producing plants, Oldenlandia affinis, Clitoria ternatea, Viola odorata, and Hybanthus enneaspermus, against the free-living model nematode Caenorhabditis elegans. We evaluated the nematicidal activity of the cyclotides kalata B1, cycloviolacin O2, and hyen D present in these extracts and found them to be active against the larvae of C. elegans. Both the plant extracts and isolated cyclotides exerted dose-dependent toxicity on the first-stage larvae of C. elegans. Isolated cyclotides caused death or damage upon interacting with the worms' mouth, pharynx, and midgut or membrane. Cycloviolacin O2 and hyen D produced bubble-like structures around the C. elegans membrane, termed blebs, implicating membrane disruption causing toxicity and death. All tested cyclotides lost their toxicity when the hydrophobic patches present on them were disrupted via a single-point mutation. The present results provide a facile assay design to measure and explore the nematicidal activities of plant extracts and purified cyclotides on C. elegans.


Assuntos
Ciclotídeos , Fabaceae , Nematoides , Violaceae , Animais , Antinematódeos/farmacologia , Caenorhabditis elegans , Ciclotídeos/farmacologia , Ciclotídeos/química , Fabaceae/química , Extratos Vegetais/química , Proteínas de Plantas/química
12.
Biomed Pharmacother ; 153: 113486, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36076504

RESUMO

Ribosomally synthesized and post-translationally modified peptides, such as plant cyclotides, are a diverse group of natural products well known as templates in drug discovery and therapeutic lead development. The cyclotide kalata B1 (kB1) has previously been discovered as immunosuppressive agent on T-lymphocytes, and a synthetic version of this peptide, [T20K]kB1 (T20K), has been effective in reducing clinical symptoms, such as inflammation and demyelination, in a mouse model of multiple sclerosis. Based on its T-cell modulatory impact we studied the effects of T20K and several analogs on the proliferation of anaplastic large cell lymphoma (ALCL), a heterogeneous group of clinically aggressive diseases associated with poor prognosis. T20K, as a prototype drug candidate, induces apoptosis and a proliferation arrest in human lymphoma T-cell lines (SR786, Mac-2a and the Jurkat E6.1) in a concentration dependent fashion, at least partially via increased STAT5 and p53 signaling. In contrary to its effect on IL-2 signaling in lymphocytes, the cytokine levels are not altered in lymphoma cells. In vivo mouse experiments revealed a promising activity of T20K on these cancer cells including decreased tumor weight and increased apoptosis. This study opens novel avenues for developing cyclotide-based drug candidates for therapy of patients with ALCL.


Assuntos
Ciclotídeos , Linfoma Anaplásico de Células Grandes , Animais , Ciclotídeos/farmacologia , Citocinas/farmacologia , Humanos , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Camundongos , Linfócitos T
13.
J Nat Prod ; 85(9): 2127-2134, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36044031

RESUMO

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.


Assuntos
Produtos Biológicos , Ciclotídeos , Proteínas de Plantas , Violaceae , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Brasil , Linhagem Celular Tumoral , Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Ciclotídeos/farmacologia , Humanos , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Espectrometria de Massas em Tandem , Violaceae/química
14.
J Biol Chem ; 298(10): 102413, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36007611

RESUMO

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.


Assuntos
Ciclotídeos , Drosophila melanogaster , Inseticidas , Proteínas de Plantas , Violaceae , Animais , Humanos , Sequência de Aminoácidos , Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Ciclotídeos/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Inseticidas/química , Inseticidas/isolamento & purificação , Inseticidas/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Violaceae/química , Eritrócitos/efeitos dos fármacos
15.
Biomed Pharmacother ; 152: 113120, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35653889

RESUMO

Circular peptides are attractive lead compounds for drug development; this study investigates the immunomodulatory effects of defined root powder extracts and isolated peptides (called cyclotides) from Carapichea ipecacuanha (Brot.) L. Andersson ('ipecac'). Changes in the viability, proliferation and function of activated human primary T cells were analysed using flow cytometry-based assays. Three distinct peptide-enriched extracts of pulverised ipecac root material were prepared via C18 solid-phase extraction and analysed by reversed-phase HPLC and mass spectrometry. These extracts induced caspase 3/7 dependent apoptosis, thus leading to a suppressed proliferation of activated T cells and a reduction of the number of cells in the G2 phase. Furthermore, the stimulated T cells had a lower activation potential and a reduced degranulation capacity after treatment with ipecac extracts. Six different cyclotides were isolated from C. ipecacuanha and an T cell proliferation inhibiting effect was determined. Furthermore, the degranulation capacity of the T cells was diminished specifically by some cyclotides. In contrast to kalata B1 and its analog T20K, secretion of IL-2 and IFN- γ was not affected by any of the caripe cyclotides. The findings add to our increased understanding of the immunomodulating effects of cyclotides, and may provide a basis for the use of ipecac extracts for immunomodulation in conditions associated with an exessive immune responses.


Assuntos
Ciclotídeos , Proliferação de Células , Ciclotídeos/farmacologia , Humanos , Ipeca/farmacologia , Ativação Linfocitária , Linfócitos , Peptídeos Cíclicos
16.
Mult Scler Relat Disord ; 64: 103958, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35716476

RESUMO

BACKGROUND: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that causes chronic inflammation. Cyclotides are small plant proteins with a wide range of biological activity, making them a target for researchers to investigate. This study was conducted to investigate the possible effects of cyclotide-rich fractions from Viola odorata as an immunomodulatory agent in an experimental autoimmune encephalomyelitis (EAE) model of MS. METHODS: At room temperature, the plant materials were subjected to maceration in methanol: dichloromethane (1:1; v/v) for 3 days. The extraction was repeated 3 times, and the final concentrated extract was partitioned 3 times by 1/2 volume of double-distilled water. The aqueous phases were separated and freeze-dried. Finally, the crude extract was fractionated by C18 silicagel using vacuum liquid chromatography, with mobile phases of 30%, 50% and 80% of ethanol: water, respectively. The 50%, and 80% fractions were analyzed by HPLC and MALDI-TOF analysis and administrated intraperitoneally to forty-five female C57BL/6 EAE-induced mice, at 5, 25, and 50 mg/kg doses. After 28 days, the animals were evaluated using EAE clinical scoring which was done every 3 days, cytokine levels, and myelination level. RESULTS: The results confirmed the presence of cyclotides in V. odorata based on their retention time and the composition of mobile phase in HPLC and the molecular weight of the peaks in MALDI-TOF analysis. It was observed that cyclotides, especially in the 80% fraction group at the dose of 50 mg/kg significantly reduced the clinical scores, inflammation, and demyelination in EAE mice compared with the normal saline group (P<0.05), and the results of this group were comparable with fingolimod (P>0.05). CONCLUSION: It could be concluded that V. odorata is a rich source of cyclotides which they could be extracted by an easily available process and also, they could be used as immunomodulatory agents in MS, with similar effects to fingolimod.


Assuntos
Ciclotídeos , Encefalomielite Autoimune Experimental , Esclerose Múltipla , Viola , Sequência de Aminoácidos , Animais , Ciclotídeos/química , Ciclotídeos/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Cloridrato de Fingolimode , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/complicações , Esclerose Múltipla/tratamento farmacológico , Viola/química , Água
17.
Methods Enzymol ; 663: 19-40, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35168789

RESUMO

Cyclotides are plant host-defense peptides that have a wide range of biological activities and have diverse potential applications in medicine and agriculture. These 27-37 amino acid peptides have a head-to-tail cyclic backbone and are built around a cystine knot core, which makes them exceptionally stable. This stability and their amenability to sequence modifications has made cyclotides attractive scaffolds in drug design, and many synthetic cyclotides have now been designed and synthesized to test their efficacy as leads for a wide range of diseases, including infectious disease, cancer, pain and multiple sclerosis. Additionally, some natural cyclotides are selectively toxic to certain cancer cell lines, opening their potential as anticancer agents, and others have insecticidal activity, with applications in crop protection. With these applications in mind, there is a need to be able to measure cyclotides in pharmaceutical or agrichemical formulations and in biological media such as blood serum, as well as to assess their potential persistence in the environment when used as agrichemical agents. This chapter describes protocols for quantifying cyclotides in biological fluids, measuring their stability, and assessing their relative cytotoxicity on various types of cells.


Assuntos
Ciclotídeos , Ciclotídeos/química , Ciclotídeos/farmacologia , Desenho de Fármacos , Plantas
18.
J Nat Prod ; 85(1): 34-46, 2022 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-35044783

RESUMO

Glioblastoma multiforme (GBM) is the most aggressive cancer originating in the brain, with a median survival of 12 months. Most patients do not respond to or develop resistance to the only effective chemotherapeutic drug, temozolomide (TMZ), used to treat gliomas. Novel treatment methods are critically needed. Cyclotides are plant peptides that may be promising adjuvants to TMZ chemotherapy. They exhibit antitumor activity and chemosensitize cells to doxorubicin in breast cancer studies. During this research, we optimized cyclotide isolation techniques, and several cyclotides (CyO2, CyO13, kalata B1, and varv peptide A) exhibited dose-dependent cytotoxicity in MTT assays with IC50 values of 2.15-7.92 µM against human brain astrocytoma cells (U-87 MG) and human bone marrow derived neuroblastoma cells (SH-SY5Y). CyO2 and varv peptide A increased TMZ-induced cell death in U-87 MG cultures alone and when coexposed with CyO2 or varv peptide A plus TMZ. Phase contrast microscopy of glioblastoma cells exposed to cyclotides alone and coexposed to TMZ indicated shrunken, granular cells with blebbing, and the most pronounced effects were observed with coexposure treatments of cyclotides and TMZ. Cumulative results provide the proof-of-concept that cyclotides may enhance TMZ chemotherapy, and in vivo pharmacokinetic investigations of cyclotides are warranted with respect to GBM.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/patologia , Ciclotídeos/farmacologia , Glioblastoma/patologia , Temozolomida/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Camundongos , Estudo de Prova de Conceito , Ensaios Antitumorais Modelo de Xenoenxerto
19.
J Am Chem Soc ; 143(44): 18481-18489, 2021 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-34723512

RESUMO

Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 1012 members based on the cyclotide scaffold of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.


Assuntos
Proteínas Sanguíneas/farmacologia , Ciclotídeos/farmacologia , Fator XIIa/metabolismo , Proteínas Sanguíneas/química , Ciclotídeos/química , Fator XIIa/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos
20.
ACS Chem Biol ; 16(11): 2373-2386, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34592097

RESUMO

The cyclotide T20K inhibits the proliferation of human immune cells and is currently in clinical trials for multiple sclerosis. Here, we provide novel functional data and mechanistic insights into structure-activity relationships of T20K. Analogs with partial or complete reduction of the cystine knot had loss of function in proliferation experiments. Similarly, an acyclic analog of T20K was inactive in lymphocyte bioassays. The lack of activity of non-native peptide analogs appears to be associated with the ability of cyclotides to interact with and penetrate cell membranes, since cellular uptake studies demonstrated fast fractional transfer only of the native peptide into the cytosol of human immune cells. Therefore, structural differences between cyclic and linear native folded peptides were investigated by NMR to elucidate structure-activity relationships. Acyclic T20K had a less rigid backbone and considerable structural changes in loops 1 and 6 compared to the native cyclic T20K, supporting the idea that the cyclic cystine knot motif is a unique bioactive scaffold. This study provides evidence that this structural motif in cyclotides governs bioactivity, interactions with and transport across biological membranes, and the structural integrity of these peptides. These observations could be useful to understand the structure-activity of other cystine knot proteins due to the structural conservation of the cystine knot motif across evolution and to provide guidance for the design of novel cyclic cysteine-stabilized molecules.


Assuntos
Ciclotídeos/química , Ciclotídeos/farmacologia , Motivos Nó de Cisteína , Imunossupressores/farmacologia , Proliferação de Células/efeitos dos fármacos , Ciclotídeos/metabolismo , Humanos , Imunossupressores/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Conformação Proteica
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