RESUMO
With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.
Assuntos
Antiprotozoários/uso terapêutico , Chalcona/metabolismo , Chalcona/farmacologia , Citosol/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Peroxidases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/farmacologia , Células Cultivadas , Chalcona/administração & dosagem , Chalcona/análogos & derivados , Citosol/enzimologia , Citosol/parasitologia , Descoberta de Drogas , Humanos , Leishmania/classificação , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Peroxidase inhibition produced by phenolic compounds as hispidin and gallic acid, alkaloids as harmine and natural extracts of Inonotus hispidus, and Marrubium vulgare were investigated in this study. No further studies have been found in this context. Thus, the results show that the phenolic and the alkaloidal extracts with the three molecules are potent inhibitors of horseradish peroxidase. Uric acid is used as a substrate reaction to finding the enzymatic inhibition for the first time. The results show that the best inhibitor is hispidin with a value of IC50 = 23 µg/ml. Moreover, Molecular docking has been carried out using the AutoDock Vina program to discuss the nature of interactions and the mechanism of inhibition between both peroxidases (horseradish and thyroid) which is performed with and without heme group for the first time. The three studied compounds were further subjected to ADEMT and Lipinski filtering analyses for drug-likeness prediction analysis. However, the results show that all the docked molecules are competitive inhibitors confirming that no further studies have been published before. Thus, hispidin is a more potent irreversible TPO inhibitor then propylthiouracil anti-thyroid drug. Its inhibition mechanism is well described through this work for the first time; which suggests is used as an anti-thyroid drug to treat hyperthyroidism. Furthermore, the studied phenolic compounds (Hispidin and Gallic acid) and one alkaloid (Harmine) are non-toxic, that bind to the receptor-binding site and catalytic dyad of peroxidases were identified from the predictive ADMET and Lipinski filter analysis.Communicated by Ramaswamy H. Sarma.
Assuntos
Alcaloides , Inibidores Enzimáticos/farmacologia , Peroxidases , Alcaloides/farmacologia , Sítios de Ligação , Simulação de Acoplamento Molecular , Peroxidases/antagonistas & inibidores , Fenóis/farmacologiaRESUMO
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). In the present age, due to the rapid increase in antibiotic resistance worldwide, TB has become a major threat to human life. Regardless of significant efforts have been inclined to improve the healthcare systems for improving diagnosis, treatment, and anticipatory measures controlling TB is challenging. To date, there are no such therapeutic chemical agents available to fight or control the bacterial drug-resistance. The catalase-peroxidase enzyme (katG) which encoded by the katG gene of Mtb is most frequently getting mutated and hence promotes Isoniazid resistance by diminishing the normal activity of katG enzyme. In the current study, an effort has been intended to find novel and therapeutically active antibacterial chemical compounds through pharmacoinformatics methodologies. Initially, the five mutant katG were generated by making mutation of Ser315 by Thr, Ile, Arg, Asn, and Gly followed by structural optimizations. About eight thousand small molecules were collected from the Asinex antibacterial library. All molecules were docked to active site of five mutant katG and wild type katG. To narrow down the chemical space several criteria were imposed including, screening for highest binding affinity towards katG proteins, compounds satisfying various criterion of drug-likeliness properties like Lipinski's rule of five (RO5), Veber's rule, absorption, distribution, metabolism, and excretion (ADME) profile, and synthetic accessibility. Finally, five molecules were found to be important antibacterial katG inhibitors. All the analyzed parameters suggested that selected molecules are promising in nature. Binding interactions analysis revealed that proposed molecules are efficient enough to form a number of strong binding interactions with katG proteins. Dynamic behavior of the proposed molecules with katG protein was evaluated through 100â¯ns molecular dynamics (MD) simulation study. Parameters calculated from the MD simulation trajectories adjudged that all molecules can form stable complexes with katG. High binding free energy of all proposed molecules definitely suggested strong affection towards the katG. Hence, it can be concluded that proposed molecules might be used as antibacterial chemical component subjected to experimental validation.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Peroxidases/antagonistas & inibidores , Antituberculosos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peroxidases/genética , Peroxidases/metabolismoRESUMO
In this work, a simulated enzyme inhibition-based strategy was transplanted from natural peroxidase-based sensing methods for colorimetric nanoperoxidase-based biothiol detection. This work might provide some new perspectives for the construction and biomimetic regulation of mimicked biological signalling systems based on nanoperoxidases.
Assuntos
Corantes Fluorescentes/química , Glutationa/análise , Mercúrio/química , Nanopartículas Metálicas/química , Peroxidases/química , Pontos Quânticos/química , Compostos de Sulfidrila/análise , Materiais Biomiméticos/química , Técnicas Biossensoriais/métodos , Carbono/química , Catálise , Colorimetria/métodos , Ouro/química , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Oxirredução , Peroxidases/antagonistas & inibidores , Propriedades de SuperfícieRESUMO
OBJECTIVES: Vandetanib¸ lenvatinib, and cabozantinib are tyrosine kinase inhibitors (TKIs) targeting VEGFR subtypes 1 and 2, EGFR and the RET-tyrosine kinase, thus considered as multiple TKIs. These TKIs have already been approved for treating patients suffering from thyroid cancer and renal cell carcinoma. Ellipticine, a DNA-damaging drug, is another anticancer agent that is effective against certain tumors of the thyroid gland, ovarian carcinoma, breast cancer and osteolytic breast cancer metastasis. Its anticancer efficiency is dictated by its oxidation with cytochrome P450 (CYP) and peroxidase enzymes. A number of studies testing the effectiveness of individual anticancer drugs, the pharmacological efficiencies of which are affected by their metabolism, alone or in a combination with other cytostatics demonstrated that such combination can have both positive and negative effects on treatment regimen. The aim of this study was to study the effect of vandetanib, lenvatinib and cabozantinib on oxidation of ellipticine which dictates its pharmacological efficiency. METHODS: Ellipticine oxidation catalyzed by hepatic microsomes, recombinant CYP enzymes and peroxidases (horseradish peroxidase, lactoperoxidase and myeloperoxidase) and the effect of TKIs (vandetanib, lenvatinib and cabozantinib) on this oxidation were analyzed by HPLC used for separation of ellipticine metabolites and quantification of their amounts formed during oxidation. RESULTS: The CYP enzymatic system oxidizes ellipticine up to five metabolites (9-hydroxy-, 12-hydroxy-, 13-hydroxy-, 7-hydroxyellipticine, and ellipticine N2- oxide), while peroxidases form predominantly ellipticine dimer. Ellipticine oxidation catalyzed by rat and human hepatic microsomes was inhibited by vandetanib and cabozantinib, but essentially no inhibition was caused by lenvatinib. Of individual CYP enzymes catalyzing oxidation of ellipticine, TKIs inhibited oxidation of ellipticine catalyzed by CYP2D6 > 2D1 > 2C9 > 3A1 > 3A4, the CYP enzymes participating in ellipticine oxidation to metabolites increasing the ellipticine anticancer efficiency. On the contrary, they have essentially no inhibition effect on ellipticine oxidation catalyzed by CYP1A1 and 1A2, which are the enzymes that predominantly detoxify this drug. All tested TKIs had essentially no effect on oxidation of ellipticine by used peroxidases. CONCLUSION: The results found demonstrate that TKIs vandetanib, lenvatinib and cabozantinib cause a decrease in oxidative activation of DNA-damaging drug ellipticine by several CYP enzymes in vitro which might lead to a decrease in its pharmacological efficiency. In contrast, they practically do not influence its detoxification catalyzed by CYP1A1, 1A2 and peroxidases. The present study indicates that tested TKIs seem not to have a potency to increase ellipticine anticancer efficiency.
Assuntos
Anilidas/farmacologia , Elipticinas/farmacocinética , Oxirredução/efeitos dos fármacos , Peroxidases/antagonistas & inibidores , Compostos de Fenilureia/farmacologia , Piperidinas/farmacologia , Piridinas/farmacologia , Quinazolinas/farmacologia , Quinolinas/farmacologia , Animais , Inibidores das Enzimas do Citocromo P-450/farmacologia , Humanos , Microssomos Hepáticos/efeitos dos fármacos , RatosRESUMO
Right ventricle (RV) remodeling is a major pathological feature in pulmonary arterial hypertension (PAH). Magnesium lithospermate B (MLB) is a compound isolated from the roots of Salvia miltiorrhiza and it possesses multiple pharmacological activities such as anti-inflammation and antioxidation. This study aims to investigate whether MLB is able to prevent RV remodeling in PAH and the underlying mechanisms. In vivo, SD rats were exposed to 10% O2 for 21 d to induce RV remodeling, which showed hypertrophic features (increases in the ratio of RV weight to tibia length, cellular size, and hypertrophic marker expression), accompanied by upregulation in expression of NADPH oxidases (NOX2 and NOX4) and vascular peroxidase 1 (VPO1), increases in hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production and elevation in phosphorylation levels of ERK; these changes were attenuated by treating rats with MLB. In vitro, the cultured H9c2 cells were exposed to 3% O2 for 24 h to induce hypertrophy, which showed hypertrophic features (increases in cellular size and hypertrophic marker expression). Administration of MLB or VAS2870 (a positive control for NOX inhibitor) could prevent cardiomyocyte hypertrophy concomitant with decreases in NOX (NOX2 and NOX4) and VPO1 expression, H2O2 and HOCl production, and ERK phosphorylation. Based on these observations, we conclude that MLB is able to prevent RV remodeling in hypoxic PAH rats through a mechanism involving a suppression of NOX/VPO1 pathway as well as ERK signaling pathway. MLB may possess the potential clinical value for PAH therapy.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Hemeproteínas/metabolismo , Hipertensão Pulmonar/fisiopatologia , NADPH Oxidases/metabolismo , Peroxidases/metabolismo , Salvia miltiorrhiza/química , Remodelação Ventricular/efeitos dos fármacos , Animais , Fator Natriurético Atrial/genética , Benzoxazóis/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/isolamento & purificação , Hemeproteínas/antagonistas & inibidores , Hipertensão Pulmonar/metabolismo , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , NADPH Oxidases/antagonistas & inibidores , Peptídeo Natriurético Encefálico/genética , Peroxidases/antagonistas & inibidores , Ratos Sprague-Dawley , Triazóis/farmacologiaRESUMO
BACKGROUND: Goodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur via disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP. METHODS: We used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score. RESULTS: We detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production in vitro. The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease. CONCLUSIONS: Anti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients.
Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Autoanticorpos/sangue , Proteínas da Matriz Extracelular/imunologia , Glomerulonefrite/imunologia , Hemorragia/imunologia , Pneumopatias/imunologia , Peroxidase/imunologia , Peroxidases/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Antimembrana Basal Glomerular/etiologia , Anticorpos Anticitoplasma de Neutrófilos/sangue , Especificidade de Anticorpos , Autoantígenos/imunologia , Criança , Estudos de Coortes , Colágeno Tipo IV/imunologia , Proteínas da Matriz Extracelular/antagonistas & inibidores , Feminino , Glomerulonefrite/etiologia , Hemorragia/etiologia , Humanos , Pneumopatias/etiologia , Masculino , Pessoa de Meia-Idade , Modelos Imunológicos , Peroxidase/antagonistas & inibidores , Peroxidases/antagonistas & inibidores , Adulto Jovem , PeroxidasinaRESUMO
Development of fast and accurate methods to discover lead compounds for drug candidates is highly important. In this study, a reliable and effective post-column on-flow biochemical assay (POBA) was established to screen potent peroxidase inhibitors from complex chemical mixtures (e.g., natural product extracts). Multiple factors such as flow rate, organic phase, detection wavelength, and reaction coil were carefully investigated. To better understand the features of POBA, another emerging technology of ultrafiltration LC-MS was used for comparison. The result showed that POBA had advantages in saving time, avoiding false positives, and improving the accuracy. To illustrate the practicality of the method, Radix Salvia Miltiorrhizae, a traditional herb for cardiovascular disease treatment, was applied as the research objective. Finally, six compounds including tanshinol, protocatechuic aldehyde, salvianolic acid D, rosmarinic acid, lithospermic acid, and salvianolic acid B were determined as novel peroxidase inhibitors. Their bioactivities were validated by microplate-based assay, molecular docking, and pharmacophore modeling. This study demonstrates a great potential of POBA in the efficient and accurate discovery of drug candidates. Graphical abstract Compared with a classical method of ultrafiltration LC-MS, the newly developed method of on-flow bioassay shows advantages in saving time, avoiding false positives and improving the accuracy.
Assuntos
Cromatografia Líquida/métodos , Inibidores Enzimáticos/isolamento & purificação , Peroxidases/antagonistas & inibidores , Compostos Fitoquímicos/isolamento & purificação , Salvia miltiorrhiza/química , Espectrometria de Massas em Tandem/métodos , Ultrafiltração/métodos , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Compostos Fitoquímicos/farmacologiaRESUMO
Peroxidase (POD) from jackfruit bulb was purified using ammonium sulfate precipitation, hydrophobic interaction and gel filtration columns. The POD was a dimer with a molecular weight of 104kDa. The Km and Vmax values for guaiacol, gallic acid and ophenylenediamine (OPD) were estimated. OPD was the most suitable substrate. The enzyme showed its maximum activity at pH5.5 and 55-60°C. The activation energy (Ea) of heat inactivation was estimated to be 206.40kJ/mol. The enthalpy, free energy and entropy values for the thermal inactivation were also determined. The POD activity was enhanced by K+, Zn2+, Ba2+, citric acid, malic acid, benzoic acid and EDTA·Na2, but inhibited by Cu2+, Ca2+, glutathione, cysteine and ascorbic acid. Chemical modification indicated a histidine residue was located in the enzyme active site. The POD activity in fruit extracts significantly decreased when heated at 80°C and 90°C. The ferric-reducing antioxidant power, ABTS radical scavenging activity and total phenolics decreased with increasing heating temperature and time.
Assuntos
Artocarpus/enzimologia , Peroxidases/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/isolamento & purificação , Catecol Oxidase/farmacologia , Cátions/farmacologia , Inibidores Enzimáticos/farmacologia , Aditivos Alimentares/farmacologia , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/farmacologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Oxirredução , Peroxidases/antagonistas & inibidores , Peroxidases/farmacologia , Fenóis/análise , Proteínas de Plantas/agonistas , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/farmacologia , Raízes de Plantas/enzimologia , Estabilidade Proteica , Especificidade por SubstratoRESUMO
Acute lung injury (ALI) is a clinical syndrome characterized by acute respiratory failure and is associated with substantial morbidity and mortality. Rhesus θ-defensin (RTD)-1 is an antimicrobial peptide with immunomodulatory activity. As airway inflammation and neutrophil recruitment and activation are hallmarks of ALI, we evaluated the therapeutic efficacy of RTD-1 in preclinical models of the disease. We investigated the effect of RTD-1 on neutrophil chemotaxis and macrophage-driven pulmonary inflammation with human peripheral neutrophils and LPS-stimulated murine alveolar macrophage (denoted MH-S) cells. Treatment and prophylactic single escalating doses were administered subcutaneously in a well-established murine model of direct endotoxin-induced ALI. We assessed lung injury by histopathology, pulmonary edema, inflammatory cell recruitment, and inflammatory cytokines/chemokines in the BAL fluid. In vitro studies demonstrated that RTD-1 suppressed CXCL8-induced neutrophil chemotaxis, TNF-mediated neutrophil-endothelial cell adhesion, and proinflammatory cytokine release in activated murine alveolar immortalized macrophages (MH-S) cells. Treatment with RTD-1 significantly inhibited in vivo LPS-induced ALI by reducing pulmonary edema and histopathological changes. Treatment was associated with dose- and time-dependent inhibition of proinflammatory cytokines (TNF, IL-1ß, and IL-6), peroxidase activity, and neutrophil recruitment into the airways. Antiinflammatory effects were demonstrated in animals receiving RTD-1 up to 12 hours after LPS challenge. Notably, subcutaneously administered RTD-1 demonstrates good peptide stability as demonstrated by the long in vivo half-life. Taken together, these studies demonstrate that RTD-1 is efficacious in an experimental model of ALI through inhibition of neutrophil chemotaxis and adhesion, and the attenuation of proinflammatory cytokines and gene expression from alveolar macrophages.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios não Esteroides/uso terapêutico , Defensinas/uso terapêutico , Infiltração de Neutrófilos/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Edema Pulmonar/tratamento farmacológico , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Adesão Celular/imunologia , Quimiocinas/biossíntese , Células Endoteliais/patologia , Humanos , Interleucina-1beta/antagonistas & inibidores , Interleucina-6/antagonistas & inibidores , Lipopolissacarídeos/toxicidade , Macaca mulatta , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Peroxidases/antagonistas & inibidores , Pneumonia/patologia , Edema Pulmonar/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Bismuth drugs have been used clinically to treat infections from Helicobacter pylori, a pathogen that is strongly related to gastrointestinal diseases even stomach cancer. Despite extensive studies, the mechanisms of action of bismuth drugs are not fully understood. Alkyl hydroperoxide reductase subunit C (AhpC) is the most abundant 2-cysteine peroxiredoxin, crucial for H. pylori survival in the host by defense of oxidative stress. Herein we show that a Bi(III) antiulcer drug (CBS) binds to the highly conserved cysteine residues (Cys49 and Cys169) with a dissociation constant (K d) of Bi(III) to AhpC of 3.0 (±1.0) × 10-24 M. Significantly the interaction of CBS with AhpC disrupts the peroxiredoxin and chaperone activities of the enzyme both in vitro and in bacterial cells, leading to attenuated bacterial survival. Moreover, using a home-made fluorescent probe, we demonstrate that Bi(III) also perturbs AhpC relocation between the cytoplasm and membrane region in decomposing the exogenous ROS. Our study suggests that disruption of redox homeostasis by bismuth drugs via interaction with key enzymes such as AhpC contributes to their antimicrobial activity.
Assuntos
Antibacterianos/farmacologia , Antiulcerosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Bismuto/farmacologia , Helicobacter pylori/efeitos dos fármacos , Peroxidases/antagonistas & inibidores , Antibacterianos/química , Antiulcerosos/química , Proteínas de Bactérias/química , Bismuto/química , Relação Dose-Resposta a Droga , Helicobacter pylori/química , Testes de Sensibilidade Microbiana , Peroxidases/química , Relação Estrutura-AtividadeRESUMO
The toxicities of azole pollutants that have widespread agricultural and industrial uses are either poorly understood or unknown, particularly with respect to how infaunal organisms are impacted by this class of persistent organic pollutant. To identify a molecular basis by which azole compounds may have unforeseen toxicity on marine annelids, we examine here their impact on the multifunctional dehaloperoxidase (DHP) hemoglobin from the terebellid polychaete Amphitrite ornata. Ultraviolet-visible and resonance Raman spectroscopic studies showed an increase in the six-coordinate low-spin heme population in DHP isoenzyme B upon binding of imidazole, benzotriazole, and benzimidazole (Kd values of 52, 82, and 110 µM, respectively), suggestive of their direct binding to the heme-Fe. Accordingly, atomic-resolution X-ray crystal structures, supported by computational studies, of the DHP B complexes of benzotriazole (1.14 Å), benzimidazole (1.08 Å), imidazole (1.08 Å), and indazole (1.12 Å) revealed two ligand binding motifs, one with direct ligand binding to the heme-Fe, and another in which the ligand binds in the hydrophobic distal pocket without coordinating the heme-Fe. Taken together, the results demonstrate a new mechanism by which azole pollutants can potentially disrupt hemoglobin function, thereby improving our understanding of their impact on infaunal organisms in marine and aquatic environments.
Assuntos
Benzimidazóis/metabolismo , Poluentes Ambientais/metabolismo , Hemoglobinas/metabolismo , Imidazóis/metabolismo , Modelos Moleculares , Peroxidases/metabolismo , Poliquetos/enzimologia , Triazóis/metabolismo , Motivos de Aminoácidos , Animais , Benzimidazóis/química , Benzimidazóis/toxicidade , Domínio Catalítico , Biologia Computacional , Poluentes Ambientais/química , Poluentes Ambientais/toxicidade , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Fungicidas Industriais/química , Fungicidas Industriais/metabolismo , Fungicidas Industriais/toxicidade , Hemoglobinas/antagonistas & inibidores , Hemoglobinas/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Imidazóis/química , Imidazóis/toxicidade , Indazóis/química , Indazóis/metabolismo , Indazóis/toxicidade , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Ligantes , Peroxidases/antagonistas & inibidores , Peroxidases/química , Praguicidas/química , Praguicidas/metabolismo , Praguicidas/toxicidade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Triazóis/química , Triazóis/toxicidadeRESUMO
There are large differences between flavonoids to protect against apoptosis, a process in which cytochrome c (Cyt c) plays a key role. In this work, we show that 7 of 13 flavonoids studied have a capacity to reduce Cyt c similar or higher than ascorbate, the flavonols quercetin, kaempferol and myricetin, flavanol epigallocatechin-gallate, anthocyanidins cyanidin and malvidin, and the flavone luteolin. In contrast, the kaempferol 3(O)- and 3,4'(O)-methylated forms, the flavanone naringenin, and also apigenin and chrysin, had a negligible reducing capacity. Equilibrium dialysis and quenching of 1,6-diphenyl-1,3,5-hexatriene fluorescence experiments showed that flavonoids did not interfere with Cyt c binding to cardiolipin (CL)/phosphatidylcholine (PC) vesicles. However, the CL-induced loss of Cyt c Soret band intensity was largely attenuated by flavonoids, pointing out a stabilizing action against Cyt c unfolding in the complex. Moreover, flavonoids that behave as Cyt c reductants also inhibited the pro-apoptotic CL-induced peroxidase activity of Cyt c, indicating that modulation of Cyt c signaling are probable mechanisms behind the protective biological activities of flavonoids. © 2016 BioFactors, 43(3):451-468, 2017.
Assuntos
Cardiolipinas/química , Citocromos c/química , Flavonoides/química , Peroxidases/química , Substâncias Redutoras/química , Lipossomas Unilamelares/química , Animais , Antocianinas/química , Ácido Ascórbico/química , Catequina/análogos & derivados , Catequina/química , Difenilexatrieno , Corantes Fluorescentes , Cavalos , Quempferóis/química , Luteolina/química , Oxirredução , Peroxidases/antagonistas & inibidores , Fosfatidilcolinas/química , Ligação Proteica , Conformação Proteica , Quercetina/química , Espectrometria de Fluorescência , Eletricidade EstáticaRESUMO
Background Phenolic compounds from Citrus are known to be a topic of many studies due to their biological properties including antioxidant activity. Methods Methanolic and aqueous extracts were isolated from Citrus leaves of different species (C. clementina, C. limon, C. hamlin, C. navel, C. aurantifolia, C. aurantium and C. grandis) harvested in Algeria. Results The results showed that aqueous extracts of all species are rich in total phenolic compounds and flavonoids (from 68.23 to 125.28âmg GAE/g DM) and (from 11.99 to 46.25âmg QE/g DM) respectively. The methanolic and aqueous extracts were examined for in vitro antioxidant properties using various antioxidant assays. For aqueous extracts, C. limon showed an important DPPH radical scavenging activity (IC50 35.35âµg/mL), and C. clementina exerted the highest ABTS radical scavenging activity (1,174.43âµM ET/g DM) and a significant ferric reducing potential (30.60âmg BHAE/g DM). For methanolic extracts, C. clementina showed the highest antioxidant activity for all the realized assays (IC50 41.85âµg/mL, 378.63âµM ET/g DM and 13.85âmg BHAE/g DM) for DPPH, ABTS radicals scavenging activities and ferric reducing potential respectively. Antiperoxidase and antipolyphenol oxidase activities of these samples were also evaluated. Conclusions In this investigation, the assessment of antiperoxidase activity proved that the leaves extracts of different species were able to inhibit peroxidase activity. However, this inhibition varied with the species and the source of these enzymes. On the other hand, the aqueous extracts of different species showed moderate inhibition of polyphenol oxidase, while no effect on these enzymes was obtained with methanolic extracts.
Assuntos
Antioxidantes/farmacologia , Citrus/química , Flavonoides/farmacologia , Reação de Maillard , Oxirredutases/antagonistas & inibidores , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Argélia , Benzotiazóis/metabolismo , Compostos de Bifenilo/metabolismo , Catecol Oxidase/antagonistas & inibidores , Peroxidases/antagonistas & inibidores , Picratos/metabolismo , Folhas de Planta/química , Especificidade da Espécie , Ácidos Sulfônicos/metabolismoRESUMO
Heme peroxidases are the most abundant type of peroxidase catalyzing a H2O2-dependent oxidation of a wide variety of substrates. They are involved in numerous processes like the innate immune response, hormone and prostaglandin synthesis and crosslinking of proteins within extracellular matrixes (ECM) as well as molecules within the cuticle and chorion of arthropods and nematodes. In the present study, a Lepeophtheirus salmonis heme peroxidase (LsHPX) 1 was characterized. Amino acids in the active site of heme peroxidases were conserved, and the predicted protein sequence showed the highest similarity to genes annotated as chorion peroxidases and genes suggested to be involved in cuticle hardening or adhesion. LsHPX1 exhibited a dynamic expression during ontogenesis and during the nauplius molting cycle. Transcripts were localized to muscle cells near the muscle-tendon junction, in nerve tissue especially at neuromuscular junctions, subcuticular epithelium, subepithelial cells facing the hemolymph, exocrine glands within the subepithelial tissue and in isolated cells within the testis. Knock-down of LsHPX1 in nauplius larvae decreased the swimming activity of emerging copepodids. Histological analysis of knock-down animals revealed increased spacing between myofibers and changes in subepithelial and exocrine gland tissue. Considering these results, the potential role of LsHPX1 in crosslinking molecules of salmon louse ECMs is discussed.
Assuntos
Proteínas de Artrópodes/metabolismo , Copépodes/enzimologia , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Heme/metabolismo , Junção Neuromuscular/metabolismo , Peroxidases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Domínio Catalítico , Sequência Conservada , Copépodes/citologia , Copépodes/crescimento & desenvolvimento , Copépodes/fisiologia , Matriz Extracelular/enzimologia , Feminino , Técnicas de Silenciamento de Genes , Estágios do Ciclo de Vida , Masculino , Muda , Junção Neuromuscular/citologia , Junção Neuromuscular/enzimologia , Especificidade de Órgãos , Oxirredução , Estresse Oxidativo , Peroxidases/antagonistas & inibidores , Peroxidases/química , Peroxidases/genética , Interferência de RNA , Especificidade por Substrato , NataçãoRESUMO
Peroxiredoxins (Prxs) are ubiquitary proteins able to play multiple physiological roles, that include thiol-dependent peroxidase, chaperone holdase, sensor of H2O2, regulator of H2O2-dependent signal cascades, and modulator of the immune response. Prxs have been found in a great number of human pathogens, both eukaryotes and prokaryotes. Gene knock-out studies demonstrated that Prxs are essential for the survival and virulence of at least some of the pathogens tested, making these proteins potential drug targets. However, the multiplicity of roles played by Prxs constitutes an unexpected obstacle to drug development. Indeed, selective inhibitors of some of the functions of Prxs are known (namely of the peroxidase and holdase functions) and are here reported. However, it is often unclear which function is the most relevant in each pathogen, hence which one is most desirable to inhibit. Indeed there are evidences that the main physiological role of Prxs may not be the same in different parasites. We here review which functions of Prxs have been demonstrated to be relevant in different human parasites, finding that the peroxidase and chaperone activities figure prominently, whereas other known functions of Prxs have rarely, if ever, been observed in parasites, or have largely escaped detection thus far.
Assuntos
Antiprotozoários/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Peroxirredoxinas/antagonistas & inibidores , Infecções por Protozoários/tratamento farmacológico , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/química , Inibidores Enzimáticos/química , Expressão Gênica , Humanos , Leishmania/efeitos dos fármacos , Leishmania/genética , Leishmania/metabolismo , Modelos Moleculares , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peroxidases/antagonistas & inibidores , Peroxidases/química , Peroxidases/genética , Peroxidases/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Plasmodium/efeitos dos fármacos , Plasmodium/genética , Plasmodium/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Infecções por Protozoários/parasitologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Schistosoma/efeitos dos fármacos , Schistosoma/genética , Schistosoma/metabolismo , Toxoplasma/efeitos dos fármacos , Toxoplasma/genética , Toxoplasma/metabolismo , Trypanosoma/efeitos dos fármacos , Trypanosoma/genética , Trypanosoma/metabolismoRESUMO
Low pH (<5.0) can significantly decrease root growth but whether this is a direct effect of H(+) or an active plant response is examined here. Tomato (Solanum lycopersicum cv Micro-Tom) roots were exposed directly or gradually to low pH through step-wise changes in pH over periods ranging from 4 to 24 h. Roots exposed gradually to pH 4.5 grew even less than those exposed directly, indicating a plant-coordinated response. Direct exposure to pH 4.0 suppressed root growth and caused high cell mortality, in contrast to roots exposed gradually, in which growth remained inhibited but cell viability was maintained. Total class III peroxidase activity increased significantly in all low pH treatments, but was not correlated with the observed differential responses. Use of the enzyme inhibitors salicylhydroxamic acid (SHAM) or diphenyleneiodonium chloride (DPI) suggest that peroxidase and, to a lesser extent, NADPH oxidase were required to prevent or reduce injury in all low pH treatments. However, a role for other enzymes, such as the alternative oxidase is also possible. The results with SHAM, but not DPI, were confirmed in tobacco BY-2 cells. Our results indicate that root growth inhibition from low pH can be part of an active plant response, and suggest that peroxidases may have a critical early role in reducing loss of cell viability and in the observed root growth constraint.
Assuntos
NADPH Oxidases/metabolismo , Peroxidases/metabolismo , Solanum lycopersicum/enzimologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/fisiologia , Proteínas Mitocondriais/metabolismo , Oniocompostos/farmacologia , Oxirredutases/metabolismo , Peroxidases/antagonistas & inibidores , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Salicilamidas/farmacologia , Nicotiana/efeitos dos fármacos , Nicotiana/enzimologia , Nicotiana/crescimento & desenvolvimento , Nicotiana/fisiologiaRESUMO
Immobilization of enzymes has been regarded as an efficient approach to develop biocatalyst with improved activity and stability characteristics under reaction conditions. In the present study, purified manganese peroxidase (MnP) from Ganoderma lucidum IBL-05 was immobilized in agar-agar support using entrapment technique. Maximum immobilization yield was accomplished at 4.0% agar-agar gel. The immobilized MnP exhibited better resistance to changes in pH and temperature than the free enzyme, with optimal conditions being pH 6.0 and 50 °C. The kinetic parameters Km and Kcat/Km for free and entrapped MnP were calculated to be 65.6 mM and 6.99 M(-1) s(-1), and 82 mM and 8.15 M(-1) s(-1), respectively. Thermo-stability was significantly improved after immobilization. After 120 h, the insolubilized MnP retained its activity up to 71.9% and 60.3% at 30 °C and 40 °C, respectively. It showed activity until 10th cycle and retained 74.3% residual activity after 3th cycle. The effects of H2O2, ionic strength and potential inhibitors on activity of free and immobilized enzyme were investigated. Moreover, the decolorization of three structurally different dyes was monitored in order to assess the degrading capability of the entrapped MnP. The decolorization efficiencies for all the tested dyes were 78.6-84.7% after 12h. The studies concluded that the toxicity of dyes aqueous solutions was significantly reduced after treatment. The remarkable catalytic, thermo-stability and re-cycling features of the agar-agar immobilized MnP display a high potential for biotechnological applications.
Assuntos
Ágar/química , Corantes/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Peroxidases/química , Peroxidases/metabolismo , Animais , Artemia/efeitos dos fármacos , Cor , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/toxicidade , Géis , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Concentração Osmolar , Peroxidases/antagonistas & inibidores , Peroxidases/toxicidade , Reishi/enzimologia , Temperatura , Têxteis , Águas Residuárias/químicaRESUMO
BACKGROUND: The Amazon is the largest rainforest in the world and is home to a rich biodiversity of medicinal plants. Several of these plants are used by the local population for the treatment of diseases, many of those with probable anti-inflammatory effect. The aim of the present investigation was to evaluate the in vitro antioxidant and anti-peroxidases potential of the ethanol extracts of five plants from the Brazilian Amazon (Byrsonima japurensis, Calycophyllum spruceanum, Maytenus guyanensis, Passiflora nitida and Ptychopetalum olacoides). METHODS: DPPH, ABTS, superoxide anion radical, singlet oxygen and the ß-carotene bleaching methods were employed for characterization of free radical scavenging activity. Also, total polyphenols were determined. Antioxidant activities were evaluated using murine fibroblast NIH3T3 cell. Inhibition of HRP and MPO were evaluated using amplex red® as susbtract. RESULTS: The stem bark extracts of C. spruceanum and M. guyanensis provided the highest free radical scavenging activities. C. spruceanum exhibited IC50 = 7.5 ± 0.9, 5.0 ± 0.1, 18.2 ± 3.0 and 92.4 ± 24.8 µg/mL for DPPH(â¢), ABTS(+â¢), O2 (-â¢) and (1)O2 assays, respectively. P. olacoides and C. spruceanum extracts also inhibited free radicals formation in the cell-based assay. At a concentration of 100 µg/mL, the extracts of C. spruceanum, B. japurensis inhibited horseradish peroxidase by 62 and 50 %, respectively. C. spruceanum, M. guyanensis, B. japurensis also inhibited myeloperoxidase in 72, 67 and 56 %, respectively. CONCLUSIONS: This work supports the folk use these species that inhibited peroxidases and exhibited significant free radical scavenging and antioxidant activities what can be related to treatment of inflammation.
Assuntos
Antioxidantes/farmacologia , Malpighiaceae/química , Maytenus/química , Olacaceae/química , Passiflora/química , Peroxidases/antagonistas & inibidores , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Brasil , Humanos , Medicina Tradicional , Camundongos , Células NIH 3T3 , Peroxidase , Fitoterapia , Plantas Medicinais/química , Polifenóis/análise , Polifenóis/farmacologiaRESUMO
The binding free energy of 4-bromophenol (4-BP), an inhibitor that binds in the internal binding site in dehaloperoxidase-hemoglobin (DHP) was calculated using Molecular Dynamics (MD) methods combined with pulling or umbrella sampling. The effects of systematic changes in the pulling speed, pulling force constant and restraint force constant on the calculated potential of mean force (PMF) are presented in this study. The PMFs calculated using steered molecular dynamics (SMD) were validated by umbrella sampling (US) in the strongly restrained regime. A series of restraint force constants ranging from 1000 down to 5 kJ/(mol nm(2)) were used in SMD simulations. This range was validated using US, however noting that weaker restraints give rise to a broader sampling of configurations. This comparison was further tested by a pulling simulation conducted without any restraints, which was observed to have a value closest to the experimentally measured free energy for binding of 4-BP to DHP based on ultraviolet-visible (UV-vis) and resonance Raman spectroscopies. The protein-inhibitor system is well suited for fundamental study of free energy calculations because the DHP protein is relatively small and the inhibitor is quite rigid. Simulation configuration structures are compared to the X-ray crystallography structures of the binding site of 4-BP in the distal pocket above the heme.