Rapid label-free detection of metal-induced Alzheimer’s amyloid β peptide aggregation by electrochemical method

Amyloid β peptide plays a critical role in the pathogenesis of Alzheimer’s disease (AD). Metal ions are highly enriched in cerebral amyloid deposits in AD and are proposed to be able to mediate Aβ conformation. Therefore, a rapid, low-cost, and sensitive detection of metal-induced Aβ aggregation and their relation to AD is clearly needed for the clinical diagnosis and treatment. In this report, we study metal-induced Aβ aggregation by a rapid, label-free electrochemical method and monitor both the aggregation kinetics and the morphology in the absence or presence of Zn (II) and Cu (II). Different electrochemical behaviors of Aβ in the absence or presence of metal ions have been observed both in the aggregation kinetics and final aggregates. Our results provide new insights into the application of the rapid, low-cost and sensitive electrochemical device for clinical diagnosis of metal effects on AD or other neurodegenerative diseases which are related to protein misfolding.     

Surface plasmon resonance biosensor for the detection of VEGFR-1—a protein marker of myelodysplastic syndromes

The surface plasmon resonance (SPR) biosensor system with dispersionless microfluidics for the direct and label-free detection of a soluble vascular endothelial growth factor receptor (sVEGFR-1) is described. The detection approach takes advantage of an affinity interaction between sVEGFR-1 and its ligand, vascular endothelial growth factor (VEGF-A), which is covalently immobilized on the surface of the SPR sensor. The ability of the immobilized VEGF-A to specifically bind the sVEGFR-1 receptor is demonstrated in a buffer. The detection of sVEGFR-1 in 2% human blood plasma is carried out by using the sequential injection approach. The detection limit of 25 ng/mL is achieved. In addition, we demonstrate that the functional surface of the sensor can be regenerated for repeated use.     

A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer’s disease drug target β-secretase

Analytical and Bioanalytical Chemistry - Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities...     

Quantitative structure-activity relationship analysis of β-amyloid aggregation inhibitors

Inhibiting the aggregation process of the β-amyloid peptide is a promising strategy in treating Alzheimer’s disease. In this work, we have collected a dataset of 80 small molecules with known inhibition levels and utilized them to develop two comprehensive quantitative structure–activity relationship models: a Bayesian model and a decision tree model. These models have exhibited high predictive accuracy: 87% of the training and test sets using the Bayesian model and 89 and 93% of the training and test sets, respectively, by the decision tree model. Subsequently these models were used to predict the activities of several new potential β-amyloid aggregation inhibitors and these predictions were indeed validated by in vitro experiments. Key chemical features correlated with the inhibition ability were identified. These include the electro-topological state of carbonyl groups, AlogP and the number of hydrogen bond donor groups. The results demonstrate the feasibility of the developed models as tools for rapid screening, which could help in the design of novel potential drug candidates for Alzheimer’s disease.     

Methodology for the synthesis of mimetics of peptide β-turns

This letter describes a scenario which provides for the stereospecific introduction of both the amino and carboxyl termini portion of a designed β-turn mimetic.     

Surface plasmon resonance imaging for affinity analysis of aptamer–protein interactions with PDMS microfluidic chips

We report on the use of PDMS multichannels for affinity studies of DNA aptamer–human Immunoglobulin E (IgE) interactions by surface plasmon resonance imaging (SPRi). The sensing surface was prepared with thiol-terminated aptamers through a self-assembling process in the PDMS channels defined on a gold substrate. Cysteamine was codeposited with the thiol aptamers to promote proper spatial arrangement of the aptamers and thus maintain their optimal binding efficiencies. Four aptamers with different nucleic acid sequences were studied to test their interaction affinity toward IgE, and the results confirmed that aptamer I (5′-SH-GGG GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CCC C-3′) has the strongest binding affinity. Control experiments were conducted with a PEG-functionalized surface and IgG was used to replace IgE in order to verify the selective binding of aptamer I to the IgE molecules. A linear concentration-dependent relationship between IgE and aptamer I was obtained, and a 2-nM detection limit was achieved. SPRi data were further analyzed by global fitting, and the dissociation constant of aptamer I–IgE complex was found to be 2.7 × 10⁻⁷ M, which agrees relatively well with the values reported in the literature. Aptamer affinity screening by SPR imaging demonstrates marked advantages over competing methods because it does not require labeling, can be used in real-time, and is potentially high-throughput. The ability to provide both qualitative and quantitative results on a multichannel chip further establishes SPRi as a powerful tool for the study of biological interactions in a multiplexed format. Figure The SPRi sensograms and thier global fits for aptamer I and IgE interactions. Insert in the difference image obtained with the PDMS microchannel flow cell for aptamer IV, III, and I (from left to right     

Aggregation state and neurotoxic properties of alzheimer β-amyloid peptide

Alzheimer's disease (AD) represents the most common form of senile dementia and represents a tremendous health problem as the world population is aging. AD is characterized by the accumulation of amyloid β-peptide (Aβ) in the brain and the loss of cholinergic neurons in the basal forebrain. Accumulation of soluble and insoluble assemblies of Aβ in the brain is a crucial event in AD pathogenesis and the presence of amyloid plaques in the brain is required for definitive identification of AD. Yet, there is no correlation between amyloid plaques and the degree of dementia. In the past two decades researchers have devoted their effort to study and explain the mechanisms involved in the pathology of this devastating disease. Studies from different areas of the natural and medical sciences have provided important information towards the elucidation of some of the pathological processes that take place in AD. An aspect of crucial importance is the aggregation state of Aβ peptide and its role in neuropathology. Here, we discuss recent studies aimed at the identification of Aβ protein aggregates, the characterization of their toxic potential and the development of therapeutic strategies that target Aβ aggregation.     

Bifunctional compounds for controlling metal-mediated aggregation of the aβ42 peptide

Abnormal interactions of Cu and Zn ions with the amyloid β (Aβ) peptide are proposed to play an important role in the pathogenesis of Alzheimer's disease (AD). Disruption of these metal-peptide interactions using chemical agents holds considerable promise as a therapeutic strategy to combat this incurable disease. Reported herein are two bifunctional compounds (BFCs) L1 and L2 that contain both amyloid-binding and metal-chelating molecular motifs. Both L1 and L2 exhibit high stability constants for Cu(2+) and Zn(2+) and thus are good chelators for these metal ions. In addition, L1 and L2 show strong affinity toward Aβ species. Both compounds are efficient inhibitors of the metal-mediated aggregation of the Aβ(42) peptide and promote disaggregation of amyloid fibrils, as observed by ThT fluorescence, native gel electrophoresis/Western blotting, and transmission electron microscopy (TEM). Interestingly, the formation of soluble Aβ(42) oligomers in the presence of metal ions and BFCs leads to an increased cellular toxicity. These results suggest that for the Aβ(42) peptide-in contrast to the Aβ(40) peptide-the previously employed strategy of inhibiting Aβ aggregation and promoting amyloid fibril dissagregation may not be optimal for the development of potential AD therapeutics, due to formation of neurotoxic soluble Aβ(42) oligomers.     

Exploring the binding mechanism of thioflavin-T to the β-amyloid peptide by blind docking method

Using blind dock method,we find that thioflavin-T(ThT) can bind to both monomers and fibrils of the full-length β-amyloid peptide(Aβ1-42) and has a higher binding affinity to the fibrils.It is shown that the hydrophobic interaction between the ligand(ThT) and substrate(Aβ1-42) are stronger than hydrogen bonds.Furthermore,ThT tends to be located near the C-terminus of Aβ monomer through hydrophobic and electrostatic interactions,while it tends to contact the residues Met35 and Gly27 of the fibril surface mainly through hydrophobic interaction.Finally,according to the docking results and ThT fluorescence assay,a kinetic equation is proposed to deduce the aggregation rate coefficient of Aβ1-42.     

Optimized method for the recombinant production of a sea anemone’s peptide

The production of the analgesic peptide HCRG21 for medical use is restricted by a number of limitations in the technology. The optimal biotechnological method scalable to industrial has been developed based on the production of a fusion protein containing a special leader, His-tag, and Smt3 sequence upstream in the peptide sequence. The resulting peptide shared its inhibitory activity to TRPV1 ion channel identical to the published early.     

Metal-amyloid-β peptide interactions: a preliminary investigation of molecular mechanisms for Alzheimer’s disease

Although humans have spent exactly 100 years combating Alzheimer’s disease (AD), the molecular mechanisms of AD remain unclear. Owing to the rapid growth of the oldest age groups of the popula-tion and the continuous increase of the incidence of AD, it has become one of the crucial problems to modern sciences. It would be impossible to prevent or reverse AD at the root without elucidating its molecular mechanisms. From the point of view of metal-amyloid-β peptide (Aβ) interactions, we review the molecular mechanisms of AD, mainly including Cu2 and Zn2 inducing the aggregation of Aβ, cata-lysing the production of active oxygen species from Aβ, as well as interacting with the ion-channel-like structures of Aβ. Moreover, the development of therapeutic drugs on the basis of metal-Aβ interactions is also briefly introduced. With the increasingly rapid progress of the molecular mechanisms of AD, we are now entering a new dawn that promises the delivery of revolutionary developments for the control of dementias.     

An enzyme-linked immunosorbent assay to compare the affinity of chemical compounds for β-amyloid peptide as a monomer

Aβ_1–42 is the proteolytic cleavage product of cleavage of the amyloid precursor protein by β- and γ-secretases. The aggregation of Aβ_1–42 plays a causative role in the development of Alzheimer’s disease. To lock Aβ_1–42 in a homogenous state, we embedded the Aβ_1–42 sequence in an unstructured region of Bcl-x_L. Both the N-terminus and the C-terminus of Aβ_1–42 were constrained in the disordered region, whereas the conjunction did not introduce any folding to Aβ_1–42 but maintained the sequence as a monomer in solution. With Bcl-x_L-Aβ₄₂, we developed an enzyme-linked immunosorbent assay to compare the affinity of compounds for monomeric Aβ_1–42. Bcl-x_L-Aβ₄₂ was coated on a microplate and this was followed by incubation with different concentrations of compounds. Compounds binding to Leu17-Val24 of Aβ_1–42 inhibited the interaction between Bcl-x_L-Aβ₄₂ and antibody 4G8. The method can not only reproduce the activities of the reported Aβ_1–42 inhibitors such as dopamine, tannin, and morin but can also differentiate decoy compounds that do not bind to Aβ_1–42. Remarkably, using this method, we discovered a new inhibitor that binds to monomeric Aβ_1–42 and inhibits Aβ_1–42 fibril formation. As the structure of Bcl-x_L-Aβ₄₂ monomer is stable in solution, the assay could be adapted for high-throughput screening with a series of antibodies that bind the different epitopes of Aβ_1–42. In addition, the monomeric form of the Aβ_1–42 sequence in Bcl-x_L-Aβ₄₂ would also facilitate the identification of Aβ_1–42 binding partners by coimmunoprecipitation, cocrystallization, surface plasmon resonance technology, or the assay as described here.     

Development of gold nanoparticle based colorimetric method for quantitatively studying the inhibitors of Cu/Zn induced β-amyloid peptide assembly

In this paper, a kind of gold nanoparticle (GNP)-based colorimetric assay has been developed for studying the reversible interaction of β-amyloid peptide (Aβ) with Cu²⁺ and Zn²⁺, and quantitatively analyzing four inhibitors (i.e., EDTA, EGTA, histidine and clioquinol) of Cu²⁺/Zn²⁺ induced Aβ assembly. The inhibition efficiencies (e.g., half maximal inhibitory concentration, IC₅₀ value) of these inhibitors could be measured in this work. As far as we know, these IC₅₀ values were reported at the first time. In this assay, the streptavidin conjugated GNPs (SA-GNPs) were employed as indicators to monitor the Cu²⁺/Zn²⁺ induced aggregating/disaggregating behaviors of biotin modified β-amyloid 1–16 peptides (Aβ_1–16(biotin)). Because of high affinity of streptavidin (SA) with biotin, the aggregating/disaggregating of Aβ_1–16(biotin) results in the significant color change of SA-GNPs. Furthermore, we demonstrate that the assay can be used as an effective tool for designing anti-dementia drugs through quantitative analysis of the interactions of four representative inhibitors with Cu²⁺/Zn²⁺ induced Aβ assembly.     

Two new sesquineolignans from the seeds of Crataegus pinnatifida and their β-amyloid aggregation inhibitory activitiy

Two new sesquineolignans, hawthornsesquinins K and L (1 and 2), were isolated from the seeds of Crataegus pinnatifida. Their structures were determined by spectroscopic analyses, including 1D, 2D NMR and HRESIMS data. All isolated compounds were tested for their β-amyloid aggregation inhibitory activity and neuroprotective effects against H₂O₂-induced damage in SH-SY5Y cells. The results indicated that compound 1 showed prominent inhibition of Aβ_1–42 aggregation and significant neuroprotective effect on H₂O₂-induced cellular damage in SH-SY5Y cells.     

A high performance method for thermodynamic study on the binding of copper ion and glycine with Alzheimer’s amyloid β peptide

The interaction of Cu²⁺ to the first 16 residues of the Alzheimer’s amyloid β peptide, Aβ(1–16) was studied by isothermal titration calorimetry at pH 7.2 and 37°C in aqueous solution. The Gholamreza Rezaei Behbehani (GRB) solvation model was used to reproduce the enthalpies of interactions of Aβ(1–16) with glycine, Gly+Aβ(1–16), and Cu²⁺ ions, Cu²⁺ +Aβ(1–16), over the whole range of Cu2+ concentrations. The binding parameters recovered from the solvation model were attributed to the structural change of Aβ(1–16) due to the glycine and Cu²⁺ interactions. It was found that there is a set of two identical binding sites for Cu²⁺ ions. p=2 indicates that the binding has positive cooperativity in the two binding sites. Aβ(1–16) structure is destabilized greatly as a result of binding to Cu²⁺ ions.     

Synthesis and evaluation of a phenylbenzothiazole-based 99mTc(CO)3-radiotracer for possible application in imaging of β-amyloid plaques in Alzheimer’s disease

The 2-phenyl benzothiazole pharmacophore is known to have high affinity for amyloid beta (Aβ) and is therefore derivatized, to [N-(4′-benzothiazol-pyridin-2-yl-methyl-amino)-acetic acid (BTPAA)] for radiolabeling with [^99mTc(CO)₃(H₂O)₃]⁺ precursor. The radiotracer, ^99mTc(CO)₃–BTPAA is evaluated in vitro and in vivo to determine its binding with the Aβ and ability to cross the blood brain barrier. The radiotracer prepared in >95 % radiochemical yield, showed ~25 % inhibition in presence of thioflavin-T, indicating its specificity towards aggregated Aβ protein. The radiotracer also showed brain uptake of 0.25 ± 0.04 % injected dose/g at 2 min post injection, indicating its ability to cross the blood brain barrier.     

Molecular docking studies of coumarin hybrids as potential acetylcholinesterase,butyrylcholinesterase, monoamine oxidase A/B and β-amyloid inhibitors for Alzheimer’s disease

Coumarins are the phytochemicals, which belong to the family of benzopyrone, that display interesting pharmacological properties. Several natural, synthetic and semisynthetic coumarin derivatives have been discovered in decades for their applicability as lead structures as drugs. Coumarin based conjugates have been described as potential AChE, BuChE, MAO and β-amyloid inhibitors. Therefore, the objective of this review is to focus on the construction of these pharmacologically important coumarin analogues with anti-Alzheimer’s activities, highlight their docking studies and structure–activity relationships based on their substitution pattern with respect to the selected positions on the chromen ring by emphasising on the research reports conducted in between year 1968 to 2017.

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Gold nanoparticle based dot-blot immunoassay for sensitively detecting Alzheimer's disease related β-amyloid peptide

A simple, sensitive gold nanoparticle (GNP)-based dot-blot immunoassay has been developed for detecting Alzheimer's disease related β-amyloid peptide 1-42 (Aβ(1-42)) down to a 50 pg mL(-1) level in aqueous solution. Practical samples (cerebrospinal fluid (CSF), cell culturing mediums and cell lysates) have been used to demonstrate the ability of the assay.     

Kinetic pathways to peptide aggregation on surfaces: the effects of β-sheet propensity and surface attraction

Mechanisms of peptide aggregation on hydrophobic surfaces are explored using molecular dynamics simulations with a coarse-grained peptide representation. Systems of peptides are studied with varying degrees of backbone rigidity (a measure of β-sheet propensity) and degrees of attraction between their hydrophobic residues and the surface. Multiple pathways for aggregation are observed, depending on the surface attraction and peptide β-sheet propensity. For the case of a single-layered β-sheet fibril forming on the surface (a dominant structure seen in all simulations), three mechanisms are observed: (a) a condensation-ordering transition where a bulk-formed amorphous aggregate binds to the surface and subsequently rearranges to form a fibril; (b) the initial formation of a single-layered fibril in the bulk depositing flat on the surface; and (c) peptides binding individually to the surface and nucleating fibril formation by individual peptide deposition. Peptides with a stiffer chiral backbone prefer mechanism (b) over (a), and stronger surface attractions prefer mechanism (c) over (a) and (b). Our model is compared to various similar experimental systems, and an agreement was found in terms of the surface increasing the degree of fibrillar aggregation, with the directions of fibrillar growth matching the crystallographic symmetry of the surface. Our simulations provide details of aggregate growth mechanisms on scales inaccessible to either experiment or atomistic simulations.