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1.
Int J Biol Macromol ; 136: 512-520, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31199971

RESUMO

Snake venom cardiotoxins (CTXs) present diverse pharmacological functions. Previous studies have reported that CTXs affect the activity of some serine proteases, namely, chymotrypsin, subtilisin, trypsin, and acetylcholinesterase. To elucidate the mode of action of CTXs, the interaction of CTXs with chymotrypsin was thus investigated. It was found that Naja atra CTX isotoxins concentration-dependently enhanced chymotrypsin activity. The capability of CTX1 and CTX5 in increasing chymotrypsin activity was higher than that of CTX2, CTX3, and CTX4. Removal of the molecular beacon-bound CTXs by chymotrypsin, circular dichroism measurement, and acrylamide quenching of Trp fluorescence indicated that CTXs bound to chymotrypsin. Chemical modification of Lys, Arg, or Met residues of CTX1 attenuated its capability to enhance chymotrypsin activity without impairing their bond with chymotrypsin. Catalytically inactive chymotrypsin retained the binding affinity for native and modified CTX1. CTX1 and chemically modified CTX1 differently altered the global conformation of chymotrypsin and inactivated chymotrypsin. Moreover, CTX1 did not reduce the interaction of 2-(p-toluidino)-naphthalene-6-sulfonate (TNS) with chymotrypsin and inactivated chymotrypsin. Together with previous results revealing that TNS can bind at the hydrophobic region of active site in chymotrypsin, our data suggest that CTXs can enhance chymotrypsin activity by binding to the region outside the enzyme's active site.


Assuntos
Cardiotoxinas/farmacologia , Quimotripsina/metabolismo , Naja naja , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cardiotoxinas/química , Cardiotoxinas/metabolismo , Quimotripsina/química , Simulação de Acoplamento Molecular , Conformação Proteica/efeitos dos fármacos
2.
Zebrafish ; 16(4): 379-387, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31145051

RESUMO

Envenomation by the Venezuelan bushmaster snake (Lachesis muta muta) (Serpentes: Viperidae) is characterized by local and cardiac alterations. This study investigates the in vivo cardiac dysfunction, tissue destruction, and cellular processes triggered by Lachesis muta muta snake crude venom and a C-type lectin (CTL)-like toxin named Mutacytin-1 (MC-1). The 28 kDa MC-1 was obtained by molecular exclusion, ion exchange, and C-18 (checking pureness) reverse-phase chromatographies. N-terminal sequencing of the first eight amino acids (NNCPQ LLM) revealed 100% identity with Mutina (CTL-like) isolated from Lachesis stenophrys, which is a Ca2+-dependent-type galactoside-binding lectin from Bothrops jararaca and CTL BpLec from Bothrops pauloensis. The cardiotoxicity in zebrafish of MC-1 was evaluated by means of specific phenotypic expressions and larvae behavior at 5, 15, 30, 40 and 60 min post-treatment. The L. muta muta venom and MC-1 also produced heart rate/rhythm alterations, circulation modifications, and the presence of thrombus and apoptotic phenomenon with pericardial damages. Acridine orange (100 µg/mL) was used to visualize apoptosis cellular process in control and treated whole embryos. The cardiotoxic alterations happened in more than 90% of all larvae under the action of L. muta muta venom and MC-1. The findings have demonstrated the potential cardiotoxicity by L. muta muta venom, suggesting the possibility of cardiovascular damages to patients after bushmaster envenoming.


Assuntos
Cardiotoxicidade/embriologia , Cardiotoxinas/farmacologia , Crotalinae , Lectinas Tipo C , Proteínas de Répteis/química , Venenos de Serpentes/química , Peixe-Zebra/embriologia , Animais , Cardiotoxinas/química , Crotalinae/embriologia , Embrião não Mamífero/efeitos dos fármacos , Lectinas Tipo C/química , Proteínas de Répteis/farmacologia
3.
Clin Pharmacol Ther ; 105(3): 614-624, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30460992

RESUMO

Anthracycline-induced cardiotoxicity (ACT) is a severe adverse drug reaction for a subset of children treated with anthracyclines as part of chemotherapy protocols. The identification of genetic markers associated with increased ACT susceptibility has clinical significance toward improving patient care and our understanding of the molecular mechanisms involved in ACT. Human-induced pluripotent stem cell-derived cardiomyocytes represent a novel approach to determine the pharmacogenomics of ACT and guide the development of genetic screening tests.


Assuntos
Antraciclinas/efeitos adversos , Antibióticos Antineoplásicos/efeitos adversos , Cardiotoxinas/efeitos adversos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Farmacogenética/tendências , Antraciclinas/química , Antibióticos Antineoplásicos/química , Cardiotoxinas/química , Criança , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo
4.
Expert Rev Proteomics ; 15(11): 873-886, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30328726

RESUMO

INTRODUCTION: Being important representatives of various proteomes, membrane-active cationic peptides (CPs) are attractive objects as lead compounds in the design of new antibacterial, anticancer, antifungal, and antiviral molecules. Numerous CPs are found in insect and snake venoms, where many of them reveal cytolytic properties. Due to advances in omics technologies, the number of such peptides is growing dramatically. Areas covered: To understand structure-function relationships for CPs in a living cell, detailed analysis of their hydrophobic/hydrophilic properties is indispensable. We consider two structural classes of membrane-active CPs: latarcins (Ltc) from spider and cardiotoxins (CTXs) from snake venoms. While the former are void off disulfide bonds and conformationally flexible, the latter are structurally rigid and cross-linked with disulfide bonds. In order to elucidate structure-activity relationships behind their antibacterial, anticancer, and hemolytic effects, the properties of these polypeptides are considered on a side-by-side basis. Expert commentary: An ever-increasing number of venom-derived membrane-active polypeptides require new methods for identification of their functional propensities and sequence-based design of novel pharmacological substances. We address these issues considering a number of the designed peptides, based either on Ltc or CTX sequences. Experimental and computer modeling techniques required for these purposes are delineated.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Cardiotoxinas/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Cardiotoxinas/química , Dissulfetos/química , Desenho de Fármacos , Hemolíticos/química , Hemolíticos/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/metabolismo , Venenos de Aranha/química , Relação Estrutura-Atividade
5.
Sci Rep ; 8(1): 10160, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29976997

RESUMO

Pharmaceutical agents despite their efficacy to treat disease can cause additional unwanted cardiovascular side effects. Cardiotoxicity is characterized by changes in either the function and/or structure of the myocardium. Over recent years, functional cardiotoxicity has received much attention, however morphological damage to the myocardium and/or loss of viability still requires improved detection and mechanistic insights. A human 3D cardiac microtissue containing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), cardiac endothelial cells and cardiac fibroblasts was used to assess their suitability to detect drug induced changes in cardiac structure. Histology and clinical pathology confirmed these cardiac microtissues were morphologically intact, lacked a necrotic/apoptotic core and contained all relevant cell constituents. High-throughput methods to assess mitochondrial membrane potential, endoplasmic reticulum integrity and cellular viability were developed and 15 FDA approved structural cardiotoxins and 14 FDA approved non-structural cardiotoxins were evaluated. We report that cardiac microtissues provide a high-throughput experimental model that is both able to detect changes in cardiac structure at clinically relevant concentrations and provide insights into the phenotypic mechanisms of this liability.


Assuntos
Imageamento Tridimensional , Miocárdio/patologia , Antineoplásicos/farmacologia , Biomarcadores/metabolismo , Cardiotoxinas/química , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Sobrevivência de Tecidos/efeitos dos fármacos
6.
J Atheroscler Thromb ; 25(7): 557-565, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29731506

RESUMO

Patients with Stage A heart failure (HF) show no HF symptoms but have related comorbid diseases with a high risk of progressing to HF. Screening for comorbid diseases warrants closer attention because of the growing interest in addressing Stage A HF as the best means of preventing eventual progression to overt HF such as Stages C and D. The identification of individuals of Stage A HF is potentially useful for the implementation of HF-prevention strategies; however, not all Stage A HF patients develop left ventricular (LV) structural heart disease or symptomatic HF, which lead to advanced HF stages. Therefore, Stage A HF requires management with the long-term goal of avoiding HF development; likewise, Stage B HF patients are ideal targets for HF prevention. Although the early detection of subclinical LV dysfunction is, thus, essential for delaying the progression to HF, the assessment of subclinical LV dysfunction can be challenging. Global longitudinal strain (GLS) as assessed by speckle-tracking echocardiography has recently been reported to be a sensitive marker of early subtle LV myocardial abnormalities, helpful for the prediction of the outcomes for various cardiac diseases, and superior to conventional echocardiographic indices. GLS reflects LV longitudinal myocardial systolic function, and can be assessed usually by means of two-dimensional speckle-tracking. This article reviews the importance of the assessment of subclinical LV dysfunction in Stage A HF patients by means of GLS, and its current potential to prevent progression to later stage HF.


Assuntos
Cardiologia/tendências , Insuficiência Cardíaca/terapia , Envelhecimento , Cardiotoxinas/química , Comorbidade , Diagnóstico por Computador , Progressão da Doença , Ecocardiografia , Ventrículos do Coração/patologia , Humanos , Hipercolesterolemia/patologia , Miocárdio/patologia , Fatores de Risco , Sístole
7.
PLoS One ; 13(4): e0195577, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29630634

RESUMO

Vandetanib, a multi-kinase inhibitor used for the treatment of various cancers, has been reported to induce several adverse cardiac effects. However, the underlying mechanisms of vandetanib-induced cardiotoxicity are unclear. This study aimed to investigate the mechanism of vandetanib-induced cardiotoxicity using intracellular electrophysiological recordings on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), rabbit Purkinje fibers, and HEK293 cells transiently expressing human ether-a-go-go-related gene (hERG; the rapidly activating delayed rectifier K+ channel, IKr), KCNQ1/KCNE1 (the slowly activating delayed rectifier K+ current, IKs), KCNJ2 (the inwardly rectifying K+ current, IK1) or SCN5A (the inward Na+ current, INa). Purkinje fiber assays and ion channel studies showed that vandetanib at concentrations of 1 and 3 µM inhibited the hERG currents and prolonged the action potential duration. Alanine scanning and in silico hERG docking studies demonstrated that Y652 and F656 in the hERG S6 domain play critical roles in vandetanib binding. In hiPSC-CMs, vandetanib markedly reduced the maximum rate of depolarization during the AP upstroke. Ion channel studies revealed that hiPSC-CMs were more sensitive to inhibition of the INa by vandetanib than in a heterogeneously expressed HEK293 cell model, consistent with the changes in the AP parameters of hiPSC-CMs. The subclasses of Class I antiarrhythmic drugs inhibited INa currents in a dose-dependent manner in hiPSC-CMs and SCN5A-encoded HEK293 cells. The inhibitory potency of vandetanib for INa was much higher in hiPSC-CMs (IC50: 2.72 µM) than in HEK293 cells (IC50: 36.63 µM). These data suggest that AP and INa assays using hiPSC-CMs are useful electrophysiological models for prediction of drug-induced cardiotoxicity.


Assuntos
Cardiotoxicidade/fisiopatologia , Cardiotoxinas/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Piperidinas/toxicidade , Ramos Subendocárdicos/efeitos dos fármacos , Ramos Subendocárdicos/fisiopatologia , Quinazolinas/toxicidade , Potenciais de Ação/efeitos dos fármacos , Animais , Cardiotoxinas/química , Canal de Potássio ERG1/química , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Fenômenos Eletrofisiológicos , Feminino , Células HEK293 , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/citologia , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Modelos Moleculares , Miócitos Cardíacos/citologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Piperidinas/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/toxicidade , Quinazolinas/química , Coelhos
8.
Artigo em Inglês | MEDLINE | ID: mdl-29382576

RESUMO

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.


Assuntos
Proteínas de Anfíbios/toxicidade , Venenos de Anfíbios/toxicidade , Bufo bufo/fisiologia , Cardiotoxinas/toxicidade , Bloqueadores Neuromusculares/toxicidade , Neurotoxinas/toxicidade , Pele/metabolismo , Proteínas de Anfíbios/química , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/metabolismo , Venenos de Anfíbios/química , Venenos de Anfíbios/isolamento & purificação , Venenos de Anfíbios/metabolismo , Animais , Bufo bufo/crescimento & desenvolvimento , Cardiotoxinas/química , Cardiotoxinas/isolamento & purificação , Cardiotoxinas/metabolismo , Feminino , Jardins , Coração/efeitos dos fármacos , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Membro Posterior , Técnicas In Vitro , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Condução Nervosa/efeitos dos fármacos , Bloqueadores Neuromusculares/química , Bloqueadores Neuromusculares/isolamento & purificação , Bloqueadores Neuromusculares/metabolismo , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Neurotoxinas/metabolismo , Parques Recreativos , Polônia , Proteômica/métodos , Ranidae , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/fisiologia , Tenebrio
9.
Molecules ; 23(2)2018 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-29463054

RESUMO

This study presents two sensitive fluorescent assays for sensing heparin on the basis of the electrostatic interaction between heparin and Naja naja atra cardiotoxin 3 (CTX3). Owing to CTX3-induced folded structure of an adenosine-based molecular beacon (MB) or a DNA aptamer against CTX3, a reduction in the fluorescent signal of the aptamer or MB 5'-end labeled with carboxyfluorescein (FAM) and 3'-end labeled with 4-([4-(dimethylamino)phenyl]azo)-benzoic acid (DABCYL) was observed upon the addition of CTX3. The presence of heparin and formation of the CTX3-heparin complex caused CTX3 detachment from the MB or aptamer, and restoration of FAM fluorescence of the 5'-FAM-and-3'-DABCYL-labeled MB and aptamer was subsequently noted. Moreover, the detection of heparin with these CTX3-aptamer and CTX3-MB sensors showed high sensitivity and selectivity toward heparin over chondroitin sulfate and hyaluronic acid regardless of the presence of plasma. The limit of detection for heparin in plasma was determined to be 16 ng/mL and 15 ng/mL, respectively, at a signal-to-noise ratio of 3. This study validates the practical utility of the CTX3-aptamer and CTX3-MB systems for determining the concentration of heparin in a biological matrix.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Cardiotoxinas/química , Heparina/isolamento & purificação , Adenosina/química , Animais , Elapidae , Fluorescência
10.
J Chem Inf Model ; 57(11): 2799-2810, 2017 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-29053272

RESUMO

Cardiotoxins (CTs) from snake venoms are a family of homologous highly basic proteins that have extended hydrophobic patterns on their molecular surfaces. CTs are folded into three ß-structured loops stabilized by four disulfide bridges. Being well-structured in aqueous solution, most of these proteins are membrane-active, although the exact molecular mechanisms of CT-induced cell damage are still poorly understood. To elucidate the structure-function relationships in CTs, a detailed knowledge of their spatial organization and local conformational dynamics is required. Protein domain motions can be either derived from a set of experimental structures or generated via molecular dynamics (MD). At the same time, traditional clustering algorithms in the Cartesian coordinate space often fail to properly take into account the local large-scale dihedral angle transitions that occur in MD simulations. This is because such perturbations are usually offset by changes in the neighboring dihedrals, thus preserving the overall protein fold. States with a "locally perturbed" backbone were found in experimental 3D models of some globular proteins and have been shown to be functionally meaningful. In this work, the possibility of large-scale dihedral angle transitions in the course of long-term MD in explicit water was explored for three CTs with different membrane activities: CT 1, 2 (Naja oxiana) and CT A3 (Naja atra). Analysis of the MD-derived distributions of backbone torsion angles revealed several important common and specific features in the structural/dynamic behavior of these proteins. First, large-amplitude transitions were detected in some residues located in the functionally important loop I region. The K5/L6 pair of residues was found to induce a perturbation of the hydrophobic patterns on the molecular surface of CTs-reversible breaking of a large nonpolar zone ("bottom") into two smaller ones and their subsequent association. Second, the characteristic sizes of these patterns perfectly coincided with the dimensions of the nonpolar zones on the surfaces of model two-component (zwitterionic/anionic) membranes. Taken together with experimental data on the CT-induced leakage of fluorescent dye from such membranes, these results allowed us to formulate a two-stage mechanism of CT-membrane binding. The principal finding of this study is that even local conformational dynamics of CTs can seriously affect their functional activity via a tuning of the membrane binding site - specific "hot spots" (like the K5/L6 pair) in the protein structure.


Assuntos
Cardiotoxinas/química , Cardiotoxinas/metabolismo , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Naja naja , Conformação Proteica em Folha beta
11.
Biomed Chromatogr ; 31(12)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28544073

RESUMO

The acute cardiotoxicity induced by Veratrum nigrum (VN) is explored by analyzing heart tissue metabolic profiles in mouse models and applying reversed-phase liquid chromatography mass spectrometry and hydrophilic interaction liquid chromatography mass spectrometry that are based on ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. An animal model of acute heart injury was established in mice via intra-gastric administration of VN. Then, electrocardiogram and echocardiograph monitoring of cardiac function and pathological examination were performed on mice in both the control and VN groups, and it was verified that acute heart injury was caused. Meanwhile, comparing the results of the control and VN groups, we detected 36 differential endogenous metabolites of heart tissue, including taurine, riboflavin, purine and lipids, which are related to many possible pathways such as purine metabolism, taurine and hypotaurine metabolism and energy metabolism. Our study provides a scientific approach for evaluating and revealing the mechanisms of VN-induced cardiotoxicity via the metabolomic strategy.


Assuntos
Cardiotoxinas/toxicidade , Cromatografia Líquida de Alta Pressão/métodos , Metaboloma/efeitos dos fármacos , Extratos Vegetais/toxicidade , Veratrum/química , Animais , Cardiotoxicidade/metabolismo , Cardiotoxinas/química , Modelos Animais de Doenças , Coração/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Espectrometria de Massas/métodos , Redes e Vias Metabólicas , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Extratos Vegetais/química
12.
J Mol Model ; 22(10): 238, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27628673

RESUMO

Cardiotoxins (CTXs) belonging to the three-finger toxin superfamily of snake venoms are one of principal toxic components and the protein toxins exhibit membrane lytic activities when the venoms are injected into victims. In the present study, complex formations between CTX VI (a P-type CTX from Naja atra) and CTX1 (an S-type CTX from Naja naja) on zwitterionic POPC bilayers (a major lipid component of cell membranes) have been studied in near physiological conditions for a total dynamic time scale of 1.35 µs using all-atom molecular dynamics (MD) simulations. Comprehensive analyses of the MD data revealed that residues such as Leu1, Lys2, Tyr11, Lys31, Asp57 and Arg58 of CTX VI, and Ala16, Lys30 and Arg58 of CTX1 were crucial for establishing interactions with the POPC bilayer. Moreover, loop I, along with globular head and loop II of CTX VI, and loop II of CTX1 were found to be the structural regions chiefly governing complex formation of the respective proteins with POPC. Rationalizations for the differential binding modes of CTXs and implications of the findings for designing small molecular inhibitors to the toxins are also discussed. Graphical Abstract Binding modes of a P-type CTX and an S-type CTX towards the POPC bilayer.


Assuntos
Cardiotoxinas/química , Membrana Celular/química , Venenos de Serpentes/química , Animais , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Naja naja/metabolismo , Conformação Proteica
13.
Chem Res Toxicol ; 29(6): 981-90, 2016 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-27104637

RESUMO

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. Although no human intoxication episodes have been reported, YTX content in shellfish is regulated by many food safety authorities due to their worldwide distribution. YTXs have been related to ultrastructural heart damage in vivo, but the functional consequences in the long term have not been evaluated. In this study, we explored the accumulative cardiotoxic potential of YTX in vitro and in vivo. Preliminary in vitro evaluation of cardiotoxicity was based on the effect on hERG (human ether-a-go-go related gene) channel trafficking. In vivo experiments were performed in rats that received repeated administrations of YTX followed by recordings of electrocardiograms, arterial blood pressure, plasmatic cardiac biomarkers, and analysis of myocardium structure and ultrastructure. Our results showed that an exposure to 100 nM YTX for 12 or 24 h caused an increase of extracellular surface hERG channels. Furthermore, remarkable bradycardia and hypotension, structural heart alterations, and increased plasma levels of tissue inhibitor of metalloproteinases-1 were observed in rats after four intraperitoneal injections of YTX at doses of 50 or 70 µg/kg that were administered every 4 days along a period of 15 days. Therefore, and for the first time, YTX-induced subacute cardiotoxicity is supported by evidence of cardiovascular function alterations related to its repeated administration. Considering international criteria for marine toxin risk estimation and that the regulatory limit for YTX has been recently raised in many countries, YTX cardiotoxicity might pose a health risk to humans and especially to people with previous cardiovascular risk.


Assuntos
Cardiotoxinas/toxicidade , Doenças Cardiovasculares/metabolismo , Coração/efeitos dos fármacos , Oxocinas/toxicidade , Animais , Células CHO , Cardiotoxicidade , Cardiotoxinas/administração & dosagem , Cardiotoxinas/química , Células Cultivadas , Cricetulus , Canal de Potássio ERG1/metabolismo , Humanos , Injeções Intraperitoneais , Conformação Molecular , Oxocinas/administração & dosagem , Oxocinas/química , Ratos , Ratos Sprague-Dawley
14.
PLoS One ; 11(1): e0147049, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26751696

RESUMO

Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury.


Assuntos
Distroglicanas/genética , Fibras Musculares Esqueléticas/fisiologia , Distrofia Muscular Animal/genética , Proteínas/genética , Regeneração , Animais , Cardiotoxinas/química , Modelos Animais de Doenças , Distroglicanas/metabolismo , Éxons , Deleção de Genes , Genótipo , Glicosilação , Laminina/metabolismo , Camundongos , Camundongos Knockout , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/metabolismo , Necrose , Transdução de Sinais , Transferases
15.
J Chem Theory Comput ; 11(6): 2560-74, 2015 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-26575555

RESUMO

We present a new method for enhanced sampling for constant-pH simulations in explicit water based on a two-dimensional (2D) replica exchange scheme. The new method is a significant extension of our previously developed constant-pH simulation method, which is based on enveloping distribution sampling (EDS) coupled with a one-dimensional (1D) Hamiltonian exchange method (HREM). EDS constructs a hybrid Hamiltonian from multiple discrete end state Hamiltonians that, in this case, represent different protonation states of the system. The ruggedness and heights of the hybrid Hamiltonian's energy barriers can be tuned by the smoothness parameter. Within the context of the 1D EDS-HREM method, exchanges are performed between replicas with different smoothness parameters, allowing frequent protonation-state transitions and sampling of conformations that are favored by the end-state Hamiltonians. In this work, the 1D method is extended to 2D with an additional dimension, external pH. Within the context of the 2D method (2D EDS-HREM), exchanges are performed on a lattice of Hamiltonians with different pH conditions and smoothness parameters. We demonstrate that both the 1D and 2D methods exactly reproduce the thermodynamic properties of the semigrand canonical (SGC) ensemble of a system at a given pH. We have tested our new 2D method on aspartic acid, glutamic acid, lysine, a four residue peptide (sequence KAAE), and snake cardiotoxin. In all cases, the 2D method converges faster and without loss of precision; the only limitation is a loss of flexibility in how CPU time is employed. The results for snake cardiotoxin demonstrate that the 2D method enhances protonation-state transitions, samples a wider conformational space with the same amount of computational resources, and converges significantly faster overall than the original 1D method.


Assuntos
Ácido Aspártico/química , Cardiotoxinas/química , Ácido Glutâmico/química , Lisina/química , Peptídeos/química , Solventes/química , Animais , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Serpentes , Termodinâmica
16.
Toxicol Sci ; 148(1): 241-60, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26259608

RESUMO

More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Current in vitro testing strategies predominantly take the form of functional assays to predict the potential for drug-induced ECG abnormalities in vivo. Cardiotoxicity can also be structural in nature, so a full and efficient assessment of cardiac liabilities for new chemical entities should account for both these phenomena. As well as providing a more appropriate nonclinical model for in vitro cardiotoxicity testing, human stem cell-derived cardiomyocytes offer an integrated system to study drug impact on cardiomyocyte structure as well as function. Employing human embryonic stem cell-derived cardiacmyocytes (hESC-CMs) on 3 assay platforms with complementary insights into cardiac biology (multielectrode array assay, electrophysiology; impedance assay, cell movement/beating; and high content analysis assay, subcellular structure) we profiled a panel of 13 drugs with well characterized cardiac liabilities (Amiodarone, Aspirin, Astemizole, Axitinib, AZT, Bepridil, Doxorubicin, E-4031, Mexiletine, Rosiglitazone, Sunitinib, Sibutramine, and Verapamil). Our data show good correlations with previous studies and reported clinical observations. Using multiparameter phenotypic profiling techniques we demonstrate the dynamic relationship that exists between functional and structural toxicity, and the benefits of this more holistic approach to risk assessment. We conclude by showing for the first time how the advent of transparent MEA plate technology enables functional and structural cardiotoxic responses to be recorded from the same cell population. This approach more directly links changes in morphology of the hESC-CMs with recorded electrophysiology signatures, offering even greater insight into the wide range of potential drug impacts on cardiac physiology, with a throughput that is more amenable to early drug discovery.


Assuntos
Cardiotoxinas/efeitos adversos , Drogas em Investigação/efeitos adversos , Miócitos Cardíacos/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Cardiotoxinas/química , Movimento Celular/efeitos dos fármacos , Tamanho do Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Criopreservação , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação/química , Impedância Elétrica , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala , Células-Tronco Embrionárias Humanas/citologia , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Dinâmica Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos/citologia , Medição de Risco/métodos , Retirada de Medicamento Baseada em Segurança , Análise Serial de Tecidos
17.
Int J Nanomedicine ; 10: 3163-70, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25995626

RESUMO

Clinical effectiveness of imatinib mesylate in cancer treatment is compromised by its off-target cardiotoxicity. In the present study, we have developed physically stable imatinib mesylate-loaded poly(lactide-co-glycolide) nanoparticles (INPs) that could sustainably release the drug, and studied its efficacy by in vitro anticancer and in vivo cardiotoxicity assays. MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay revealed that INPs are more cytotoxic to MCF-7 breast cancer cells compared to the equivalent concentration of free imatinib mesylate. Wistar rats orally administered with 50 mg/kg INPs for 28 days showed no significant cardiotoxicity or associated changes. Whereas, increased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels, and reduced white blood cell, red blood cell, and hemoglobin content were observed in the animals administered with free drug. While the histological sections from hearts of animals that received INPs did not show any significant cardiotoxic symptoms, loss of normal architecture and increased cytoplasmic vacuolization were observed in the heart sections of animals administered with free imatinib mesylate. Based on these results, we conclude that nano-encapsulation of imatinib mesylate increases its efficacy against cancer cells, with almost no cardiotoxicity.


Assuntos
Antineoplásicos , Cardiotoxinas , Mesilato de Imatinib , Nanopartículas/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Cardiotoxinas/química , Cardiotoxinas/farmacocinética , Cardiotoxinas/farmacologia , Cardiotoxinas/toxicidade , Humanos , Mesilato de Imatinib/química , Mesilato de Imatinib/farmacocinética , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/toxicidade , Células MCF-7 , Ratos , Ratos Wistar
18.
Dev Biol ; 402(1): 72-80, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25794679

RESUMO

Each skeletal muscle contains a fixed ratio of fast and slow myofibers that are distributed in a stereotyped pattern to achieve a specific motor function. How myofibers are specified during development and regeneration is poorly understood. Here we address this question using transgenic reporter mice that indelibly mark the myofiber lineages based on activation of fast or slow myosin. Lineage tracing indicates that during development all muscles have activated the fast myosin gene Myl1, but not the slow myosin gene Myh7, which is activated in all slow but a subset of fast myofibers. Similarly, most nascent myofibers do not activate Myh7 during fast muscle regeneration, but the ratio and pattern of fast and slow myofibers are restored at the completion of regeneration. At the single myofiber level, most mature fast myofibers are heterogeneous in nuclear composition, manifested by mosaic activation of Myh7. Strikingly, Myh7 is activated in a subpopulation of proliferating myoblasts that co-express the myogenic progenitor marker Pax7. When induced to differentiate, the Myh7-activated myoblasts differentiate more readily than the non-activated myoblasts, and have a higher tendency, but not restricted, to become slow myotubes. Together, our data reveal significant nuclear heterogeneity within a single myofiber, and challenge the conventional view that myosin genes are only expressed after myogenic differentiation. These results provide novel insights into the regulation of muscle fiber type specification.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fibras Musculares de Contração Lenta/metabolismo , Músculos/citologia , Músculos/metabolismo , Mioblastos/citologia , Cadeias Pesadas de Miosina/metabolismo , Animais , Cardiotoxinas/química , Diferenciação Celular , Núcleo Celular/metabolismo , Separação Celular , Células Cultivadas , Citometria de Fluxo , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculos/patologia , Miosinas/química , Fator de Transcrição PAX7/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/citologia
19.
Mol Inform ; 34(10): 698-701, 2015 10.
Artigo em Inglês | MEDLINE | ID: mdl-27490970

RESUMO

The blockage of the hERG K(+) channels is closely associated with lethal cardiac arrhythmia. The notorious ligand promiscuity of this channel earmarked hERG as one of the most important antitargets to be considered in early stages of drug development process. Herein we report on the development of an innovative and freely accessible web server for early identification of putative hERG blockers and non-blockers in chemical libraries. We have collected the largest publicly available curated hERG dataset of 5,984 compounds. We succeed in developing robust and externally predictive binary (CCR≈0.8) and multiclass models (accuracy≈0.7). These models are available as a web-service freely available for public at http://labmol.farmacia.ufg.br/predherg/. Three following outcomes are available for the users: prediction by binary model, prediction by multi-class model, and the probability maps of atomic contribution. The Pred-hERG will be continuously updated and upgraded as new information became available.


Assuntos
Cardiotoxinas/química , Simulação por Computador , Bases de Dados de Compostos Químicos , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/química , Animais , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/metabolismo , Cardiotoxinas/toxicidade , Canal de Potássio ERG1/metabolismo , Humanos
20.
Protein Eng Des Sel ; 27(3): 83-8, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24421342

RESUMO

The inhibition of ErbB2 by the use of human antibodies can be a valuable strategy for the treatment of breast and gastric cancer. Trastuzumab, a humanized anti-ErbB2 antibody in clinical use, is effective but can engender resistance as well as cardiotoxicity. ImmunoRNases, made up of a human anti-ErbB2 scFv and human pancreatic ribonucleases (HP-RNases), have been engineered to overcome the limits of other immunotoxins, such as immunogenicity and nonspecific toxicity. Here, we report that a novel anti-ErbB2 immunoRNase, called Erb-HPDDADD-RNase, obtained by fusing Erbicin, a human ErbB2-directed scFv, with an HP-RNase variant that resists the cytosolic inhibitor protein, binds with high affinity to a panel of ErbB2-positive gastric tumor cells and inhibits their growth more than does the parental immunoRNase, which is not resistant to the inhibitor. Moreover, Erb-HP-DDADD-RNase is endowed with antiproliferative activity for trastuzumab-resistant cancer cells both in vitro and in vivo that is more potent than that of the parental immunoRNase. Importantly, Erb-HP-DDADD-RNase does not show cardiotoxic effects in vitro on human cardiomyocytes and does not impair cardiac function in a mouse model. Thus, Erb-HP-DDADD-RNase could fulfil the therapeutic need of cancer patients ineligible for trastuzumab treatment due to primary or acquired trastuzumab resistance or to cardiac dysfunction.


Assuntos
Antineoplásicos/farmacologia , Cardiotoxinas/toxicidade , Receptor ErbB-2/química , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/toxicidade , Ribonuclease Pancreático/química , Animais , Anticorpos Monoclonais Humanizados , Antineoplásicos/química , Cardiotoxinas/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Neoplasias Experimentais/patologia , Engenharia de Proteínas , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Ribonuclease Pancreático/genética , Trastuzumab
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