ABSTRACT
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.
Subject(s)
Amyloid beta-Peptides/drug effects , Biosensing Techniques , Amyloid beta-Peptides/chemistry , Electrochemical Techniques/instrumentation , Electrochemical Techniques/methods , Humans , Immunochemistry/instrumentation , Immunochemistry/methods , MicroelectrodesABSTRACT
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.
Subject(s)
Acetylcholinesterase/metabolism , Cholinesterase Inhibitors/analysis , Cholinesterase Inhibitors/pharmacology , Electrochemical Techniques/instrumentation , Indans/analysis , Indans/pharmacology , Piperidines/analysis , Piperidines/pharmacology , Animals , Donepezil , Electrodes , Equipment Design , Erythrocytes/enzymology , Humans , Inhibitory Concentration 50 , Mice , Mice, Inbred C57BL , MiniaturizationABSTRACT
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.
Subject(s)
Acetylcholine/chemistry , Alzheimer Disease , Biological Products/chemistry , Drug Delivery Systems/methods , Triazines/chemistry , Acetylcholine/administration & dosage , Alzheimer Disease/drug therapy , Alzheimer Disease/pathology , Amyloid beta-Peptides/chemistry , Biological Products/administration & dosage , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line, Tumor , Humans , Peptide Fragments/chemistry , Protein Structure, Secondary , Triazines/administration & dosageABSTRACT
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.
