RESUMO
BACKGROUND: Resistant infectious diseases caused by gram-negative bacteria are among the most serious worldwide health problems. Antimicrobial peptides (AMPs) have been explored as promising antibacterial, antibiofilm, and anti-infective candidates to address these health challenges. MAJOR CONCLUSIONS: Here we report the potent antibacterial effect of the peptide PaDBS1R6 on clinical bacterial isolates and identify an immunomodulatory peptide fragment incorporated within it. PaDBS1R6 was evaluated against Acinetobacter baumannii and Escherichia coli clinical isolates and had minimal inhibitory concentration (MIC) values from 8 to 32 µmol L-1. It had a rapid bactericidal effect, with eradication showing within 3 min of incubation, depending on the bacterial strain tested. In addition, PaDBS1R6 inhibited biofilm formation for A. baumannii and E. coli and was non-toxic toward healthy mammalian cells. These findings are explained by the preference of PaDBS1R6 for anionic membranes over neutral membranes, as assessed by surface plasmon resonance assays and molecular dynamics simulations. Considering its potent antibacterial activity, PaDBS1R6 was used as a template for sliding-window fr agmentation studies (window size = 10 residues). Among the sliding-window fragments, PaDBS1R6F8, PaDBS1R6F9, and PaDBS1R6F10 were ineffective against any of the bacterial strains tested. Additional biological assays were conducted, including nitric oxide (NO) modulation and wound scratch assays, and the R6F8 peptide fragment was found to be active in modulating NO levels, as well as having strong wound healing properties. GENERAL SIGNIFICANCE: This study proposes a new concept whereby peptides with different biological properties can be derived by the screening of fragments from within potent AMPs.
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Ziconotide (ω-conotoxin MVIIA) is an approved analgesic for the treatment of chronic pain. However, the need for intrathecal administration and adverse effects have limited its widespread application. Backbone cyclization is one way to improve the pharmaceutical properties of conopeptides, but so far chemical synthesis alone has been unable to produce correctly folded and backbone cyclic analogues of MVIIA. In this study, an asparaginyl endopeptidase (AEP)-mediated cyclization was used to generate backbone cyclic analogues of MVIIA for the first time. Cyclization using six- to nine-residue linkers did not perturb the overall structure of MVIIA, and the cyclic analogues of MVIIA showed inhibition of voltage-gated calcium channels (CaV 2.2) and substantially improved stability in human serum and stimulated intestinal fluid. Our study reveals that AEP transpeptidases are capable of cyclizing structurally complex peptides that chemical synthesis cannot achieve and paves the way for further improving the therapeutic value of conotoxins.
Assuntos
Conotoxinas , ômega-Conotoxinas , Humanos , ômega-Conotoxinas/farmacologia , ômega-Conotoxinas/uso terapêutico , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Conotoxinas/farmacologia , Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologiaRESUMO
Gomesin is a cationic antimicrobial peptide which is isolated from the haemocytes of the Brazilian tarantula Acanthoscurria gomesiana and can be produced chemically by Fmoc solid-phase peptide synthesis. Gomesin exhibits a range of biological activities, as demonstrated by its toxicity against therapeutically relevant pathogens such as Gram-positive or Gram-negative bacteria, fungi, cancer cells, and parasites. In recent years, a cyclic version of gomesin has been used for drug design and development as it is more stable than native gomesin in human serum and can penetrate and enter cancer cells. It can therefore interact with intracellular targets and has the potential to be developed as a drug lead for to treat cancer, infectious diseases, and other human diseases. This review provides a perspective on the discovery, structure-activity relationships, mechanism of action, biological activity, and potential clinical applications of gomesin.
Assuntos
Peptídeos Antimicrobianos , Neoplasias , Humanos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Neoplasias/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while ß/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that ß/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides.
Assuntos
Neurotoxinas , Peptídeos , Toxinas Biológicas , Urticaceae , Humanos , Neurotoxinas/química , Dor , Técnicas de Patch-Clamp , Peptídeos/química , Peptídeos/toxicidade , Toxinas Biológicas/química , Urticaceae/química , Canais de Sódio Disparados por Voltagem/efeitos dos fármacosRESUMO
Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead, and an orally active analgesic is demonstrated.
Assuntos
Produtos Biológicos , Domesticação , Proteínas de Plantas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Plantas/metabolismo , Peptídeos/metabolismo , Produtos Biológicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Cyclotides are a class of ribosomally-synthesized plant peptides that function in plants as a defense against insects and fungal pathogens. Their unique structure comprises a cyclized peptide backbone threaded by three disulfide bonds, that imparts structural stability, a desirable quality for peptide-based therapeutics or insecticides. Producing these peptides synthetically is challenging due to the amount of chemical waste produced and inefficiency of folding certain cyclotides. Thus, it is desirable to develop a means to access cyclotide biosynthesis in their native hosts, cultured in defined conditions, at both laboratory and commercial scale. Here we developed suspension cell cultures from two species previously unexplored for cyclotide production in suspension cells, Clitoria ternatea L., Hybanthus enneaspermus F. Muell., as well as with Oldenlandia affinis (Roem. & Schult.) DC., a species reported previously to accumulate cyclotides in cell suspensions. We assessed the growth rate, cyclotide production and gene expression for the various species. We found that while many cyclotides had reduced expression in Oldenlandia affinis suspension cells when compared to plant organs, those in Clitoria ternatea and Hybanthus enneaspermus maintained or increased expression levels. The cyclotides that continued to be expressed in suspension cultures shared similar sequence and biophysical properties as a group, regardless of phylogenetic origin of the host. Of particular interest was the discovery of inducibility by NaCl of cyclotide expression in O. affinis, cycloviolacin O2 expression in O. affinis, and the scale up of cycloviolacin O2 production in H. enneaspermus. Together the results presented here highlight the utility of plant cell suspensions as modalities to produce macrocyclic peptides.
Assuntos
Ciclotídeos , Sequência de Aminoácidos , Ciclotídeos/genética , Agricultura Molecular , Filogenia , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , SuspensõesRESUMO
Angiogenesis is important for tumor growth, and accordingly, targeting angiogenesis has become an important pathway for antitumor therapy. A novel proapoptotic peptide, CIGB-300 (P15-Tat), has been shown to be involved in the casein kinase II phosphorylation pathway, conferring it with antiangiogenic activity. Cyclic peptides have been widely used as scaffolds in drug design studies due to their high stability and favorable biopharmaceutical properties. Here, we chose two very stable cyclic trypsin inhibitors, MCoTI-II and SFTI-1, as frameworks to incorporate the bioactive epitope P15 into various backbone loops. NMR studies revealed that all re-engineered analogs had similar secondary structures to their native cyclic frameworks. One key analog, MCoP15, displayed significant improvement for inhibiting human umbilical vein endothelial cell migration, was nontoxic, and had higher stability than the P15 epitope alone. Overall, the results show the value of P15 being engineered into cyclic trypsin inhibitor scaffolds for improving antiangiogenic activity and stability. More broadly, the study highlights the versatility of cyclic peptide frameworks in drug design for antiangiogenic therapies.
Assuntos
Inibidores da Angiogênese/química , Colágeno/química , Ciclotídeos/química , Neovascularização Patológica/tratamento farmacológico , Fragmentos de Peptídeos/química , Peptídeos Cíclicos/química , Inibidores da Tripsina/química , Sequência de Aminoácidos , Inibidores da Angiogênese/metabolismo , Caseína Quinase II/metabolismo , Movimento Celular/efeitos dos fármacos , Colágeno/metabolismo , Desenho de Fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Inibidores da Tripsina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Lactate dehydrogenase 5 (LDH5) is overexpressed in metastatic tumors and is an attractive target for anticancer therapy. Small-molecule drugs have been developed to target the substrate/cofactor sites of LDH5, but none has reached the clinic to date, and alternative strategies remain almost unexplored. Combining rational and computer-based approaches, we identified peptidic sequences with high affinity toward a ß-sheet region that is involved in protein-protein interactions (PPIs) required for the activity of LDH5. To improve stability and potency, these sequences were grafted into a cyclic cell-penetrating ß-hairpin peptide scaffold. The lead grafted peptide, cGmC9, inhibited LDH5 activity in vitro in low micromolar range and more efficiently than the small-molecule inhibitor GNE-140. cGmC9 inhibits LDH5 by targeting an interface unlikely to be inhibited by small-molecule drugs. This lead will guide the development of new LDH5 inhibitors and challenges the landscape of drug discovery programs exclusively dedicated to small molecules.
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Inibidores Enzimáticos/farmacologia , Lactato Desidrogenase 5/antagonistas & inibidores , Peptídeos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Sítios de Ligação , Sangue/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Humanos , Lactato Desidrogenase 5/química , Lactato Desidrogenase 5/metabolismo , Masculino , Simulação de Dinâmica Molecular , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Estabilidade ProteicaRESUMO
Cyclotides are plant-derived peptides found within five families of flowering plants (Violaceae, Rubiaceae, Fabaceae, Solanaceae, and Poaceae) that have a cyclic backbone and six conserved cysteine residues linked by disulfide bonds. Their presence within the Violaceae species seems ubiquitous, yet not all members of other families produce these macrocyclic peptides. The genus Palicourea Aubl. (Rubiaceae) contains hundreds of neotropical species of shrubs and small trees; however, only a few cyclotides have been discovered hitherto. Herein, five previously uncharacterized Möbius cyclotides within Palicourea sessilis and their pharmacological activities are described. Cyclotides were isolated from leaves and stems of this plant and identified as pase A-E, as well as the known peptide kalata S. Cyclotides were de novo sequenced by MALDI-TOF/TOF mass spectrometry, and their structures were solved by NMR spectroscopy. Because some cyclotides have been reported to modulate immune cells, pase A-D were assayed for cell proliferation of human primary activated T lymphocytes, and the results showed a dose-dependent antiproliferative function. The toxicity on other nonimmune cells was also assessed. This study reveals that pase cyclotides have potential for applications as immunosuppressants and in immune-related disorders.
Assuntos
Ciclotídeos/efeitos dos fármacos , Ciclotídeos/metabolismo , Fabaceae/química , Linfócitos/metabolismo , Solanaceae/química , Violaceae/química , Brasil , Ciclotídeos/química , Humanos , Linfócitos/química , Linfócitos/efeitos dos fármacos , Magnoliopsida , Espectrometria de Massas , Folhas de Planta/química , Folhas de Planta/metabolismoRESUMO
Agelaia-MPI and protonectin are antimicrobial peptides isolated from the wasp Parachartergus fraternus that show antimicrobial and neuroactive activities. Previously, two analogues of these peptides, neuroVAL and protonectin-F, were designed to reduce nonspecific toxicity and improve potency. Here, the three-dimensional structures of neuroVAL, protonectin and protonectin-F were determined by using circular dichroism and NMR spectroscopy. Antibacterial, antifungal, cytotoxic and hemolytic activities were tested for the parent peptides and analogues. All peptides showed moderate antimicrobial activity against Gram-positive bacteria, with agelaia-MPI being the most active. Protonectin and protonectin-F were found to be toxic to cancerous and noncancerous cell lines. Internalization experiments revealed that these peptides accumulate inside both cell types. By contrast, neuroVAL was nontoxic to all tested cells and was able to enter cells without accumulating. In summary, neuroVAL has potential as a nontoxic cell-penetrating peptide, while protonectin-F needs further modification to realize its potential as an antitumor peptide.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Vespas/química , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antifúngicos/síntese química , Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Linhagem Celular , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Anticancer chemo- and targeted therapies are limited in some cases due to strong side effects and/or drug resistance. Peptides have received renascent interest as anticancer therapeutics and are currently being considered as alternatives and/or as complementary to biologics and small-molecule drugs. Gomesin, a disulfide-rich host defense peptide expressed in the Brazilian spider Acanthoscurria gomesiana selectively targets and disrupts cancer cell membranes. In the current study, we employed a range of biophysical methodologies with model membranes and bioassays to investigate the use of a cyclic analogue of gomesin as a drug scaffold to internalize cancer cells. We found that cyclic gomesin can internalize cancer cells via endocytosis and direct membrane permeation. In addition, we designed an improved non-disruptive and non-toxic cyclic gomesin analogue by incorporating D-amino acids within the scaffold. This improved analogue retained the ability to enter cancer cells and can be used as a scaffold to deliver drugs. Efforts to investigate the internalization mechanism used by host defense peptides, and to improve their stability, potency, selectivity and ability to permeate cancer cell membranes will increase the opportunities to repurpose peptides as templates for designing alternative anticancer therapeutic leads.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes , Membrana Celular/metabolismo , Sistemas de Liberação de Medicamentos , Neoplasias/metabolismo , Aranhas/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Artrópodes/química , Proteínas de Artrópodes/farmacocinética , Proteínas de Artrópodes/farmacologia , Membrana Celular/patologia , Células HeLa , Humanos , Células MCF-7 , Neoplasias/tratamento farmacológico , Neoplasias/patologiaRESUMO
Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a kcat/Km of 620 mM-1 s-1, making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.
Assuntos
Biocatálise , Cisteína Endopeptidases/metabolismo , Inibidores da Tripsina/metabolismo , Sequência de Aminoácidos , Ciclização , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Proteínas de Plantas/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Momordica trypsin inhibitors (TIs) such as those isolated from the seeds of the gac fruit, Momordica cochinchinensis (MCoTI-I and MCoTI-II), are widely used as scaffolds for drug design studies. To more effectively exploit these molecules in the development of therapeutics, there is a need for wider discovery of the natural sequence diversity among TIs from other species in the Momordica subfamily. Here we report the discovery of the encoding gene and six TIs from the seeds of the spiny gourd, Momordica dioica, four of which possess novel sequences (Modi 1, 3, 5, and 6) and two (Modi 2 and 4) of which are known peptides (TI-14, TI-17) previously identified in Momordica subangulata. Modi 6 is an acyclic peptide featuring a pyrrolidone carboxylic acid modification, whereas the remaining five TIs are cyclic. All Modi peptides display similar overall structures and trypsin inhibitory activities. No toxicity was observed for these peptides when tested against cancer and insect cells. All Modi peptides were exceptionally stable over 24 h in human serum, indicating a dual strategy to stabilize the peptides in nature, either head-to-tail cyclization or N-pyrolation, which suggests these peptides might be excellent candidates as scaffolds for epitope stabilization in drug design studies.
Assuntos
Momordica/química , Peptídeos Cíclicos/isolamento & purificação , Peptídeos/isolamento & purificação , Inibidores da Tripsina/isolamento & purificação , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Estabilidade de Medicamentos , Humanos , Espectroscopia de Ressonância Magnética , Peptídeos/química , Peptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
Cyclotides are macrocyclic cystine-knotted peptides most commonly found in the Violaceae plant family. Although Rinorea is the second-largest genera within the Violaceae family, few studies have examined whether or not they contain cyclotides. To further our understanding of cyclotide diversity and evolution, we examined the cyclotide content of two Rinorea species found in Southeast Asia: R. virgata and R. bengalensis. Seven cyclotides were isolated from R. virgata (named Rivi1-7), and a known cyclotide (cT10) was found in R. bengalensis. Loops 2, 5, and 6 of Rivi1-4 contained sequences not previously seen in corresponding loops of known cyclotides, thereby expanding our understanding of the diversity of cyclotides. In addition, the sequence of loop 2 of Rivi3 and Rivi4 were identical to some related noncyclic "acyclotides" from the Poaceae plant family. As only acyclotides, but not cyclotides, have been reported in monocotyledons thus far, our findings support an evolutionary link between monocotyledon-derived ancestral cyclotide precursors and dicotyledon-derived cyclotides. Furthermore, Rivi2 and Rivi3 had comparable cytotoxic activities to the most cytotoxic cyclotide known to date: cycloviolacin O2 from Viola odorata; yet, unlike cycloviolacin O2, they did not show hemolytic activity. Therefore, these cyclotides represent novel scaffolds for use in future anticancer drug design.
Assuntos
Ciclotídeos/química , Violaceae/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Extratos Vegetais/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Gomesin, a disulfide-rich antimicrobial peptide produced by the Brazilian spider Acanthoscurria gomesiana, has been shown to be potent against Gram-negative bacteria and to possess selective anticancer properties against melanoma cells. In a recent study, a backbone cyclized analogue of gomesin was shown to be as active but more stable than its native form. In the current study, we were interested in improving the antimicrobial properties of the cyclic gomesin, understanding its selectivity toward melanoma cells and elucidating its antimicrobial and anticancer mode of action. Rationally designed analogues of cyclic gomesin were examined for their antimicrobial potency, selectivity toward cancer cells, membrane-binding affinity, and ability to disrupt cell and model membranes. We improved the activity of cyclic gomesin by â¼10-fold against tested Gram-negative and Gram-positive bacteria without increasing toxicity to human red blood cells. In addition, we showed that gomesin and its analogues are more toxic toward melanoma and leukemia cells than toward red blood cells and act by selectively targeting and disrupting cancer cell membranes. Preference toward some cancer types is likely dependent on their different cell membrane properties. Our findings highlight the potential of peptides as antimicrobial and anticancer leads and the importance of selectively targeting cancer cell membranes for drug development.
Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Aranhas/química , Animais , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fungos/efeitos dos fármacos , Humanos , Leucemia/tratamento farmacológico , Bicamadas Lipídicas/metabolismo , Melanoma/tratamento farmacológico , Micoses/tratamento farmacológicoRESUMO
Peptide analogues derived from bioactive hormones such as somatostatin or certain growth factors have great potential as angiogenesis inhibitors for cancer applications. In an attempt to combat emerging drug resistance many FDA-approved anti-angiogenesis therapies are co-administered with cytotoxic drugs as a combination therapy to target multiple signaling pathways of cancers. However, cancer therapies often encounter limiting factors such as high toxicities and side effects. Here, we combined two anti-angiogenic epitopes that act on different pathways of angiogenesis into a single non-toxic cyclic peptide framework, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II), and subsequently assessed the anti-angiogenic activity of the novel compound. We hypothesized that the combination of these two epitopes would elicit a synergistic effect by targeting different angiogenesis pathways and result in improved potency, compared to that of a single epitope. This novel approach has resulted in the development of a potent, non-toxic, stable and cyclic analogue with nanomolar potency inhibition in in vitro endothelial cell migration and in vivo chorioallantoic membrane angiogenesis assays. This is the first report to use the MCoTI-II framework to develop a 2-in-1 anti-angiogenic peptide, which has the potential to be used as a form of combination therapy for targeting a wide range of cancers.
Assuntos
Inibidores da Angiogênese/química , Ciclotídeos/química , Epitopos/química , Momordica/química , Neoplasias/tratamento farmacológico , Peptídeos Cíclicos/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Membrana Corioalantoide/química , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Eritrócitos/citologia , Hemólise , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Meliteno/química , Neovascularização Patológica , Codorniz , Transdução de Sinais/efeitos dos fármacos , Tripsina/químicaRESUMO
Cyclotides are cyclic peptides from plants in the Violaceae, Rubiaceae, Fabaceae, Cucurbitaceae, and Solanaceae families. They are sparsely distributed in most of these families, but appear to be ubiquitous in the Violaceae, having been found in every plant so far screened from this family. However, not all geographic regions have been examined and here we report the discovery of cyclotides from a Viola species from South-East Asia. Two novel cyclotides (Visu 1 and Visu 2) and two known cyclotides (kalata S and kalata B1) were identified in V. sumatrana. NMR studies revealed that kalata S and kalata B1 had similar secondary structures. Their biological activities were determined in cytotoxicity assays; both had similar cytotoxic activity and were more toxic to U87 cells compared with other cell lines. Overall, the study strongly supports the ubiquity of cyclotides in the Violaceae and adds to our understanding of their distribution and cytotoxic activity.
Assuntos
Citotoxinas , Peptídeos Cíclicos , Proteínas de Plantas , Viola/química , Linhagem Celular , Citotoxinas/química , Citotoxinas/isolamento & purificação , Citotoxinas/farmacologia , Humanos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Estrutura Secundária de ProteínaRESUMO
The SET protein is a promising drug target in cancer therapy, because of its ability to inhibit the function of the tumor suppressor gene protein phosphatase 2A (PP2A). COG peptides, derived from apolipoprotein E (apoE), are potent antagonists of SET; they induce cytotoxicity in cancer cells upon binding to intracellular SET and modulate the nuclear factor kappa B (NF-κB) signaling pathway. However, the therapeutic potential of COG peptides is limited, because of their poor proteolytic stability and low bioavailability. In this study, the COG peptide, COG1410, was stabilized by grafting it onto the ultrastable cyclic peptide scaffold, Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The grafted MCoTI-II peptides were cytotoxic to a cancer cell line and showed high stability in human serum. The most potent grafted MCoTI-II peptide inhibited lipopolysaccharide (LPS)-mediated activation of NF-κB in murine macrophages. Overall, this study demonstrates the application of the MCoTI-II scaffold for the development of stable peptide drugs for cancer therapy.
Assuntos
Chaperonas de Histonas/antagonistas & inibidores , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Apolipoproteínas E/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclotídeos/química , Ciclotídeos/farmacologia , Proteínas de Ligação a DNA , Humanos , Imageamento por Ressonância Magnética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Proteína Fosfatase 2/metabolismoRESUMO
Tumour formation is dependent on nutrient and oxygen supply from adjacent blood vessels. Angiogenesis inhibitors can play a vital role in controlling blood vessel formation and consequently tumour progression by inhibiting endothelial cell proliferation, sprouting and migration. The primary aim of the present study was to design cyclic thrombospondin-1 (TSP-1) mimetics using disulfide-rich frameworks for anti-angiogenesis therapies and to determine whether these peptides have better potency than the linear parent peptide. A short anti-angiogenic heptapeptide fragment from TSP-1 (GVITRIR) was incorporated into two cyclic disulfide-rich frameworks, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1). The cyclic peptides were chemically synthesized and folded in oxidation buffers, before being tested in a series of in vitro evaluations. Incorporation of the bioactive heptapeptide fragment into the cyclic frameworks resulted in peptides that inhibited microvascular endothelial cell migration, and had no toxicity against normal primary human endothelial cells or cancer cells. Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum. The present study has demonstrated a novel approach to stabilize the active region of TSP-1. The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics.