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1.
Virus Res ; 278: 197867, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31972246

RESUMO

Infections produced by hepaciviruses have been associated with liver disease in horses. Currently, at least three viruses belonging to the Flaviviridae family are capable of producing a chronic infection in equines: non-primate hepacivirus (NPHV), Theiler's disease-associated virus (TDAV), and equine pegivirus (EPgV). The RNA-dependent RNA polymerases of viruses (RdRp) (NS5 protein), from the flavivirus family, use de novo RNA synthesis to initiate synthesis. The two antiviral drugs currently used to treat hepatitis C (HCV), sofosbuvir and dasabuvir, act on the viral NS5B polymerase as nucleoside and non-nucleoside inhibitors, respectively. Both drugs have shown significant clinical inhibition of viral response. In this work, we aimed to model the NS5B polymerase of the equine hepacivirus (EHCV) subtypes 1 and 2, TDAV and EPgV, to assess whether current direct-acting antiviral drugs against HCV interact with these proteins. Crystal structures of HCV-NS5B were used as templates for modeling target sequences in both conformations (open and closed). Also, molecular docking of sofosbuvir and dasabuvir were performed to predict their possible binding modes at the modeled NS5B polymerase binding sites. We observed that the NS5B models of the EHCV and EPgV shared well-conserved 3D structures to HCV-NS5B and other RdRps, suggesting functional conservation. Interactions of EHCV subtypes 1, 2 and TDAV polymerases with sofosbuvir showed a similar molecular interaction pattern compared to HCV-NS5B, while interactions with dasabuvir were less conserved. In silico studies of molecular interactions between these modeled structures and sofosbuvir suggest that this compound could be efficient in combating equine pathogens, thus contributing to animal welfare.

3.
Viruses ; 11(8)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366046

RESUMO

Hepatitis C virus genotype 1a (HCV-1a) comprises clades I and II. The Q80K polymorphism is found predominantly in clade I but rarely in clade II. Here, we investigated whether natural polymorphisms in HCV-1a clade II entailed structural protein changes when occurrence of the Q80K variant was simulated. Based on HCV-1a clade I and II protein sequences, the structure of the HCV-1a Q80K mutant NS3-4A was obtained by comparative modeling. Its physicochemical properties were studied by molecular dynamics simulations and network analysis. Results demonstrate that, in the presence of the K80 variant, clade II protease polymorphisms A91 and S/G174 led to variations in hydrogen bond occupancies. Structural analyses revealed differences in (i) flexibility of the H57 catalytic residue on the NS3 protease and (ii) correlations between amino acids on the NS3 protease and the NS4A cofactor. The latter indicated possible destabilization of interactions, resulting in increased separation of these proteins. The present findings describe how the relationships between different HCV-1a NS3 protease amino acid residues could affect the appearance of viral variants and the existence of distinct genetic barriers to HCV-1a isolates.

4.
PLoS One ; 14(4): e0215508, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30998730

RESUMO

Brain-derived neurotrophic factor (BDNF) plays an important role in neurogenesis and synapse formation. The V66M is the most prevalent BDNF mutation in humans and impairs the function and distribution of BDNF. This mutation is related to several psychiatric disorders. The pro-region of BDNF, particularly position 66 and its adjacent residues, are determinant for the intracellular sorting and activity-dependent secretion of BDNF. However, it has not yet been fully elucidated. The present study aims to analyze the effects of the V66M mutation on BDNF structure and function. Here, we applied nine algorithms, including SIFT and PolyPhen-2, for functional and stability prediction of the V66M mutation. The complete theoretical model of BNDF was generated by Rosetta and validated by PROCHECK, RAMPAGE, ProSa, QMEAN and Verify-3D algorithms. Structural alignment was performed using TM-align. Phylogenetic analysis was performed using the ConSurf server. Molecular dynamics (MD) simulations were performed and analyzed using the GROMACS 2018.2 package. The V66M mutation was predicted as deleterious by PolyPhen-2 and SIFT in addition to being predicted as destabilizing by I-Mutant. According to SNPeffect, the V66M mutation does not affect protein aggregation, amyloid propensity, and chaperone binding. The complete theoretical structure of BDNF proved to be a reliable model. Phylogenetic analysis indicated that the V66M mutation of BDNF occurs at a non-conserved position of the protein. MD analyses indicated that the V66M mutation does not affect the BDNF flexibility and surface-to-volume ratio, but affects the BDNF essential motions, hydrogen-bonding and secondary structure particularly at its pre and pro-domain, which are crucial for its activity and distribution. Thus, considering that these parameters are determinant for protein interactions and, consequently, protein function; the alterations observed throughout the MD analyses may be related to the functional impairment of BDNF upon V66M mutation, as well as its involvement in psychiatric disorders.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Simulação por Computador , Transtornos Mentais/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Medicina de Precisão , Substituição de Aminoácidos , Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/genética , Feminino , Humanos , Masculino , Relação Estrutura-Atividade
5.
Sci Rep ; 9(1): 4197, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862893

RESUMO

Riboswitches are RNA sensors that affect post-transcriptional processes through their ability to bind to small molecules. Thiamine pyrophosphate (TPP) riboswitch class is the most widespread riboswitch occurring in all three domains of life. Even though it controls different genes involved in the synthesis or transport of thiamine and its phosphorylated derivatives in bacteria, archaea, fungi, and plants, the TPP aptamer has a conserved structure. In this study, we aimed at understanding differences in the structural dynamics of TPP riboswitches from Escherichia coli and Arabidopsis thaliana, based on their crystallographic structures (TPPswec and TPPswat, respectively) and dynamics in aqueous solution, both in apo and holo states. A combination of Molecular Dynamics Simulations and Network Analysis empowered to find out slight differences in the dynamical behavior of TPP riboswitches, although relevant for their dynamics in bacteria and plants species. Our results suggest that distinct interactions in the microenvironment surrounding nucleotide U36 of TPPswec (and U35 in TPPswat) are related to different responses to TPP. The network analysis showed that minor structural differences in the aptamer enable enhanced intramolecular communication in the presence of TPP in TPPswec, but not in TPPswat. TPP riboswitches of plants present subtler and slower regulation mechanisms than bacteria do.

6.
J Gen Virol ; 99(4): 536-548, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29469689

RESUMO

Southeastern Brazil has been suffering a rapid expansion of a severe sylvatic yellow fever virus (YFV) outbreak since late 2016, which has reached one of the most populated zones in Brazil and South America, heretofore a yellow fever-free zone for more than 70 years. In the current study, we describe the complete genome of 12 YFV samples from mosquitoes, humans and non-human primates from the Brazilian 2017 epidemic. All of the YFV sequences belong to the modern lineage (sub-lineage 1E) of South American genotype I, having been circulating for several months prior to the December 2016 detection. Our data confirm that viral strains associated with the most severe YF epidemic in South America in the last 70 years display unique amino acid substitutions that are mainly located in highly conserved positions in non-structural proteins. Our data also corroborate that YFV has spread southward into Rio de Janeiro state following two main sylvatic dispersion routes that converged at the border of the great metropolitan area comprising nearly 12 million unvaccinated inhabitants. Our original results can help public health authorities to guide the surveillance, prophylaxis and control measures required to face such a severe epidemiological problem. Finally, it will also inspire other workers to further investigate the epidemiological and biological significance of the amino acid polymorphisms detected in the Brazilian 2017 YFV strains.


Assuntos
Febre Amarela/virologia , Vírus da Febre Amarela/genética , Brasil/epidemiologia , Surtos de Doenças , Genoma Viral , Genômica , Genótipo , Humanos , Modelos Moleculares , Filogenia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Febre Amarela/epidemiologia , Vírus da Febre Amarela/química , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/isolamento & purificação
8.
Mem Inst Oswaldo Cruz ; 112(9): 617-625, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28902287

RESUMO

BACKGROUND: Leishmanolysins have been described as important parasite virulence factors because of their roles in the infection of promastigotes and resistance to host's defenses. Leishmania (Viannia) braziliensis contains several leishmanolysin genes in its genome, especially in chromosome 10. However, the functional impact of such diversity is not understood, but may be attributed partially to the lack of structural data for proteins from this parasite. OBJECTIVES: This works aims to compare leishmanolysin sequences from L. (V.) braziliensis and to understand how the diversity impacts in their structural and dynamic features. METHODS: Leishmanolysin sequences were retrieved from GeneDB. Subsequently, 3D models were built using comparative modeling methods and their dynamical behavior was studied using molecular dynamic simulations. FINDINGS: We identified three subgroups of leishmanolysins according to sequence variations. These differences directly affect the electrostatic properties of leishmanolysins and the geometry of their active sites. We identified two levels of structural heterogeneity that might be related to the ability of promastigotes to interact with a broad range of substrates. MAIN CONCLUSION: Altogether, the structural plasticity of leishmanolysins may constitute an important evolutionary adaptation rarely explored when considering the virulence of L. (V.) braziliensis parasites.


Assuntos
Variação Genética , Leishmania braziliensis/genética , Metaloendopeptidases/genética , Conformação Proteica , Cromossomos , Humanos , Modelos Moleculares
9.
Mem. Inst. Oswaldo Cruz ; 112(9): 617-625, Sept. 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-894879

RESUMO

BACKGROUND Leishmanolysins have been described as important parasite virulence factors because of their roles in the infection of promastigotes and resistance to host's defenses. Leishmania (Viannia) braziliensis contains several leishmanolysin genes in its genome, especially in chromosome 10. However, the functional impact of such diversity is not understood, but may be attributed partially to the lack of structural data for proteins from this parasite. OBJECTIVES This works aims to compare leishmanolysin sequences from L. (V.) braziliensis and to understand how the diversity impacts in their structural and dynamic features. METHODS Leishmanolysin sequences were retrieved from GeneDB. Subsequently, 3D models were built using comparative modeling methods and their dynamical behavior was studied using molecular dynamic simulations. FINDINGS We identified three subgroups of leishmanolysins according to sequence variations. These differences directly affect the electrostatic properties of leishmanolysins and the geometry of their active sites. We identified two levels of structural heterogeneity that might be related to the ability of promastigotes to interact with a broad range of substrates. MAIN CONCLUSION Altogether, the structural plasticity of leishmanolysins may constitute an important evolutionary adaptation rarely explored when considering the virulence of L. (V.) braziliensis parasites.


Assuntos
Humanos , Leishmania braziliensis/genética , Metaloendopeptidases/genética , Conformação Proteica , Variação Genética , Modelos Moleculares
10.
Eur J Med Chem ; 126: 72-83, 2017 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-27744189

RESUMO

Malaria remains one of the most serious global infectious diseases. An important target for antimalarial chemotherapy is the enzyme dihydroorotate dehydrogenase from Plasmodium falciparum (PfDHODH), which is responsible for the conversion of dihydroorotate to orotate in the de novo pyrimidine biosynthetic pathway. In this study, we have designed and synthesized fifteen 7-arylpyrazolo[1,5-a]pyrimidine derivatives using ring bioisosteric replacement and molecular hybridization of functional groups based on the highly active 5-methyl-N-(naphthalen-2-yl)-2-(trifluoromethyl)- [1,2,4]triazolo[1,5-a]pyrimidin-7-amine. The compounds were tested against Plasmodium falciparum, as antimalarials in mice with P. berghei, and as inhibitors of PfDHODH. Thirteen compounds were found to be active against P. falciparum, with IC50 values ranging from 1.2 ± 0.3 to 92 ± 26 µM in the anti-HRP2 and hypoxanthine assays. Four compounds showed the highest selective index (SI), which is a ratio between cytotoxicity and activity in vitro. The inhibition of PfDHODH showed that compound 30 (R2 = CH3; R5 = CF3; Ar = 7-ß-naphthyl) displayed higher and selective inhibitory activity, with IC50 = 0.16 ± 0.01 µM, followed by 25 (R2 = CH3; R5 = CH3; Ar = 7-ß-Naphthyl) and 19 (R2 = CF3; R5 = CF3; Ar = 7-ß-naphthyl), with IC50 = 4 ± 1 µM and 6 ± 1 µM, respectively. The trifluoromethyl group at the 2- or 5-positions of the pyrazolo[1,5-a]pyrimidine ring led to increased drug activity. The docking results agreed with the values obtained from enzymatic assays.


Assuntos
Antimaláricos/farmacologia , Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Antimaláricos/química , Antimaláricos/metabolismo , Antimaláricos/toxicidade , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Humanos , Camundongos , Simulação de Acoplamento Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Conformação Proteica , Pirimidinas/metabolismo , Pirimidinas/toxicidade
11.
J Chem Inf Model ; 56(12): 2495-2506, 2016 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-28024405

RESUMO

In this work, we propose a deep learning approach to improve docking-based virtual screening. The deep neural network that is introduced, DeepVS, uses the output of a docking program and learns how to extract relevant features from basic data such as atom and residues types obtained from protein-ligand complexes. Our approach introduces the use of atom and amino acid embeddings and implements an effective way of creating distributed vector representations of protein-ligand complexes by modeling the compound as a set of atom contexts that is further processed by a convolutional layer. One of the main advantages of the proposed method is that it does not require feature engineering. We evaluate DeepVS on the Directory of Useful Decoys (DUD), using the output of two docking programs: Autodock Vina1.1.2 and Dock 6.6. Using a strict evaluation with leave-one-out cross-validation, DeepVS outperforms the docking programs, with regard to both AUC ROC and enrichment factor. Moreover, using the output of Autodock Vina1.1.2, DeepVS achieves an AUC ROC of 0.81, which, to the best of our knowledge, is the best AUC reported so far for virtual screening using the 40 receptors from the DUD.


Assuntos
Desenho Assistido por Computador , Desenho de Fármacos , Simulação de Acoplamento Molecular/métodos , Redes Neurais de Computação , Software , Algoritmos , Humanos , Ligantes , Proteínas/metabolismo , Curva ROC
12.
J Mol Graph Model ; 70: 30-39, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27649549

RESUMO

Cysteine proteinase B (CPB) is a significant virulence factor for Leishmania infections. Upon processing from its zymogen form, it happens a release of the immunomodulatory CPB C-terminal extension (cyspep) into the cytoplasm of the macrophage. Epitopes derived from this fragment were shown to influence the proportion of lymphocytes CD8+ upon infection, favoring the parasite escaping from the host́s immune system. At present, there is no available structural data of cyspep, which impairs a proper understanding of its biological functions. Here, we attempted to build molecular models for this fragment and subsequently evaluate their stabilities in aqueous solution from molecular dynamics simulations analysis. Characterization of our models obtained with distinct techniques (comparative modeling, threading, and ab initio) indicates a prevalence of ß-sheets in agreement with consensus secondary structure predictions. Simulation data supported this finding since the formation of new strands, along with a rapid disruption of helical content, were observed. Overall, this study provides a rationalization of epitope mapping data and an improved understanding of cyspep antigenicity.


Assuntos
Cisteína Proteases/química , Leishmania/enzimologia , Simulação de Dinâmica Molecular , Proteínas de Protozoários/química , Sequência de Aminoácidos , Dissulfetos/metabolismo , Estrutura Secundária de Proteína , Alinhamento de Sequência , Soluções , Fatores de Tempo
13.
Cad. saúde colet., (Rio J.) ; 24(1): 63-69, jan.-mar. 2016. tab, graf
Artigo em Português | LILACS-Express | ID: lil-781540

RESUMO

Resumo Objetivo Descrever o estágio do envelhecimento populacional no município do Rio de Janeiro. Métodos Estudo ecológico tendo como unidades de observação os 160 bairros que compõem o município, utilizando indicadores sociais e demográficos construídos a partir de informações do Censo 2010. Realizou-se análise exploratória por meio de mapas temáticos e determinou-se a dependência espacial pelo Índice de Moran Global. Para agrupar bairros em estágios semelhantes do envelhecimento foi realizada uma análise de agrupamento a partir do método K-means. Resultados Encontraram-se três grupos de bairros em estágios diferentes de envelhecimento populacional, identificando-se uma tendência espacial no sentido oeste-leste com os bairros da “Zona Sul” se encontrando no estágio mais avançado de envelhecimento. Conclusão O estudo identificou as diferenças no processo de envelhecimento populacional e na composição etária dos bairros, apontando para a necessidade de políticas de saúde pública específicas que contemplem as particularidades desse processo em cada localidade, visando garantir um envelhecimento sustentável.


Abstract Objective Describe the stage of population aging in the city of Rio de Janeiro. Methods Ecological study with the 160 city neighborhoods as observational units, using social and demographic indicators built with information from the 2010 census. The exploratory analysis was undertaken with thematic maps, and the spatial dependence was measured with the Global Moran’s Index. K-means clustering was used for grouping neighborhoods with similar aging stages. Results Three neighborhood clusters in different stages of population aging were found and a spatial trend in the west-east direction was identified, with neighborhoods in the ‘South Zone’ in a more advanced stage of population aging. Conclusion The study identified differences in the population aging process and in the age composition of neighborhoods, indicating the need for specific public health policies that allow for the particularities of this process in each location, aiming a sustainable population aging.

14.
Proteins ; 84(4): 473-87, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26798994

RESUMO

New strategies to control Leishmania disease demand an extensive knowledge about several aspects of infection including the understanding of its molecular events. In murine models, cysteine proteinase B from Leishmania amazonensis promotes regulation of immune response, and fragments from its C-terminus extension (cyspep) can play a decisive role in the host-parasite interaction. The interaction between cyspep-derived peptides and major histocompatibility complex (MHC) proteins is a crucial factor in Leishmania infections. Seven cyspep-derived peptides, previously identified as capable of interacting with H-2 (murine) MHC class I proteins, were studied in this work. We established a protocol to simulate the unbinding of these peptides from the cleft of H-2 receptors. From the simulations, we estimated the corresponding free energy of dissociation (ΔGd ) and described the molecular events that occur during the exit of peptides from the cleft. To test the reliability of this method, we first applied it to a calibration set of four crystallographic MHC/peptide complexes. Next, we explored the unbinding of the seven complexes mentioned above. Results were consistent with ΔGd values obtained from surface plasmon resonance (SPR) experiments. We also identified some of the primary interactions between peptides and H-2 receptors, and we detected three regions of influence for the interaction. This pattern was systematically observed for the peptides and helped determine a minimum distance for the real interaction between peptides and H-2 proteins occurring at ∼ 25 Å.


Assuntos
Cisteína Proteases/química , Epitopos/química , Antígenos de Histocompatibilidade Classe I/química , Leishmania braziliensis/química , Peptídeos/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Cisteína Proteases/genética , Cisteína Proteases/imunologia , Epitopos/genética , Epitopos/imunologia , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Leishmania braziliensis/patogenicidade , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Simulação de Dinâmica Molecular , Peptídeos/genética , Peptídeos/imunologia , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Ressonância de Plasmônio de Superfície , Termodinâmica
15.
Mem Inst Oswaldo Cruz ; 110(7): 847-64, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26560977

RESUMO

Reverse transcriptase (RT) is a multifunctional enzyme in the human immunodeficiency virus (HIV)-1 life cycle and represents a primary target for drug discovery efforts against HIV-1 infection. Two classes of RT inhibitors, the nucleoside RT inhibitors (NRTIs) and the nonnucleoside transcriptase inhibitors are prominently used in the highly active antiretroviral therapy in combination with other anti-HIV drugs. However, the rapid emergence of drug-resistant viral strains has limited the successful rate of the anti-HIV agents. Computational methods are a significant part of the drug design process and indispensable to study drug resistance. In this review, recent advances in computer-aided drug design for the rational design of new compounds against HIV-1 RT using methods such as molecular docking, molecular dynamics, free energy calculations, quantitative structure-activity relationships, pharmacophore modelling and absorption, distribution, metabolism, excretion and toxicity prediction are discussed. Successful applications of these methodologies are also highlighted.


Assuntos
Fármacos Anti-HIV/química , Desenho Assistido por Computador , Desenho de Fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/química , HIV-1/enzimologia , Humanos , Modelos Biológicos , Estrutura Molecular , Relação Quantitativa Estrutura-Atividade , Inibidores da Transcriptase Reversa/química
16.
Mem. Inst. Oswaldo Cruz ; 110(7): 847-864, Nov. 2015. graf
Artigo em Inglês | LILACS | ID: lil-764593

RESUMO

Reverse transcriptase (RT) is a multifunctional enzyme in the human immunodeficiency virus (HIV)-1 life cycle and represents a primary target for drug discovery efforts against HIV-1 infection. Two classes of RT inhibitors, the nucleoside RT inhibitors (NRTIs) and the nonnucleoside transcriptase inhibitors are prominently used in the highly active antiretroviral therapy in combination with other anti-HIV drugs. However, the rapid emergence of drug-resistant viral strains has limited the successful rate of the anti-HIV agents. Computational methods are a significant part of the drug design process and indispensable to study drug resistance. In this review, recent advances in computer-aided drug design for the rational design of new compounds against HIV-1 RT using methods such as molecular docking, molecular dynamics, free energy calculations, quantitative structure-activity relationships, pharmacophore modelling and absorption, distribution, metabolism, excretion and toxicity prediction are discussed. Successful applications of these methodologies are also highlighted.


Assuntos
Humanos , Fármacos Anti-HIV/química , Desenho Assistido por Computador , Desenho de Fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1 , Inibidores da Transcriptase Reversa/farmacologia , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/química , HIV-1 , Modelos Biológicos , Estrutura Molecular , Relação Quantitativa Estrutura-Atividade , Inibidores da Transcriptase Reversa/química
17.
Mol Pharmacol ; 88(5): 949-61, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26330549

RESUMO

Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88- and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-ß-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-ß knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.


Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Limoninas/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Antígeno 96 de Linfócito/metabolismo , Macrófagos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Sítios de Ligação , Células Cultivadas , Citocinas/biossíntese , Dinoprostona/biossíntese , Relação Dose-Resposta a Droga , Limoninas/metabolismo , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/química , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Receptores de Interleucina-1/fisiologia , Receptor 4 Toll-Like/fisiologia
18.
Antimicrob Agents Chemother ; 59(4): 1910-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25583728

RESUMO

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm(2)), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm(2)) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm(2)). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound's effects on the parasite.


Assuntos
Antiprotozoários/farmacologia , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/enzimologia , Naftoquinonas/farmacologia , Inibidores de Serino Proteinase/farmacologia , Animais , Antipaína/farmacologia , Simulação por Computador , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Serina Endopeptidases/metabolismo
19.
Mem. Inst. Oswaldo Cruz ; 109(3): 315-323, 06/2014. tab, graf
Artigo em Inglês | LILACS | ID: lil-711722

RESUMO

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.


Assuntos
Animais , Humanos , Masculino , Camundongos , Nitrorredutases/efeitos dos fármacos , Tiadiazóis , Triazóis , Tripanossomicidas , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Nitrorredutases/metabolismo , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tiadiazóis/química , Tiadiazóis/metabolismo , Tiadiazóis/farmacologia , Tiadiazóis/toxicidade , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologia , Triazóis/toxicidade , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Tripanossomicidas/toxicidade , Trypanosoma cruzi/efeitos dos fármacos
20.
Mem Inst Oswaldo Cruz ; 109(3): 315-23, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24676659

RESUMO

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.


Assuntos
Nitrorredutases/efeitos dos fármacos , Tiadiazóis , Triazóis , Tripanossomicidas , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Animais , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Masculino , Camundongos , Nitrorredutases/metabolismo , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tiadiazóis/química , Tiadiazóis/metabolismo , Tiadiazóis/farmacologia , Tiadiazóis/toxicidade , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologia , Triazóis/toxicidade , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Tripanossomicidas/toxicidade , Trypanosoma cruzi/efeitos dos fármacos
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