RESUMO
A novel electrochemical immunosensor fabricated from gold compact disc electrodes was designed for rapid evaluation of aggregation processes that lead to the formation of oligomeric and fibrillar states of amyloid-beta(1-42) (Aß(1-42)) during Alzheimer's disease. Conformation-specific antibodies were immobilized on the surface of the gold electrode using a 3,3'-dithiobis (sulfosuccinimidyl) propionate (DTSSP) linker. Surface binding events were analyzed by electrochemical impedance spectroscopy (EIS) in which the formation of an antigen-antibody complex was quantified as a function of charge transfer resistance using a [Fe(CN)6](3-/4-) redox probe. The effectiveness of novel sym-triazine-derived aggregation modulators (TAE-1, TAE-2) to reduce the population of toxic oligomers was evaluated. Aß fibril formation was validated by thioflavin T (ThT) fluorescence, whereas oligomer formation was investigated by MALDI. Antigen detection by EIS was further supported by immuno dot blot assays for oligomeric and fibrillar components. Docking simulations of the aggregation modulators TAE-1 and TAE-2 with Aß(1-42) fibrils performed using Autodock Vina suggest a mechanism for the improved aggregation inhibition observed for TAE-2. The results demonstrate the utility and convenience of impedance immunosensing as an analytical tool for rapid and comprehensive evaluation of effective Aß aggregation modulating agents.
Assuntos
Peptídeos beta-Amiloides/efeitos dos fármacos , Técnicas Biossensoriais , Peptídeos beta-Amiloides/química , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Humanos , Imunoquímica/instrumentação , Imunoquímica/métodos , MicroeletrodosRESUMO
The growing interest in membrane interactions of amyloidogenic proteins indicates that lipid binding and the regulation of membrane potential are critical to the onset and progression of neurodegenerative diseases such as Parkinson's (PD), Alzheimer's (AD), and prion diseases. Advancing the understanding of this field requires the application of varied biophysical and biological techniques designed to probe the characteristics and underlying mechanisms of membrane-peptide interactions. Therefore, the development of a rapid cytotoxicity evaluation system using a membrane potential-sensitive bis-oxonol fluorescent dye, DiBAC4(3) is reported here. The exposure of C-terminal truncated α-synuclein 119 (α-Syn119) and amyloid-ß(1-42) (Aß(1-42)) to U2-OS cell cultures resulted in an immediate, significant, and concentration-dependent increase in fluorescence response of DiBAC4(3). This response was strongly correlated with the cytotoxicity of α-Syn119 and Aß(1-42) as determined by conventional CC8 and ATP assays. Furthermore, the capacity of well-defined polyphenolic antioxidants (i.e., pyrroloquinoline quinone (PQQ), baicalein, (-)-epigallocatechin-3-gallate (EGCG), and myricetin) to mitigate amyloid-induced cytotoxicity was evaluated using the developed biosensing system. We envisage that this work would accelerate the development of a rapid and cost-effective high-throughput screening platform in drug discovery for AD and PD.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Técnicas Biossensoriais , Corantes Fluorescentes/química , Fragmentos de Peptídeos/antagonistas & inibidores , alfa-Sinucleína/antagonistas & inibidores , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/química , Barbitúricos/química , Catequina/análogos & derivados , Catequina/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Flavanonas/química , Humanos , Isoxazóis/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/toxicidade , Tiobarbitúricos/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
A number of human protein misfolding disorders, including Alzheimer's disease (AD), are closely related to the accumulation of ß-sheet-rich amyloid fibrils or aggregates. Neuronal toxicity in AD has been linked to the interactions of amyloid-ß (Aß) with metals, especially Zn(2+), Cu(2+), and Fe(3+), which leads to the production of reactive oxygen species. Nucleation-dependent Aß aggregation, or "seeding", is thought to propagate fibril formation. In this surface plasmon resonance imaging (SPRi) study, we have shown that the fibril seeds formed with the incubation of Aß in the presence of metals are better at promoting monomer elongation compared to Aß alone or in the presence of a well-described polyphenol, (-)-epigallocatechin-3-gallate (EGCG). This is a novel attempt to simultaneously monitor the effects of multiple modulators on fibril elongation using a single chip. EGCG was shown in transmission electron microscopy (TEM) and thioflavin T (ThT) studies to promote the formation of off-pathway, highly stable unstructured oligomers, supporting the SPRi results. These findings suggest that SPRi provides a promising platform as a screening tool for small molecules that can affect the aggregation pathways in neurodegenerative diseases.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Catequina/análogos & derivados , Metais/química , Ressonância de Plasmônio de Superfície , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Benzotiazóis , Catequina/química , Catequina/metabolismo , Cobre/química , Compostos Férricos/química , Humanos , Íons/química , Cinética , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Tiazóis/química , Tiazóis/metabolismo , Zinco/químicaRESUMO
Several severe neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and prion-associated transmissible spongiform encephalopathies, have been linked to dysregulation of specific proteins capable of self-assembly into deleterious fibrillar aggregates termed amyloids. A wide range of analytical techniques has been used to clarify the mechanisms of these protein-misfolding processes, in the hope of developing effective therapeutic treatment. Most of these studies have relied heavily on conventional methods of protein characterization, notably circular dichroism spectroscopy, thioflavin T fluorescence, transmission electron microscopy, and atomic force microscopy, which are particularly suitable for monitoring later-stage aggregate formation. Although electrochemical methods of protein detection have existed for some time, they have only recently gained prominence as a powerful tool for studying the early stages of protein aggregation during which the more toxic soluble amyloid species form. Electrochemical detection methods include direct detection of intrinsic redox-active amino acid residues, protein-catalyzed hydrogen evolution, use of extrinsic ß-sheet binding mediators, and impedance spectroscopy. In this review, we evaluate the use of electrochemistry for study of protein aggregation related to neurodegenerative disorders.
Assuntos
Técnicas Eletroquímicas/métodos , Doenças Neurodegenerativas/diagnóstico , Proteínas/química , Humanos , Doenças Neurodegenerativas/metabolismo , Proteínas/metabolismoRESUMO
The rapid diagnosis of Legionellosis is crucial for the effective treatment of this disease. Currently, most clinical laboratories utilize rapid immunoassays that are sufficient for the detection of Legionella serogroup 1, but not other clinically relevant serogroups. In this report, the development of a disposable immunochip system is described in connection with electrochemical impedance spectroscopy and fluorescence microscopy. The immunochips were prepared by covalently immobilizing fluorophore-conjugated L. pneumophilaantibodies on Au chips. The analytical performance of the immunochips was optimized as a prescreening tool for L. pneumophila. The versatile immunochips described here can be easily adapted for the monitoring of all Legionella serogroups in clinical and environmental samples.
Assuntos
Espectroscopia Dielétrica , Legionella pneumophila , Legionelose/diagnóstico , Técnicas Analíticas Microfluídicas , Humanos , Imunoquímica , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/tendências , Microscopia de FluorescênciaRESUMO
Alzheimer's disease (AD) is marked by the accumulation of neuronal plaques from insoluble amyloid-beta (Aß) peptides. Growing evidence for the role of Aß oligomers in neuronal cell cytotoxicity and pathogenesis has prompted the development of novel techniques to better understand the early stages of aggregation. Near infrared (NIR) optical trapping was applied to characterize the early stages of Aß aggregation in the presence of a ß-sheet intercalating dye, Congo Red (CR), as the fluorescent marker. The integration of fluorescence analysis with NIR optical trapping has provided a new outlook into the first two hours of Aß aggregation.
Assuntos
Peptídeos beta-Amiloides/química , Pinças Ópticas , Fragmentos de Peptídeos/química , Vermelho Congo/química , Técnicas Eletroquímicas , Cinética , Lasers , Espectrometria de FluorescênciaRESUMO
Alzheimer's disease (AD) is associated with the formation and deposition of amyloid fibrils. A better understanding of the oligomeric intermediates on the pathway to fibrilization is highly desired, but efficient methods for their detection are lacking. We have studied the interfacial properties of amyloid peptides (Abeta-40 and Abeta-42) and the course of their aggregation in vitro in the presence of the benzothiazole dyes Thioflavin T (4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline) chloride, ThT) and BTA-1 ([2-(4'-(methylamino)phenyl) benzothiazole]) using electrochemical techniques. The intercalative properties of these dyes between the beta-sheets of amyloids have been well-documented using fluorescence-based systems, but their electrochemistry is reported here for the first time. ThT is positively charged and water-soluble, whereas BTA-1 is neutral and hydrophobic. Immediate and significantly different electrochemical characteristics of these dyes were observed in the presence of amyloid peptides. A decrease of the BTA-1 oxidation signal was observed upon incubation with Abeta-40. Incubation of BTA-1 with Abeta-42 results in an increased rate of exponential decay, which was in agreement with the known rapid aggregation properties of Abeta-42. The aggregation of amyloid peptides with ThT resulted in an unexpected increase in signal after 24 h of incubation, consistent for both peptides. The results of the electrochemical trials were confirmed using simultaneous fluorescence analysis of the same incubated amyloid samples. The very early changes in the interfacial behavior of the amyloid peptides after the first few minutes of incubation were attributed to the fast oligomerization of the peptides with the disruption of the intercalative properties of the benzothiazole dyes between the beta-sheets. The subsequent changes in the electrochemical signals can be related to the onset of intercalation between the fibrils. Our results demonstrate the utility of electrochemical oxidation signals of the benzothiazole dyes as a new and simple tool for the investigation of amyloid formation related to the AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/biossíntese , Benzotiazóis/química , Técnicas Eletroquímicas/métodos , Peptídeos beta-Amiloides/química , Humanos , OxirreduçãoRESUMO
The misfolding of amyloid-beta (Aß) peptide is one of the pathological hallmarks of Alzheimer's disease (AD). Polyphenols are strong antioxidants and metal chelators, with characteristics that are of beneficial therapeutic values for their development as candidates targeting neurodegenerative and metal-induced diseases. We have demonstrated here the electrochemical properties of a green tea component, (-)-epigallocatechin-3-gallate (EGCG), and its potent activity on Aß peptides. Characterization of early interactions (≤48 h) between EGCG and Aß was conducted using square wave voltammetry (SWV). The interaction of Cu(ii) ions with the Tyr-10 residue of Aß was shown to be affected by surrounding His residues. Morphological changes due to the binding of EGCG and Cu(II) were also elucidated using transmission electron microscopy (TEM). Electroanalytical techniques are promising for facilitating the investigation of metals and flavonoids in drug screening studies.
Assuntos
Peptídeos beta-Amiloides/química , Catequina/análogos & derivados , Cobre/química , Catequina/química , Cobre/farmacologia , Microscopia Eletrônica de Transmissão , Ligação Proteica/efeitos dos fármacosRESUMO
The utility of a simple, low-cost detection platform for label-free electrochemical characterization of acetylcholinesterase (AChE) inhibition is demonstrated as a potential tool for screening of small-molecule therapeutic agents for Alzheimer's disease (AD). Technique validation was performed against the standard Ellman's colorimetric assay using the clinically established cholinesterase inhibitor (ChEI), Donepezil (Aricept(®)). Electrochemical measurements were obtained by differential pulse voltammetry (DPV) performed using a portable potentiostat system for detection of the enzymatic product, thiocholine (TCh), by direct oxidation on unmodified gold screen-printed electrodes. The IC50 profiles for Donepezil measured in vitro were found to be comparable between both colorimetric and electrochemical detection methods for the analysis of purified human erythrocyte-derived AChE (28±7 nM by DPV; 26±8 nM by Ellman's method). The selectivity of this unmodified electrode system was compared to a range of biological sulfur-containing compounds including cysteine, homocysteine, glutathione and methionine as well as ascorbic acid. Preliminary studies also demonstrated the potential applicability of this electrochemical technique for the analysis of Donepezil in crude cholinesterase samples from anterior cortex homogenates of C57BL/6J mice.
Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/análise , Inibidores da Colinesterase/farmacologia , Técnicas Eletroquímicas/instrumentação , Indanos/análise , Indanos/farmacologia , Piperidinas/análise , Piperidinas/farmacologia , Animais , Donepezila , Eletrodos , Desenho de Equipamento , Eritrócitos/enzimologia , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos C57BL , MiniaturizaçãoRESUMO
The bioactivities of two novel compounds (TAE-1 and TAE-2) that contain a sym-triazine scaffold with acetylcholine-like substitutions are examined as promising candidate agents against Alzheimer's disease. Inhibition of amyloid-ß fibril formation in the presence of Aß1-42, evaluated by Thioflavin T fluorescence, demonstrated comparable or improved activity to a previously reported pentapeptide-based fibrillogenesis inhibitor, iAß5p. Destabilization of Aß1-42 assemblies by TAE-1 and TAE-2 was confirmed by scanning electron microscopy imaging. sym-Triazine inhibition of acetylcholinesterase (AChE) activity was observed in cytosol extracted from differentiated human SH-SY5Y neuronal cells and also using human erythrocyte AChE. The sym-triazine derivatives were well tolerated by these cells and promoted beneficial effects on human neurons, upregulating expression of synaptophysin, a synaptic marker protein, and MAP2, a neuronal differentiation marker.
Assuntos
Acetilcolina/química , Doença de Alzheimer , Produtos Biológicos/química , Sistemas de Liberação de Medicamentos/métodos , Triazinas/química , Acetilcolina/administração & dosagem , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Produtos Biológicos/administração & dosagem , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Triazinas/administração & dosagemRESUMO
Alzheimer's disease (AD) is a complex neurodegenerative disorder marked by numerous causative factors of disease progression, termed pathologies. We report here the synthesis of a small library of novel sym-triazine compounds designed for targeted modulation of multiple pathologies related to AD, specifically human acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and Aß aggregation. Rational targeting of AChE was achieved by the incorporation of acetylcholine substrate analogues into a sym-triazine core in either a mono-, di-, or trisubstituted regime. A subset of these derivatives demonstrated improved activity compared to several commercially available cholinesterase inhibitors. High AChE/BuChE selectivity was characteristic of all derivatives, and AChE steady-state kinetics indicated a mixed-type inhibition mechanism. Further integration of multiple hydrophobic phenyl units allowed for improved ß-sheet intercalation into amyloid aggregates. Several highly effective structures exhibited fibril inhibition greater than the previously reported ß-sheet-disrupting penta-peptide, iAß5p, evaluated by thioflavin T fluorescence spectroscopy and transmission electron microscopy. Highly effective sym-triazines were shown to be well tolerated by differentiated human neuronal cells, as demonstrated by the absence of adverse effects on cellular viability at a wide range of concentrations. Parallel targeting of multiple pathologies using sym-triazines is presented here as an effective strategy to address the complex, multifactorial nature of AD progression.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/efeitos dos fármacos , Amiloide/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Triazinas/farmacologia , Acetilcolinesterase/efeitos dos fármacos , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/metabolismo , Butirilcolinesterase/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores da Colinesterase/síntese química , Humanos , Concentração Inibidora 50 , Microscopia Eletrônica de Transmissão , Neurônios/efeitos dos fármacos , Triazinas/síntese químicaRESUMO
The STAT3 gene is abnormally active in glioblastoma (GBM) and is a critically important mediator of tumor growth and therapeutic resistance in GBM. Thus, for poorly treated brain cancers such as gliomas, astrocytomas, and glioblastomas, which harbor constitutively activated STAT3, a STAT3-targeting therapeutic will be of significant importance. Herein, we report a most potent, small molecule, nonphosphorylated STAT3 inhibitor, 31 (SH-4-54) that strongly binds to STAT3 protein (K D = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells (BTSCs) and effectively suppresses STAT3 phosphorylation and its downstream transcriptional targets at low nM concentrations. Moreover, in vivo, 31 exhibited blood-brain barrier permeability, potently controlled glioma tumor growth, and inhibited pSTAT3 in vivo. This work, for the first time, demonstrates the power of STAT3 inhibitors for the treatment of BTSCs and validates the therapeutic efficacy of a STAT3 inhibitor for GBM clinical application.
RESUMO
The development of generic inhibitors in order to control the formation of amyloid fibrils and early oligomers is still an unmet medical need. Here, we demonstrate the applicability of electrochemical analysis for the detection of ß-sheet breaker peptide ligands that act as excellent inhibitors of Alzheimer's disease (AD) amyloid-ß (Aß) fibrils and oligomers in vitro. As the case study, a well-defined ß-sheet breaker pentapeptide (LPFFD, FibIII) was utilized with Aß(1-42) peptides. Square wave voltammetry (SWV) measurements were confirmed with simultaneous fluorescence analysis of the same incubated Aß samples using a well-known fluorescent marker of ß-sheet formation, Thioflavin T (ThT). Significant changes in the electrochemical signals were observed for the interaction of the Aß oligomers with FibIII at the early stages of aggregation. The electrochemical approach, in principle, allowed monitoring ß-sheet breaker-Aß interactions on the time scale of aggregation in a label-free and cost-effective format using screen-printed carbon strip (SPCS) electrodes.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Eletroquímica/métodos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Sequência de Aminoácidos , Humanos , Cinética , Ligantes , Oligopeptídeos/metabolismo , Oxirredução , Estrutura Secundária de Proteína , Espectrometria de FluorescênciaRESUMO
Au electrodeposition has been performed on carbon electrodes to fabricate Au nanostructures on the working electrode of screen-printed carbon chips. The amount of Au deposited was characterized using the raw reflection spectra obtained by detecting UV-Vis spectrum reflected from the electrode surface. SEM images at different deposition time points provided evidence for the growth of Au nanostructures. The increase in deposition time led to an increase in the intensity of the spectrum obtained for both chips. The acquired optical properties of the carbon chips provide a promising platform for simultaneous optical and electrochemical measurements.
RESUMO
The detection of trace levels of paraoxon and carbofuran was achieved utilizing differential pulse voltammetry (DPV) on gold disposable electrochemical printed (DEP) chips. The nanostructured gold surface of the chips enables highly sensitive oxidation of the thiocholine (TCh) product even in the absence of costly surface modifications. The inhibition of AChE activity at varying insecticide concentrations was detected with low detection limits of 10 ppb (36 nM) for paraoxon and 8 ppb (18 nM) for carbofuran. Fine-tuning of the experimental conditions will allow for the application of unmodified DEP gold chips for inexpensive on-field detection of AChE inhibition by various insecticides at or below the allowable concentrations set by European and North American regulation standards.