Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros

Intervalo de ano de publicação
1.
J. venom. anim. toxins incl. trop. dis ; 24: 30, 2018. tab, graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-976028

Resumo

Drug repurposing has been an interesting and cost-effective approach, especially for neglected diseases, such as Chagas disease. Methods: In this work, we studied the activity of the antidepressant drug sertraline against Trypanosoma cruzi trypomastigotes and intracellular amastigotes of the Y and Tulahuen strains, and investigated its action mode using cell biology and in silico approaches. Results: Sertraline demonstrated in vitro efficacy against intracellular amastigotes of both T. cruzi strains inside different host cells, including cardiomyocytes, with IC50 values between 1 to 10 µM, and activity against bloodstream trypomastigotes, with IC50 of 14 µM. Considering the mammalian cytotoxicity, the drug resulted in a selectivity index of 17.8. Sertraline induced a change in the mitochondrial integrity of T. cruzi, resulting in a decrease in ATP levels, but not affecting reactive oxygen levels or plasma membrane permeability. In silico approaches using chemogenomic target fishing, homology modeling and molecular docking suggested the enzyme isocitrate dehydrogenase 2 of T. cruzi (TcIDH2) as a potential target for sertraline. Conclusions: The present study demonstrated that sertraline had a lethal effect on different forms and strains of T. cruzi, by affecting the bioenergetic metabolism of the parasite. These findings provide a starting point for future experimental assays and may contribute to the development of new compounds.(AU)


Assuntos
Trypanosoma cruzi , Técnicas In Vitro , Sertralina , Reposicionamento de Medicamentos
2.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 24: 30, Nov. 29, 2018. ilus, tab, graf
Artigo em Inglês | VETINDEX | ID: vti-19371

Resumo

Background: Drug repurposing has been an interesting and cost-effective approach, especially for neglected diseases, such as Chagas disease. Methods: In this work, we studied the activity of the antidepressant drug sertraline against Trypanosoma cruzi trypomastigotes and intracellular amastigotes of the Y and Tulahuen strains, and investigated its action mode using cell biology and in silico approaches. Results: Sertraline demonstrated in vitro efficacy against intracellular amastigotes of both T. cruzi strains inside different host cells, including cardiomyocytes, with IC50 values between 1 to 10 M, and activity against bloodstream trypomastigotes, with IC50 of 14 M. Considering the mammalian cytotoxicity, the drug resulted in a selectivity index of 17.8. Sertraline induced a change in the mitochondrial integrity of T. cruzi, resulting in a decrease in ATP levels, but not affecting reactive oxygen levels or plasma membrane permeability. In silico approaches using chemogenomic target fishing, homology modeling and molecular docking suggested the enzyme isocitrate dehydrogenase 2 of T. cruzi (TcIDH2) as a potential target for sertraline. Conclusions: The present study demonstrated that sertraline had a lethal effect on different forms and strains of T. cruzi, by affecting the bioenergetic metabolism of the parasite. These findings provide a starting point for future experimental assays and may contribute to the development of new compounds.(AU)


Assuntos
Trypanosoma cruzi , Sertralina/análise , Doença de Chagas/tratamento farmacológico , Doenças Negligenciadas
3.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 24: 1-11, 2018. graf, ilus
Artigo em Inglês | VETINDEX | ID: vti-18170

Resumo

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.(AU)


Assuntos
Humanos , Animais , Venenos Elapídicos/isolamento & purificação , Elapidae , Motilidade dos Espermatozoides , Fármacos para a Fertilidade Masculina , Sêmen , Reações Bioquímicas , Espectrometria de Massas em Tandem/métodos
4.
J. venom. anim. toxins incl. trop. dis ; 24: 1-11, 2018. graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1484741

Resumo

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.


Assuntos
Humanos , Animais , Elapidae , Fármacos para a Fertilidade Masculina , Motilidade dos Espermatozoides , Sêmen , Venenos Elapídicos/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Reações Bioquímicas
5.
Tese em Inglês | VETTESES | ID: vtt-220141

Resumo

A raiva é uma doença zoonótica que afeta principalmente a população pobre de países em desenvolvimento. Embora exista uma vacina eficiente contra o vírus, muitas das pessoas nas áreas mais afetadas pela doença podem não ter conhecimento da vacina e de sua necessidade, não conseguem chegar a uma área onde as vacinas estão prontamente disponíveis, ou não podem pagar pelo alto custo da vacinação e da imunoglobulina. Este projeto teve como objetivo encontrar um tratamento potencial para a doença quando ela já atingiu seus estágios mais avançados, de modo que aqueles que não têm acesso à vacinação ainda tenham uma chance de sobreviver à esta doença letal. Este projeto teve como foco a capacidade da bioinformática de encontrar potenciais ligantes que pudessem inativar ou bloquear todas as cinco proteínas ou raiva, de modo que fossem incapazes de se ligar aos receptores do hospedeiro, ou de danificar o sistema imunológico dos pacientes. Todas as cinco proteínas da raiva de dezenove cepas diferentes (noventa e cinco proteínas) foram modeladas por meio de modelagem de homologia, e vinte e seis ligantes que passaram pela regra de cinco de Lipinski foram escolhidos. A primeira etapa de docagem foi docar todas as 95 proteínas por meio de uma docagem cega com cada um dos 26 ligantes para reduzir o número de ligantes e analisar potenciais sítios ativos. Após a conclusão de todos as docagens cegas, foram escolhidos dezessete ligantes para a docagem ativa, que considerou resíduos específicos (encontrados na literatura, ferramentas de bioinformática e análise da docagem cega) como potenciais sítios ativos de cada proteína. Os resultados da docagem cega também foram visualizados e analisados quanto às energias de ligação, bem como a localização das conexões. O número de vezes que cada um dos resíduos dos potenciais sítios ativos foi acessado também foi analisado. Os ligantes que tiveram os melhores resultados gerais foram reduzidos a quatro e tiveram suas estruturas e farmacologia analisadas no contexto da infecção por raiva. Os sítios ativos potenciais também foram analisados e limitados aos sítios ativos mais prováveis para cada proteína.


Rabies is a zoonotic disease that mainly affects poor population in developing countries. While there is an efficient vaccine for it, many of those in the areas most affected by the disease can be unaware of the vaccine and their need for it, may be unable to reach an area where the vaccines are readily available, or may be unable to pay for the high cost of vaccination and immunoglobulin. This project aimed to find a potential treatment for the disease when it has already reached its later stages, so those who cant access vaccination still have a chance of surviving this lethal disease. This project focused on the bioinformatics capability of finding potential ligands that could inactivate or block all five proteins or rabies, so that they would be unable to bind to host receptors, or to damage the immune system of patients. All five proteins of rabies from nineteen different strains (ninety-five proteins) were modeled through homology modeling, and twenty-six drug-like ligands that passed Lipinskis rule of five were chosen. The first docking step was to put all ninety-five proteins through a blind docking with each of the twenty-six ligands to narrow down the number of ligands and analyze potential active sites. After all the blind dockings were concluded, seventeen ligands were chosen for the active site docking, which also considered specific residues (found from literature, bioinformatic tools, and analysis of blind docking) as potential active sites of each protein. The blind docking results were also visualized and analyzed for both the binding energies of each binding as well as the location of the connections. The number of times each of the residues from the potential active sites were accessed were also analyzed. The ligands that had the best results overall were narrowed down to four and analyzed both for their structure and pharmacology in the context of rabies infection. Potential active sites were also analyzed and narrowed down to the most likely active sites for each protein.

6.
Tese em Português | VETTESES | ID: vtt-216540

Resumo

As salmoneloses são doenças de grande importância em todo o mundo. São causadas por bactérias do gênero Salmonella, um dos principais agentes causadores de Doenças Transmitidas por Alimentos, sendo a carne de frango e ovos, as principais fontes de contaminação. A administração de antimicrobianos tem sido uma prática que contribui no tratamento e prognóstico das salmoneloses. Contudo, é preocupante o surgimento de sorotipos resistentes a diversas classes de antibióticos, justificando a busca por novas alternativas de tratamento. Nesse contexto, as enzimas Alanina Racemase e a D-alanina, D-alanina ligase (Dal-Dal-ligase), apresentam-se como alvos promissores, devido ao papel essencial na biossíntese do peptidoglicano presente nestas bactérias. O objetivo deste trabalho foi determinar a estrutura da enzima Dal-Dal-ligase de Salmonella spp. com o propósito de identificar novos inibidores com capacidade antimicrobiana contra cepas desta bactéria. A estrutura da enzima Dal-Dal-ligase na presença dos co-fatores Mg2+ e ATP, mais o substrato D-alanil, D-alanina, foi modelada por homologia, a qual foi utilizada em simulações de varredura virtual com moléculas obtidas do banco de dados do Zinc. Foram selecionadas 23 moléculas, as quais foram filtradas com sucesso pelos critérios ADMETox. Duas moléculas: SaDD01 e SaDD02 foram adquiridas para ensaios in vitro contra cepas ATCCs e isolados clínicos resistentes. Nenhuma das moléculas apresentou atividade antibacteriana isoladamente até o limite de 512 g.mL1. Entretanto, quando testados em sinergismo com a D-cicloserina ambas as moléculas apresentaram-se como potencializadores da atividade deste antimicrobiano, diminuindo a Concentração Inibitória Mínima pela metade. Estes resultados sugerem um caminho para o desenvolvimento de novas drogas contra as salmoneloses.


Salmonelloses are diseases of great importance all over the world. They are caused by bacteria of genus Salmonella, one of the main Foodborne Diseases causing agents, where the chicken eggs and meat are the main sources of contamination. The administration of antimicrobials has been a practice that contributes to the treatment and prognosis of salmonellosis. However, the arising of serotypes resistant to several classes of antibiotics is a concerning matter which justify the search for new alternatives of treatment. In this context, the enzymes Alanine Racemase and D-alanine, D-alanine ligase (Dal-Dal-ligase) are promising targets, due to their essential role in the biosynthesis of peptidoglycan in these bacteria. The objective of this work was to modeling the structure of the enzyme Dal-Dal-ligase of Salmonella spp. aiming the identification of new inhibitors with antimicrobial activity against strains of these bacteria. The structure of the Dal-Dal-ligase enzyme in the presence of the cofactors Mg2+ and ATP plus the D-alanyl, D-alanine substrate was modeled by homology in order to be used on virtual screening simulations using molecules obtained from Zinc database. Twenty three molecules were selected and successfully filtered by the ADMETox criteria. Two molecules: SaDD01 and SaDD02 were purchased for in vitro assays against ATCC strains and also clinical resistant isolates. None of the molecules presented antibacterial activity alone up to the limit of 512 g.mL-1. However, when they were tested in synergism with D-cycloserine both molecules exhibit potentiation effect for the activity of this antimicrobial, where the Minimal Inhibitory Concentration were reduced by half. These results suggest a pathway for the development of new drugs against salmonellosis.

7.
Artigo em Inglês | VETINDEX | ID: vti-444244

Resumo

Entomopathogenic fungus Verticillium lecanii is a promising whitefly and aphid control agent. Chitinases secreted by this insect pathogen have considerable importance in the biological control of some insect pests. An endochitinase gene Vlchit1 from the fungus was cloned and overexpressed in Escherichia coli. The Vlchit1 gene not only contains an open reading frame (ORF) which encodes a protein of 423 amino acids (aa), but also is interrupted by three short introns. A homology modelling of Vlchit1 protein showed that the chitinase Vlchit1 has a (/)8 TIM barrel structure. Overexpression test and Enzymatic activity assay indicated that the Vlchit1 is a functional enzyme that can hydrolyze the chitin substrate, so the Vlchit1 gene can service as a useful gene source for genetic manipulation leading to strain improvement of entomopathogenic fungi or constructing new transgenic plants with resistance to various fungal and insects pests.


O fungo entomopatogênico Verticillium lecanii é um agente promissor no controle da mosca-branca e do pulgão. As quitinases secretadas por esse patógeno de insetos têm uma grande importância no controle biológico de doenças causadas por insetos. Um gene de endoquitinase Vlchit1 desse fungo foi clonado e expresso em Escherichia coli. O gene Vlchit contém não apenas um ORF que codifica uma proteína de 423 aminoácidos, mas também é interrompido por três pequenos introns. A modelagem de homologia da proteína Vlchit1indicou que a quitinase Vlchit1 tem uma estrutura (/) 8 TIM barrel. Testes de expressão e de atividade enzimática indicaram que Vlchit1 é uma enzima funcional que hidroliza quitina, portanto o gene Vlchit pode ser um gene útil para manipulação genética para melhoramento de cepas de fungos entomopatogênicos ou para a construção de novas plantas transgênicas com resistência a várias doenças causadas por fungos e insetos.

8.
Jaboticabal; s.n; 23/02/2011. 118 p.
Tese em Português | VETTESES | ID: vtt-4758

Resumo

As PKSs tipo II são complexos enzimáticos envolvidos na produção de policetídeos. Estes são metabólitos secundários que possuem papel-chave no desencadeamento das respostas bioquímicas que levam à diversificação fisiológica frente a fatores adversos, fazem parte da composição de pigmentos de esporos microbianos, além de comporem a estrutura química de importantes antibióticos explorados pela indústria farmacêutica. Foi feita a prospecção de novos ?clusters? gênicos para PKSs tipo II em uma biblioteca metagenômica de 9.320 clones, através de amplificação por PCR com ?primers? degenerados para uma região gênica conservada. Dentre três clones encontrados, um foi sequenciado pela estratégia de shotgun, resultando em um consensus de 22.988 pares de bases. Foi feita a predição funcional de ORFs por homologia obtida com a ferramenta Blast (NCBI) e também a identificação de domínios enzimáticos pelo PRODOM e PFAM. Dentre os resultados da predição por homologia de domínios enzimáticos foi possível identificar ORFs homologas à: reguladores gênicos (LacI e LuxR); carboidrases (Lacases, ?-glucosidases, Quitinases/Celulases); transferases (O-metil-transferases, Acíl-transferases); pks mínima; ciclases, aromatases, hidroxilases, mono-oxigenases e transportadores ABC. Alguns ?clusters? enzimáticos podem ainda atuar em degradação de celulose e lignina. As enzimas candidatas ao core biossíntético do ?cluster? (pks mínima) foram submetidas à modelagem de estrutura tridimensional in silico. Os modelos 3D foram avaliados quanto a acurácia, utilizando as ferramentas: Verify3D, Qmean, Procheck, Rampage e Swiss-Model. Uma vez validados, as KSA e CLF foram submetidas a simulações de ?docking? enzimático


The type II PKSs are enzyme complexes involved in the production of polyketides. These are secondary metabolites that have key role in triggering the biochemical responses that lead to physiological diversification against adverse factors are part of the pigment composition of microbial spores, and compose the chemical structure of most antibiotics exploited by the pharmaceutical industry. Some clusters could act in enzymatic degradation of cellulose and lignin. It was made a prospection for new clusters gene for type II PKSs in a metagenomic library of 9320 clones by PCR amplification with degenerate primers for a conserved gene region. Among three clones found, one was sequenced by subcloning and shotgun strategy, resulting in a consensus of 22,988 base pairs. It was made the prediction of functional homology ORFs by Blast (NCBI) and also identifying areas for enzymatic Prodom and PFAM. Among them were found homologous to: ABC transporters; minimal PKS, cyclasea; Aromatasea; Hydroxylases; Mono-oxygenases; O-methyl-transferases; Multicopper oxidase/laccase, ?-glucosidase, chitinase/ Cellulases; Peptidases; Acyl-transferases, in addition of gene regulators similar to LacI and LuxR. The biosynthetic enzymes candidates for the core of the cluster (minimal pks) underwent three-dimensional structure modeling in silico. The 3D models were evaluated for accurate, using the tools of 'Assessing "Verify3D, Qmean, Procheck, Rampage and Swiss-Model. Once validated, the KSA and CLF were subjected to simulated docking enzyme between them

9.
Artigo em Inglês | VETINDEX | ID: vti-443055

Resumo

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins, which are able to interfere with hemostasis. They share significant similarity in their primary structures with C-type lectins of other animals, and also present a conserved carbohydrate recognition domain (CRD). A very well studied sv C-type lectin is the heterodimeric toxin, convulxin (CVX), from the venoms of South American rattlesnakes, Crotalus durissus terrificus and C. d. cascavella. It consists of two subunits, alfa (CVXalpha , 13.9 kDa) and beta (CVXbeta , 12.6 kDa), joined by inter and intra-chain disulfide bounds, and is arranged in a tetrameric alpha4beta4 conformation. Convulxin is able to activate platelet and induce their aggregation by acting via p62/GPVI collagen receptor. Several cDNA precursors, homolog of CVX subunits, were cloned by PCR homology screening. As determined by computational analysis, one of them, named crotacetin beta subunit, was predicted as a polypeptide with a tridimensional conformation very similar to other subunits of convulxin-like snake toxins. Crotacetin was purified from C. durissus venoms by gel permeation and reverse phase high performance liquid chromatography. The heterodimeric crotacetin is expressed in the venoms of several C. durissus subspecies, but it is prevalent in the venom of C. durissus cascavella. As inferred from homology modeling, crotacetin induces platelet aggregation but noticeably exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria.

10.
Braz. j. biol ; 84: e268133, 2024. tab, graf, ilus
Artigo em Inglês | VETINDEX | ID: biblio-1439672

Resumo

Phalaenopsis amabilis (L.) Blume commonly called Moth Orchid (Orchidaceae) is a natural orchid species designated as the National Flower of Indonesia for its beautiful flower shape and long-lasting flowering period. Basically, P. amabilis has a long vegetative phase that cause late flowering, about 2 to 3 years for flowering, hence a method to shorten vegetative period is desired. The latest technological approach that can be used to accelerate flowering of P. amabilis is the CRISPR/Cas9 genome editing method to inactivate the GAI (Gibberellic Acid Insensitive) gene as a mutant gene that can accelerate the regulation of FLOWERING TIME (FT) genes flowering biosynthesis pathway. The approach that needs to be taken is to silence the GAI gene with a knockout system which begins with identifying and characterizing the GAI target gene in the P. amabilis which will be used as a single guide RNA. CRISPR/Cas9 mediated knockout efficiency is highly dependent on the properties of the sgRNA used. SgRNA consists of a target sequence, determining its specificity performance. We executed phylogenetic clustering for the PaGAI protein with closely related orchid species such as Dendrobium capra, Dendrobium cultivars and Cymbidium sinensis. SWISS-Model as tool webserver for protein structure homology modeling. Results show that P. amabilis has a specific domain with the occurrence of point mutations in the two conservative domains. Therefore, a single guide RNA reconstruction needs to be implemented.


Phalaenopsis amabilis (L.) Blume, comumente chamada de orquídea mariposa (Orchidaceae), é uma espécie natural de orquídea designada como a flor nacional da Indonésia por seu belo formato de flor e período de floração duradouro. Basicamente, P. amabilis tem uma longa fase vegetativa que causa floração tardia, cerca de 2 a 3 anos para a floração, portanto, um método para encurtar o período vegetativo é desejado. A mais recente abordagem tecnológica que pode ser utilizada para acelerar a floração de P. amabilis é o método de edição do genoma CRISPR/Cas9 para inativar o GAI (Gibberellic Acid Insensitive) que pode ser usado como um gene mutante para acelerar a regulação da floração dos genes FLOWERING TIME (FT), via de biossíntese. Para isto, a melhor abordagem é silenciar o GAI gene com um sistema knockout que deve ser iniciado com a identificação e caracterização do gene alvo GAI no P. amabilis, e que, posteriormente, será utilizado como um único RNA guia. A eficiência de nocaute mediada por CRISPR/Cas9 é altamente dependente das propriedades do sgRNA usados. O SgRNA consiste em uma sequência alvo, determinando seu desempenho de especificidade. Executamos agrupamento filogenético para a proteína PaGAI com espécies de orquídeas intimamente relacionadas, como Dendrobium capra, Dendrobium cultivars e Cymbidium sinensis. SWISS-Model foi utilizado como ferramenta webserver para modelagem de homologia de estruturas de proteínas. Os resultados mostram que P. amabilis possui um domínio específico com ocorrência de mutações pontuais nos dois domínios conservativos. Portanto, uma única reconstrução de RNA guia precisa ser implementada.


Assuntos
Orchidaceae/genética , Flores , Desenvolvimento Vegetal , Mutação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA