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1.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443451

RESUMO

Diabetes mellitus is characterized by tissue oxidative damage and impaired microcirculation, as well as worsened erythrocyte properties. Measurements of erythrocyte deformability together with determination of nitric oxide (NO) production and osmotic resistance were used for the characterization of erythrocyte functionality in lean (control) and obese Zucker diabetic fatty (ZDF) rats of two age categories. Obese ZDF rats correspond to prediabetic (younger) and diabetic (older) animals. As antioxidants were suggested to protect erythrocytes, we also investigated the potential effect of quercetin (20 mg/kg/day for 6 weeks). Erythrocyte deformability was determined by the filtration method and NO production using DAF-2DA fluorescence. For erythrocyte osmotic resistance, we used hemolytic assay. Erythrocyte deformability and NO production deteriorated during aging-both were lower in older ZDF rats than in younger ones. Three-way ANOVA indicates improved erythrocyte deformability after quercetin treatment in older obese ZDF rats only, as it was not modified or deteriorated in both (lean and obese) younger and older lean animals. NO production by erythrocytes increased post treatment in all experimental groups. Our study indicates the potential benefit of quercetin treatment on erythrocyte properties in condition of diabetes mellitus. In addition, our results suggest potential age-dependency of quercetin effects in diabetes that deserve additional research.


Assuntos
Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Eritrócitos/metabolismo , Quercetina/uso terapêutico , Animais , Antioxidantes , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Deformação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Óxido Nítrico/metabolismo , Osmose , Estresse Oxidativo , Quercetina/farmacologia , Ratos Zucker
2.
Molecules ; 26(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34443534

RESUMO

Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression.


Assuntos
Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Oligoquetos/química , Peptídeo Hidrolases/farmacologia , Trombose/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Bases de Dados de Proteínas , Eritrócitos/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/química , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ativador de Plasminogênio Tecidual/metabolismo , Peixe-Zebra
3.
Int J Mol Sci ; 22(12)2021 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-34203038

RESUMO

Heavy metals are toxic environmental pollutants associated with severe ecological and human health risks. Among them is mercury (Hg), widespread in air, soil, and water, due to its peculiar geo-biochemical cycle. The clinical consequences of Hg exposure include neurotoxicity and nephrotoxicity. Furthermore, increased risk for cardiovascular diseases is also reported due to a direct effect on cardiovascular tissues, including endothelial cells, recently identified as important targets for the harmful action of heavy metals. In this review, we will discuss the rationale for the potential use of erythrocytes as a surrogate model to study Hg-related toxicity on the cardiovascular system. The toxic effects of Hg on erythrocytes have been amply investigated in the last few years. Among the observed alterations, phosphatidylserine exposure has been proposed as an underlying mechanism responsible for Hg-induced increased proatherogenic and prothrombotic activity of these cells. Furthermore, following Hg-exposure, a decrease in NOS activity has also been reported, with consequent lowering of NO bioavailability, thus impairing endothelial function. An additional mechanism that may induce a decrease in NO availability is the generation of an oxidative microenvironment. Finally, considering that chronic Hg exposure mainly occurs through contaminated foods, the protective effect of dietary components is also discussed.


Assuntos
Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/fisiopatologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Metais Pesados/toxicidade , Animais , Vasos Sanguíneos/metabolismo , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/fisiopatologia , Suscetibilidade a Doenças , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Poluentes Ambientais/efeitos adversos , Humanos , Mercúrio/toxicidade , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos
4.
Molecules ; 26(13)2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34279413

RESUMO

Malaria i a serious health problem caused by Plasmodium spp. that can be treated by an anti-folate pyrimethamine (PYR) drug. Deferiprone (DFP) is an oral iron chelator used for the treatment of iron overload and has been recognized for its potential anti-malarial activity. Deferiprone-resveratrol hybrids (DFP-RVT) have been synthesized to present therapeutic efficacy at a level which is superior to DFP. We have focused on determining the lipophilicity, toxicity and inhibitory effects on P. falciparum growth and the iron-chelating activity of labile iron pools (LIPs) by DFP-RVT. According to our findings, DFP-RVT was more lipophilic than DFP (p < 0.05) and nontoxic to blood mononuclear cells. Potency for the inhibition of P. falciparum was PYR > DFP-RVT > DFP in the 3D7 strain (IC50 = 0.05, 16.82 and 47.67 µM, respectively) and DFP-RVT > DFP > PYR in the K1 strain (IC50 = 13.38, 42.02 and 105.61 µM, respectively). The combined treatment of DFP-RVT with PYR additionally enhanced the PYR activity in both strains. DFP-RVT dose-dependently lowered LIP levels in PRBCs and was observed to be more effective than DFP at equal concentrations. Thus, the DFP-RVT hybrid should be considered a candidate as an adjuvant anti-malarial drug through the deprivation of cellular iron.


Assuntos
Antimaláricos/farmacologia , Deferiprona/farmacologia , Eritrócitos/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Resveratrol/farmacologia , Antioxidantes/farmacologia , Eritrócitos/parasitologia , Humanos , Quelantes de Ferro/farmacologia , Malária Falciparum/parasitologia
5.
Biomolecules ; 11(7)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34208829

RESUMO

BACKGROUND: Blood transfusion remains a key treatment for managing occlusive episodes and painful crises in sickle-cell disease (SCD). In that clinical context, red blood cells (RBCs) from donors and transfused to patients, may be affected by plasma components in the recipients' blood. Senescence lesion markers appear on the red cells after transfusion, shortening the RBC lifespan in circulation. In the specific context of SCD, senescence signals can also trigger the occlusive painful events, typical of the disease. This work follows through our previous data that described a RBC senescence process, rapidly detected after challenge with SCD pathological plasmas. In this clinical context, we wanted here to further explore the characteristics and physiologic consequences of AA RBC lesions associated with senescence, as lesions caused by RBCs after transfusion may have adverse consequences for SCD patients. METHODS: Plasma samples from SCD patients, with acute symptoms (n = 20) or steady-state disease (n = 34) were co-incubated with donor AA RBCs from blood units for 24 to 48 h. Specific markers signing RBC senescence were quantified after the incubation with SCD plasma samples. The physiologic in-flow adhesion was investigated on senescent RBCs, an in vitro technic into biochips that mimic adherence of RBCs during the occlusive events of SCD. RESULTS: Senescence markers on AA RBCs, together with their in-flow adhesion to the plasma-bridging protein thrombospondin, were associated with the clinical status of the SCD patients from whom plasma was obtained. In these experiments, the highest values were obtained for SCD acute plasma samples. Adhesion of senescent RBCs into biochips, which is not reversed by a pre-treatment with recombinant Annexin V, can be reproduced with the use of chemical agents acting on RBC membrane channels that regulate either Ca2+ entry or modulating RBC hydration. CONCLUSION: We found that markers on red cells are correlated, and that the senescence induced by SCD plasma provokes the adhesion of RBCs to the vessel wall protein thrombospondin. In-flow adhesion of senescent red cells after plasma co-incubations can be reproduced with the use of modulators of RBC membrane channels; activating the Piezo1 Ca2+ mechanosensitive channel provokes RBC adhesion of normal (non-senescent) RBCs, while blocking the Ca2+-dependent K+ Gardos channel, can reverse it. Clinically modulating the RBC adhesion to vascular wall proteins might be a promising avenue for the treatment of painful occlusive events in SCD.


Assuntos
Anemia Falciforme/metabolismo , Senescência Celular/fisiologia , Eritrócitos/metabolismo , Doença Aguda , Adulto , Anemia Falciforme/sangue , Adesão Celular , Transfusão de Eritrócitos/métodos , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Masculino , Plasma/química , Plasma/metabolismo
6.
Am J Physiol Heart Circ Physiol ; 321(2): H400-H411, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34213392

RESUMO

Therapeutic agents that increase the Hb affinity for oxygen (O2) could, in theory, lead to decreased O2 release from Hb and impose a hypoxic risk to tissues. In this study, GBT1118, an allosteric modifier of Hb affinity for O2, was used to assess the impact of increasing Hb affinity for O2 on brain tissue oxygenation, blood pressure, heart rate, O2 delivery, and tolerance to hypoxia in Townes transgenic sickle cell disease (SCD) mice. Brain oxygenation and O2 delivery were studied during normoxia and severe hypoxic challenges. Chronic treatment with GBT1118 increased Hb affinity for O2, reducing the Po2 for 50% HbO2 saturation (P50) in SCD mice from 31 mmHg to 18 mmHg. This treatment significantly reduced anemia, increasing hematocrit by 33%, improved cardiac output (CO), and O2 delivery and extraction. Chronically increasing Hb affinity for O2 with GBT1118 preserved cortical O2 tension during normoxia, improved cortical O2 tension during hypoxia, and increased tolerance to severe hypoxia in SCD mice. Independent of hematological changes induced by chronic treatment, a single dose of GBT1118 significantly improved tolerance to hypoxia, highlighting the benefits of increasing Hb affinity for O2 and consequently reducing sickling of RBCs in blood during hypoxia in SCD.NEW & NOTEWORTHY Chronic pharmacologically increased hemoglobin affinity for oxygen in sickle cell disease mice alleviated hematological consequences of sickle cell disease, increasing RBC half-life, hematocrit, and hemoglobin concentration, while also decreasing reticulocyte count. Additionally, chronically increased hemoglobin affinity for oxygen significantly improved survival as well as cortical tissue oxygenation in sickle cell disease mice during hypoxia, suggesting that oxygen delivery and utilization is improved by increased hemoglobin affinity for oxygen.


Assuntos
Anemia Falciforme/metabolismo , Benzaldeídos/farmacologia , Córtex Cerebral/metabolismo , Eritrócitos/efeitos dos fármacos , Fármacos Hematológicos/farmacologia , Hemoglobina Falciforme/efeitos dos fármacos , Hipóxia/metabolismo , Niacinamida/análogos & derivados , Oxigênio/metabolismo , Regulação Alostérica , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Hematócrito , Hemoglobina Falciforme/metabolismo , Camundongos , Camundongos Transgênicos , Niacinamida/farmacologia , Pressão Parcial
7.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202704

RESUMO

The aim of this study was to evaluate the effect of everolimus, a mammalian target of rapamycin (mTOR) inhibitor, on red blood cell parameters in the context of iron homeostasis in patients with tuberous sclerosis complex (TSC) and evaluate its effect on cell size in vitro. Everolimus has a significant impact on red blood cell parameters in patients with TSC. The most common alteration was microcytosis. The mean MCV value decreased by 9.2%, 12%, and 11.8% after 3, 6, and 12 months of everolimus treatment. The iron level declined during the first 3 months, and human soluble transferrin receptor concentration increased during 6 months of therapy. The size of K562 cells decreased when cultured in the presence of 5 µM everolimus by approximately 8%. The addition of hemin to the cell culture with 5 µM everolimus did not prevent any decrease in cell size. The stage of erythroid maturation did not affect the response to everolimus. Our results showed that the mTOR inhibitor everolimus caused red blood cell microcytosis in vivo and in vitro. This effect is not clearly related to a deficit of iron and erythroid maturation. This observation confirms that mTOR signaling plays a complex role in the control of cell size.


Assuntos
Tamanho Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adolescente , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Criança , Pré-Escolar , Índices de Eritrócitos , Eritrócitos/metabolismo , Everolimo/administração & dosagem , Everolimo/efeitos adversos , Everolimo/farmacologia , Citometria de Fluxo , Humanos , Ferro/metabolismo , Células K562 , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos
8.
ACS Appl Mater Interfaces ; 13(23): 27430-27444, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34060800

RESUMO

Life-threatening invasive fungal infections represent an urgent threat to human health worldwide. The limited set of antifungal drugs has critical constraints such as resistance development and/or adverse side effects. One approach to overcome these limitations is to mimic naturally occurring antifungal peptides called defensins. Inspired by their advantageous amphiphilic properties, a library of 35 synthetic, linear, ternary polyacrylamides was prepared by controlled/living radical polymerization. The effect of the degree of polymerization (20, 40, and 100) and varying hydrophobic functionalities (branched, linear, cyclic, or aromatic differing in their number of carbons) on their antifungal activity was investigated. Short copolymers with a calculated log P of ∼1.5 revealed optimal activity against the major human fungal pathogen Candida albicans and other pathogenic fungal species with limited toxicity to mammalian host cells (red blood cells and fibroblasts). Remarkably, selected copolymers outperformed the commercial antifungal drug amphotericin B, with respect to the therapeutic index, highlighting their potential as novel antifungal compounds.


Assuntos
Resinas Acrílicas/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Biblioteca de Peptídeos , Resinas Acrílicas/química , Animais , Humanos , Camundongos , Testes de Sensibilidade Microbiana
9.
Methods Mol Biol ; 2326: 155-165, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097267

RESUMO

This chapter describes, in detail, the operational principles and experimental design to analyze the premature death of human red blood cells (RBCs; erythrocytes). Necrosis (i.e., hemolysis), eryptosis, and necroptosis are the three types of cell death thus far known to exist in RBCs, and distinctive markers of each are well established. Here, methods based on flow cytometry are presented in an easily reproducible form. Moreover, manipulation of incubation medium to promote or inhibit certain physiological phenomena, along with a step-by-step approach to examine membrane scrambling, cell volume, surface complexity, calcium activity, oxidative stress, and signal transduction pathways are also discussed.


Assuntos
Eriptose , Eritrócitos/citologia , Citometria de Fluxo/métodos , Hemólise , Necroptose , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Hemólise/efeitos dos fármacos , Humanos , Necroptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Testes de Toxicidade/métodos
10.
Mol Biochem Parasitol ; 244: 111392, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34171456

RESUMO

Plasmodium falciparum gametocytes modify the mechanical properties of their erythrocyte host to persist for several weeks in the blood circulation and to be available for mosquitoes. These changes are tightly regulated by the plasmodial phosphodiesterase delta that decreases both the stiffness and the permeability of the infected host cell. Here, we address the effect of the phosphodiesterase inhibitor tadalafil on deformability and permeability of gametocyte-infected erythrocytes. We show that this inhibitor drastically increases isosmotic lysis of gametocyte-infected erythrocytes and impairs their ability to circulate in an in vitro model for splenic retention. These findings indicate that tadalafil represents a novel drug lead potentially capable of blocking malaria parasite transmission by impacting gametocyte circulation.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Gametogênese/efeitos dos fármacos , Estágios do Ciclo de Vida/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Tadalafila/farmacologia , Fenômenos Biomecânicos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Deformação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Eritrócitos/ultraestrutura , Feminino , Expressão Gênica , Interações Hospedeiro-Parasita/efeitos dos fármacos , Interações Hospedeiro-Parasita/genética , Humanos , Estágios do Ciclo de Vida/genética , Masculino , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Reprodução Assexuada/efeitos dos fármacos
12.
Transfusion ; 61(7): 2054-2063, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33960433

RESUMO

BACKGROUND: Daratumumab, a human anti-CD38 monoclonal antibody used to treat multiple myeloma, interferes with pretransfusion testing and can mask alloantibodies. Incidence of alloimmunization in patients on daratumumab has not been well characterized, and optimal transfusion guidelines regarding prophylactic antigen matching, accounting for both patient safety and efficiency, have not been well established for these patients. METHODS: Records of patients who received daratumumab between January 1, 2014 and July 2, 2019 were reviewed. Daratumumab interference with pretransfusion testing was managed by testing with reagent red blood cells (RBCs) treated with 0.2 M dithiothreitol. When daratumumab was present during antibody testing, patients were transfused with RBC units prophylactically matched for D, C, c, E, e, and K antigens per hospital policy. RESULTS: Out of 90 patients identified, 52 received a total of 638 RBC transfusions (average of 12.3 units per patient, SD 17.2, range 1-105, median 5 among those transfused). Alloantibodies existing before daratumumab initiation were identified in seven patients. No new alloantibodies were detected in any patients after starting daratumumab treatment. CONCLUSIONS: The incidence of alloimmunization in patients receiving daratumumab is low. Whether this is due to the effect of daratumumab, underlying pathophysiology, or other factors, is unknown. Because these patients require a large number of RBC transfusions overall and have little observed alloimmunization, phenotype matching (beyond RhD) may be unnecessary. Since the use of dithiothreitol cannot rule out the presence of anti-K, we recommend transfusion of ABO-compatible units, prophylactically matched for the D and K antigens only.


Assuntos
Anticorpos Monoclonais/imunologia , Antineoplásicos Imunológicos/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Artefatos , Incompatibilidade de Grupos Sanguíneos/sangue , Tipagem e Reações Cruzadas Sanguíneas , Transfusão de Sangue , Eritrócitos/imunologia , Isoanticorpos/sangue , Mieloma Múltiplo/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aloenxertos , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/uso terapêutico , Antígenos de Grupos Sanguíneos/imunologia , Incompatibilidade de Grupos Sanguíneos/diagnóstico , Incompatibilidade de Grupos Sanguíneos/epidemiologia , Tipagem e Reações Cruzadas Sanguíneas/métodos , Terapia Combinada , Ditiotreitol/farmacologia , Eritrócitos/efeitos dos fármacos , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Incidência , Isoanticorpos/biossíntese , Isoanticorpos/imunologia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Transplante Autólogo
13.
Chem Biodivers ; 18(6): e2100221, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34033215

RESUMO

Several synthetic approaches (aminomethylation, alkylation, condensation, etc.) have been used to synthesize derivatives based on the sesamol (1), natural phenol. The set of methods, including the study of antioxidant activity (AOA) by the ability to inhibit the initiated oxidation of animal lipids, radical scavenging activity, Fe2+ -chelation ability, as well as a comparative assessment of membrane-protective activity under the conditions of H2 O2 -induced hemolysis of mice red blood cells (RBCs), was used to analyze the antioxidant potential of the synthesized compounds. The synthesized derivatives have demonstrated different activity in the listed test systems, and we have identified compounds which appear to be most promising for a detailed study of their pharmacological properties.


Assuntos
Antioxidantes/farmacologia , Benzodioxóis/farmacologia , Fenóis/farmacologia , Animais , Antioxidantes/síntese química , Antioxidantes/química , Benzodioxóis/síntese química , Benzodioxóis/química , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Camundongos , Modelos Moleculares , Estrutura Molecular , Estresse Oxidativo/efeitos dos fármacos , Fenóis/síntese química , Fenóis/química
14.
Fish Physiol Biochem ; 47(4): 1073-1085, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34021418

RESUMO

In this study, substance P, an antioxidant peptide of tachykinin, was identified using bioinformatics tools from the earlier established muscle transcriptome of a freshwater murrel Channa striatus and the peptide was named RM12. The antioxidant properties of RM12 were screened using various colorimetric assays. The toxicity of RM12 was experimented using fish erythrocytes, and it is observed that the maximum concentration (320 µM) of RM12 was found to have 15 or 20% of hemolytic activity; however, it was not significant with other tested concentrations (10, 20, 40, 80, and 160 µM). Further, the in vivo antioxidant properties of RM12 were experimented on zebrafish embryo, the intracellular ROS level was estimated by 5 mM H2O2 stress in the zebrafish embryo, and inhibition of apoptosis was evaluated. The antioxidant enzymes were extracted from the H2O2-stressed zebrafish embryo, and the intracellular ROS was eliminated due to RM12. Collectively, the experiment showed that the substance P from the freshwater murrel C. striatus possessed potent antioxidant properties; thus, it can further be focused to develop it as antioxidant molecule in aquaculture organisms.


Assuntos
Antioxidantes/farmacologia , Eritrócitos/efeitos dos fármacos , Peixes/metabolismo , Substância P/farmacologia , Animais , Compostos de Bifenilo/metabolismo , Catalase/metabolismo , Embrião não Mamífero/metabolismo , Eritrócitos/metabolismo , Feminino , Peixes/embriologia , Água Doce , Hemólise/efeitos dos fármacos , Radical Hidroxila/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Picratos/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo
15.
J Med Chem ; 64(11): 7359-7370, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34032114

RESUMO

Novel antibacterial agents capable of efficiently sterilizing intracellular Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) but with low cytotoxicity and low resistance development are quite appealing. In this work, three Ru(II) complexes with photolabile ligands were explored to realize such a goal. Complex 3 (5 µM) can inhibit more than 90% growth of S. aureus/MRSA that has invaded in J774A.1 cells upon visible light irradiation, being much more efficient than vancomycin. In similar conditions, negligible dark- and phototoxicity were found toward the host cells. The bactericidal activity is highly correlated with DNA covalent binding by the Ru(II) fractions generated after ligand photodissociation. Moreover, S. aureus quickly developed resistance toward vancomycin, while negligible resistance toward complex 3 even after 700 generations was obtained. These appealing results may pave a new way for fighting against intracellular antibiotic-resistant pathogens.


Assuntos
Antibacterianos/farmacologia , Complexos de Coordenação/química , Luz , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Rutênio/química , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , DNA/química , DNA/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Ligantes , Fotólise , Coelhos
16.
Biomolecules ; 11(4)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917850

RESUMO

In a large variety of organisms, antimicrobial peptides (AMPs) are primary defenses against pathogens. BP100 (KKLFKKILKYL-NH2), a short, synthetic, cationic AMP, is active against bacteria and displays low toxicity towards eukaryotic cells. BP100 acquires a α-helical conformation upon interaction with membranes and increases membrane permeability. Despite the volume of information available, the action mechanism of BP100, the selectivity of its biological effects, and possible applications are far from consensual. Our group synthesized a fluorescent BP100 analogue containing naphthalimide linked to its N-terminal end, NAPHT-BP100 (Naphthalimide-AAKKLFKKILKYL-NH2). The fluorescence properties of naphthalimides, especially their spectral sensitivity to microenvironment changes, are well established, and their biological activities against transformed cells and bacteria are known. Naphthalimide derived compounds are known to interact with DNA disturbing related processes as replication and transcription, and used as anticancer agents due to this property. A wide variety of techniques were used to demonstrate that NAPHT-BP100 bound to and permeabilized zwitterionic POPC and negatively charged POPC:POPG liposomes and, upon interaction, acquired a α-helical structure. Membrane surface high peptide/lipid ratios triggered complete permeabilization of the liposomes in a detergent-like manner. Membrane disruption was driven by charge neutralization, lipid aggregation, and bilayer destabilization. NAPHT-BP100 also interacted with double-stranded DNA, indicating that this peptide could also affect other cellular processes besides causing membrane destabilization. NAPHT-BP100 showed increased antibacterial and hemolytic activities, compared to BP100, and may constitute an efficient antimicrobial agent for dermatological use. By conjugating BP100 and naphthalimide DNA binding properties, NAPHT-BP100 bound to a large extent to the bacterial membrane and could more efficiently destabilize it. We also speculate that peptide could enter the bacteria cell and interact with its DNA in the cytoplasm.


Assuntos
Anti-Infecciosos/química , Lipossomos/química , Naftalimidas/química , Oligopeptídeos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Dicroísmo Circular , DNA/química , DNA/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Escherichia coli/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipossomos/metabolismo , Testes de Sensibilidade Microbiana , Oligopeptídeos/síntese química , Permeabilidade/efeitos dos fármacos , Conformação Proteica em alfa-Hélice , Espectrometria de Fluorescência , Staphylococcus aureus/efeitos dos fármacos , Termodinâmica
17.
J Med Chem ; 64(9): 6397-6409, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33901399

RESUMO

Herein, relationships between the structures of 1-aminoethyl-substituted chromenes and their antimalarial activities were thoroughly investigated. At first, the methyl moiety in the side chain was removed to eliminate chirality. The hydrogenation state of the benzopyran system, the position of the phenolic OH moiety, and the distance of the basic amino moiety toward both aromatic rings were varied systematically. 1-Benzopyran-5-ol 8b (IC50 = 10 nM), 1-benzopyran-7-ol 9c (IC50 = 38 nM), and the aminoalcohol 19c (IC50 = 17 nM) displayed antiplasmodial activity with IC50 values below 50 nM. To identify the mechanism of action, inhibition of three key enzymes by 9c was investigated. 9c was not able to reduce the number of Plasmodia in erythrocytes of mice. This low in vivo activity was explained by fast clearance from blood plasma combined with rapid biotransformation of 9c. Three main metabolites of 9c were identified by liquid chromatography-mass spectrometry (LC-MS) methods.


Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Benzopiranos/química , Benzopiranos/farmacologia , Produtos Biológicos/química , Plasmodium/efeitos dos fármacos , Alquilação , Animais , Antimaláricos/síntese química , Benzopiranos/síntese química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Técnicas de Química Sintética , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Cinética , Camundongos , Relação Estrutura-Atividade
18.
Artigo em Inglês | MEDLINE | ID: mdl-33839052

RESUMO

Snakebite is classified as a priority Neglected Tropical Disease by the World Health Organization. Understanding the pathology of individual snake venom toxins is of great importance when developing more effective snakebite therapies. Snake venoms may induce a range of pathologies, including haemolytic activity. Although snake venom-induced erythrocyte lysis is not the primary cause of mortality, haemolytic activity can greatly debilitate victims and contributes to systemic haemotoxicity. Current assays designed for studying haemolytic activity are not suitable for rapid screening of large numbers of toxic compounds. Consequently, in this study, a high-throughput haemolytic assay was developed that allows profiling of erythrocyte lysis, and was validated using venom from a number of medically important snake species (Calloselasma rhodostoma, Daboia russelii, Naja mossambica, Naja nigricollis and Naja pallida). The assay was developed in a format enabling direct integration into nanofractionation analytics, which involves liquid chromatographic separation of venom followed by high-resolution fractionation and subsequent bioassaying (and optional proteomics analysis), and parallel mass spectrometric detection. Analysis of the five snake venoms via this nanofractionation approach involving haemolytic assaying provided venom-cytotoxicity profiles and enabled identification of the toxins responsible for haemolytic activity. Our results show that the elapid snake venoms (Naja spp.) contained both direct and indirect lytic toxins, while the viperid venoms (C. rhodostoma and D. russelii) only showed indirect lytic activities, which required the addition of phospholipids to exert cytotoxicity on erythrocytes. The haemolytic venom toxins identified were mainly phospholipase A2s and cytotoxic three finger toxins. Finally, the applicability of this new analytical method was demonstrated using a conventional snakebite antivenom treatment and a small-molecule drug candidate to assess neutralisation of venom cytotoxins.


Assuntos
Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Nanotecnologia/métodos , Venenos de Serpentes , Animais , Fracionamento Químico , Cromatografia Líquida , Humanos , Espectrometria de Massas , Fosfolipases A2 , Venenos de Serpentes/química , Venenos de Serpentes/toxicidade , Serpentes
19.
ACS Appl Mater Interfaces ; 13(14): 16062-16074, 2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33797891

RESUMO

Gram-negative bacteria are covered by both an inner cytoplasmic membrane (IM) and an outer membrane (OM). Antimicrobial peptides (AMPs) must first permeate through the OM and cell wall before attacking the IM to cause cytoplasmic leakage and kill the bacteria. The bacterial OM is an asymmetric bilayer with the outer leaflet primarily composed of lipopolysaccharides (LPSs) and the inner leaflet composed of phospholipids (PLs). Two cationic α-helical AMPs were designed to target Gram-negative bacteria, a full peptide G(IIKK)3I-NH2 (G3), and a hydrophobic lipopeptide C8-G(IIKK)2I-NH2 (C8G2, with C8 denoting the octanoyl chain). LPS dominates OM functions as the first line of defense against antibiotics, thereby reducing drug susceptibility. This work explores how the two AMPs interact with LPS through several carefully chosen OM models that facilitated measurements from solid-state nuclear magnetic resonance (ss-NMR), small-angle neutron scattering (SANS), and neutron reflectivity (NR). The results revealed that G3 molecules bound preferably to the LPS head region and functioned as bridge molecules to reassemble the dislocated lipids into bilayer stacks. In contrast, C8G2 lipopeptides could quickly penetrate into the central region of the OM to cause direct removal of some membrane lipids. Different structural disruptions implicated different antimicrobial efficacies from these AMPs. The demonstration of the structural features underlying different susceptibilities of the OM to AMPs offers a useful route for the future development of strain-specific AMPs against antimicrobial-resistant pathogens.


Assuntos
Parede Celular/química , Bactérias Gram-Negativas/química , Proteínas Citotóxicas Formadoras de Poros/química , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Bicamadas Lipídicas , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Conformação Proteica
20.
PLoS Pathog ; 17(4): e1009442, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33886685

RESUMO

Malaria, caused by infection with Plasmodium parasites, remains a significant global health concern. For decades, genetic intractability and limited tools hindered our ability to study essential proteins and pathways in Plasmodium falciparum, the parasite associated with the most severe malaria cases. However, recent years have seen major leaps forward in the ability to genetically manipulate P. falciparum parasites and conditionally control protein expression/function. The conditional knockdown systems used in P. falciparum target all 3 components of the central dogma, allowing researchers to conditionally control gene expression, translation, and protein function. Here, we review some of the common knockdown systems that have been adapted or developed for use in P. falciparum. Much of the work done using conditional knockdown approaches has been performed in asexual, blood-stage parasites, but we also highlight their uses in other parts of the life cycle and discuss new ways of applying these systems outside of the intraerythrocytic stages. With the use of these tools, the field's understanding of parasite biology is ever increasing, and promising new pathways for antimalarial drug development are being discovered.


Assuntos
Antimaláricos/farmacologia , Eritrócitos/efeitos dos fármacos , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Eritrócitos/parasitologia , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/genética , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/genética , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/metabolismo
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