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1.
J Nat Prod ; 83(6): 1817-1828, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32437150

RESUMO

Viola is the largest genus in the Violaceae plant family and is known for its ubiquitous natural production of cyclotides. Many Viola species are used as medicinal herbs across Asia and are often consumed by humans in teas for the treatment of diseases, including ulcers and asthma. Previous studies reported the isolation of cyclotides from Viola species in many countries in the hope of discovering novel compounds with anti-cancer activities; however, Viola species from Vietnam have not been investigated to date. Here, the discovery of cyclotides from three Viola species (V. arcuata, V. tonkinensis, and V. austrosinensis) collected in the northern mountainous region of Vietnam is reported. Ten cyclotides were isolated from these three Viola species: four are novel and six were previously reported to be expressed in other plants. The structures of three of the new bracelet cyclotides are similar to that of cycloviolacin O2. Because cycloviolacin O2 has previously been shown to have potent activity against a wide range of cancer cell lines including HeLa (human cervical cancer cells) and PC-3 (human prostate cancer cells), the cancer cytotoxicity of the cyclotides isolated from V. arcuata was assessed. All tested cyclotides were cytotoxic against cancer cells, albeit to varying degrees. The sequences discovered in this study significantly expand the understanding of cyclotide diversity, especially in comparison with other cyclotides found in plants from the Asian region.

2.
J Biol Chem ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32414842

RESUMO

Cyclotides are plant-derived peptides characterized by a ~30-amino-acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and one known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, M, and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

3.
J Med Chem ; 62(17): 8140-8151, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31411881

RESUMO

Diverse peptides have been evaluated for their activity against pathogenic microorganisms. Here, five mastoparan variants were designed based on mastoparan-L, among which two (R1 and R4) were selected for in-depth analysis. Mastoparan-L (parent/control), R1, and R4 inhibited susceptible/resistant bacteria at concentrations ranging from 2 to 32 µM, whereas only R1 and R4 eradicated Pseudomonas aeruginosa biofilms at 16 µM. Moreover, the toxic effects of mastoparan-L toward mammalian cells were drastically reduced in both variants. In skin infections, R1 at 64 µM was the most effective variant, reducing P. aeruginosa bacterial counts 1000 times on day 4 post-infection. Structurally, all of the peptides showed varying levels of helicity and structural stability in aqueous and membrane-like conditions, which may affect the different bioactivities observed here. By computationally modifying the physicochemical properties of R1 and R4, we reduced the cytotoxicity and optimized the therapeutic potential of these mastoparan-like peptides both in vitro and in vivo.

4.
ACS Infect Dis ; 5(7): 1081-1086, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31016969

RESUMO

Bacterial biofilms and associated infections represent one of the biggest challenges in the clinic, and as an alternative to counter bacterial infections, antimicrobial peptides have attracted great attention in the past decade. Here, ten short cationic antimicrobial peptides were generated through a sliding-window strategy on the basis of the 19-amino acid residue peptide, derived from a Pyrobaculum aerophilum ribosomal protein. PaDBS1R6F10 exhibited anti-infective potential as it decreased the bacterial burden in murine Pseudomonas aeruginosa cutaneous infections by more than 1000-fold. Adverse cytotoxic and hemolytic effects were not detected against mammalian cells. The peptide demonstrated structural plasticity in terms of its secondary structure in the different environments tested. PaDBS1R6F10 represents a promising antimicrobial agent against bacteria infections, without harming human cells.

5.
ACS Infect Dis ; 4(12): 1727-1736, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30346140

RESUMO

Computer-aided screening of antimicrobial peptides (AMPs) is a promising approach for discovering novel therapies against multidrug-resistant bacterial infections. Here, we functionally and structurally characterized an Escherichia coli-derived AMP (EcDBS1R5) previously designed through pattern identification [α-helical set (KK[ILV](3)[AILV])], followed by sequence optimization. EcDBS1R5 inhibited the growth of Gram-negative and Gram-positive, susceptible and resistant bacterial strains at low doses (2-32 µM), with no cytotoxicity observed against non-cancerous and cancerous cell lines in the concentration range analyzed (<100 µM). Furthermore, EcDBS1R5 (16 µM) acted on Pseudomonas aeruginosa pre-formed biofilms by compromising the viability of biofilm-constituting cells. The in vivo antibacterial potential of EcDBS1R5 was confirmed as the peptide reduced bacterial counts by two-logs 2 days post-infection using a skin scarification mouse model. Structurally, circular dichroism analysis revealed that EcDBS1R5 is unstructured in hydrophilic environments, but has strong helicity in 2,2,2-trifluoroethanol (TFE)/water mixtures (v/v) and sodium dodecyl sulfate (SDS) micelles. The TFE-induced nuclear magnetic resonance structure of EcDBS1R5 was determined and showed an amphipathic helical segment with flexible termini. Moreover, we observed that the amide protons for residues Met2-Ala8, Arg10, Ala13-Ala16, and Trp19 in EcDBS1R5 are protected from the solvent, as their temperature coefficients values are more positive than -4.6 ppb·K-1. In summary, this study reports a novel dual-antibacterial/antibiofilm α-helical peptide with therapeutic potential in vitro and in vivo against clinically relevant bacterial strains.


Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/química , Biofilmes/efeitos dos fármacos , Escherichia coli/química , Infecções por Pseudomonas/tratamento farmacológico , Animais , Dicroísmo Circular , Desenho Assistido por Computador , Desenho de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia
6.
Biochemistry ; 54(31): 4863-76, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26174911

RESUMO

Enterocin NKR-5-3B, one of the multiple bacteriocins produced by Enterococcus faecium NKR-5-3, is a 64-amino acid novel circular bacteriocin that displays broad-spectrum antimicrobial activity. Here we report the identification, characterization, and three-dimensional nuclear magnetic resonance solution structure determination of enterocin NKR-5-3B. Enterocin NKR-5-3B is characterized by four helical segments that enclose a compact hydrophobic core, which together with its circular backbone impart high stability and structural integrity. We also report the corresponding structural gene, enkB, that encodes an 87-amino acid precursor peptide that undergoes a yet to be described enzymatic processing that involves adjacent cleavage and ligation of Leu(24) and Trp(87) to yield the mature (circular) enterocin NKR-5-3B.


Assuntos
Bacteriocinas/química , Enterococcus faecium/química , Bacteriocinas/biossíntese , Bacteriocinas/genética , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína
7.
Future Med Chem ; 6(15): 1617-28, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25406003

RESUMO

BACKGROUND: Chlorotoxin is a small scorpion peptide that inhibits glioma cell migration. We investigated the importance of a major component of chlorotoxin's chemical structure - four disulfide bonds - to its tertiary structure and biological function. RESULTS: Five disulfide bond analogs of chlorotoxin were synthesized, with l-α-aminobutyric acid residues replacing each or all of the disulfide bonds. Chemical oxidation and circular dichroism experiments revealed that Cys III-VII and Cys V-VIII were essential for native structure formation. Cys I-IV and Cys II-VI were important for stability of enzymatic proteolysis but not for the inhibition of human umbilical vein endothelial cell migration. CONCLUSION: The disulfide bonds of chlorotoxin are important for its structure and stability and have a minor role in its activity against cell migration.


Assuntos
Dissulfetos/química , Venenos de Escorpião/química , Alquilação , Sequência de Aminoácidos , Movimento Celular/efeitos dos fármacos , Dicroísmo Circular , Ciclização , Células Endoteliais da Veia Umbilical Humana , Humanos , Dados de Sequência Molecular , Oxirredução , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Venenos de Escorpião/síntese química , Venenos de Escorpião/toxicidade
8.
J Biol Chem ; 289(10): 6627-38, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24425873

RESUMO

Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Cisteína/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Conotoxinas/química , Ciclização , Ciclotídeos/química , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica
9.
Biopolymers ; 100(5): 480-91, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23893608

RESUMO

Cyclotides are a family of naturally occurring backbone-cyclized macrocyclic mini-proteins from plants that have a knotted trio of intramolecular disulfide bonds. Their structural features imbue cyclotides with extraordinary stability against degradation at elevated temperatures or in the presence of proteolytic enzymes. The plasticity of their intracysteine loop sequences is exemplified by the more than 250 natural cyclotides sequenced to date, and this tolerance to sequence variation, along with their diverse bioactivities, underpins the suitability of the cyclic cystine knot motif as a valuable drug design scaffold and research tool for protein engineering studies. Here, we review the recent literature on applications of cyclotides for the stabilization of peptide epitopes and related protein engineering studies. Possible future directions in this field are also described.


Assuntos
Ciclotídeos , Desenho de Fármacos , Sequência de Aminoácidos , Ciclotídeos/genética , Modelos Moleculares , Dados de Sequência Molecular , Engenharia de Proteínas
10.
J Biol Chem ; 288(19): 13885-96, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23548907

RESUMO

BACKGROUND: Sunflower trypsin inhibitor-1 (SFTI-1) and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) are potent protease inhibitors comprising a cyclic backbone. RESULTS: Elucidation of structure-activity relationships for SFTI-1 and MCoTI-II was used to design inhibitors with enhanced inhibitory activity. CONCLUSION: An analog of MCoTI-II is one of the most potent inhibitors of matriptase. SIGNIFICANCE: These results provide a solid basis for the design of selective peptide inhibitors of matriptase with therapeutic potential. The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase.


Assuntos
Peptídeos Cíclicos/química , Proteínas de Plantas/química , Serina Endopeptidases/química , Inibidores de Serino Proteinase/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Helianthus/química , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Momordica/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/genética , Proteínas de Plantas/síntese química , Proteínas de Plantas/genética , Ligação Proteica , Relação Estrutura-Atividade , Propriedades de Superfície
11.
Blood ; 118(25): 6709-17, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22039263

RESUMO

Fragments from the extracellular matrix proteins laminin and osteopontin and a sequence from VEGF have potent proangiogenic activity despite their small size (< 10 residues). However, these linear peptides have limited potential as drug candidates for therapeutic angiogenesis because of their poor stability. In the present study, we show that the therapeutic potential of these peptides can be significantly improved by "grafting" them into cyclic peptide scaffolds. Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1), naturally occurring, plant-derived cyclic peptides of 34 and 14 residues, respectively, were used as scaffolds in this study. Using this approach, we have designed a peptide that, in contrast to the small peptide fragments, is stable in human serum and at nanomolar concentration induces angiogenesis in vivo. This is the first report of using these scaffolds to improve the activity and stability of angiogenic peptide sequences and is a promising approach for promoting angiogenesis for therapeutic uses.


Assuntos
Proteínas Angiogênicas/química , Dissulfetos/química , Peptídeos Cíclicos/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Ciclotídeos/química , Ciclotídeos/genética , Ciclotídeos/farmacologia , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Hemólise/efeitos dos fármacos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/farmacologia , Conformação Proteica , Estabilidade Proteica , Ratos
12.
J Nat Prod ; 72(8): 1453-8, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19711988

RESUMO

The plant Momordica cochinchinensis has traditionally been used in Chinese medicine to treat a variety of illnesses. A range of bioactive molecules have been isolated from this plant, including peptides, which are the focus of this study. Here we report the isolation and characterization of two novel peptides, MCoCC-1 and MCoCC-2, containing 33 and 32 amino acids, respectively, which are toxic against three cancer cell lines. The two peptides are highly homologous to one another, but show no sequence similarity to known peptides. Elucidation of the three-dimensional structure of MCoCC-1 suggests the presence of a cystine knot motif, also found in a family of trypsin inhibitor peptides from this plant. However, unlike its structural counterparts, MCoCC-1 does not inhibit trypsin. MCoCC-1 has a well-defined structure, characterized mainly by a triple-stranded antiparallel beta-sheet, but unlike the majority of cystine knot proteins MCoCC-1 contains a disordered loop presumably as a result of flexibility in a localized region of the molecule. Of the cell lines tested, MCoCC-1 is the most toxic against a human melanoma cell line (MM96L) and is nonhemolytic to human erythrocytes. The role of these peptides within the plant remains to be determined.


Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Momordica/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação , Peptídeos/química , Peptídeos/isolamento & purificação , Plantas Medicinais/química , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Sementes/química , Homologia de Sequência de Aminoácidos , Inibidores da Tripsina/química , Vietnã
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