Inhibit Amyloid Aggregation Research Articles

Pharmacophore Modeling and 3D-QSAR Study of Indole and Isatin Derivatives as Antiamyloidogenic Agents Targeting Alzheimer's Disease.

Thirty-six novel indole-containing compounds, mainly 3-(2-phenylhydrazono) isatins and structurally related 1H-indole-3-carbaldehyde derivatives, were synthesized and assayed as inhibitors of beta amyloid (Aβ) aggregation, a hallmark of pathophysiology of Alzheimer’s disease. The newly synthesized molecules spanned their IC50 values from sub- to two-digit micromolar range, bearing further information into structure-activity relationships. Some of the new compounds showed interesting multitarget activity, by inhibiting monoamine oxidases A and B. A cell-based assay in tau overexpressing bacterial cells disclosed a promising additional activity of some derivatives against tau aggregation. The accumulated data of either about ninety published and thirty-six newly synthesized molecules were used to generate a pharmacophore hypothesis of antiamyloidogenic activity exerted in a wide range of potencies, satisfactorily discriminating the ‘active’ compounds from the ‘inactive’ (poorly active) ones. An atom-based 3D-QSAR model was also derived for about 80% of ‘active’ compounds, i.e., those achieving finite IC50 values lower than 100 μM. The 3D-QSAR model (encompassing 4 PLS factors), featuring acceptable predictive statistics either in the training set (n = 45, q2 = 0.596) and in the external test set (n = 14, r2ext = 0.695), usefully complemented the pharmacophore model by identifying the physicochemical features mainly correlated with the Aβ anti-aggregating potency of the indole and isatin derivatives studied herein.

Study of the effect of ascorbic acid on Aβ42 peptide aggregation

AbstractBackgroundAscorbic acid (AA) is an antioxidant molecule found at high concentrations in the brain. A negative correlation was found in Alzheimer’s disease (AD) patients between the decrease in AA brain levels and the accumulation of amyloid plaques indicating that AA plays some function in amyloid aggregation, however, no studies have elucidated its mechanism of action so far, which can greatly contribute to the development of new therapeutic strategies for AD.MethodTo investigate AA as inhibitor of amyloid aggregation, amyloid‐beta 1‐42 (Aβ42) samples containing or not AA were incubated in pro‐aggregation conditions and analyzed by atomic force microscopy (AFM) and fluorescence spectroscopy using Thioflavin T. The mechanism of action presented by AA was evaluated using isothermal titration calorimetry (ITC) technique to investigate its binding to Aβ42 monomers.ResultFluorescence analyses pointed out to a decrease in fibril formation for samples incubated with AA at a concentration found in brains of healthy patients. After 72 h, these samples presented almost 80% fewer β‐sheet structures than samples without the antioxidant molecule. The fluorescence results were corroborated by AFM images, indicating that AA inhibited the amyloid aggregation in the fibrillar form. ITC measurements showed a negative cooperative binding between AA and Aβ42 monomers. In this interaction model for multiple sites, the binding of AA molecule in the first site of Aβ42 decreases the affinity constant (Kd) for the binding in the second site (Kd1>Kd2).ConclusionOur study revealed that ascorbic acid is able to inhibit the aggregation of Aβ42 peptides into fibrils through the direct binding to the monomers. Based on the thermodynamic parameters, we observed that the interaction is spontaneous and exothermic. This finding highlights ascorbic acid as a potential candidate for Alzheimer’s disease treatment and the mechanism of action provides novel insights for the inhibition of amyloid aggregation.

Thermodynamic and kinetic design principles for amyloid-aggregation inhibitors

Understanding the mechanism of action of compounds capable of inhibiting amyloid-fibril formation is critical to the development of potential therapeutics against protein-misfolding diseases. A fundamental challenge for progress is the range of possible target species and the disparate timescales involved, since the aggregating proteins are simultaneously the reactants, products, intermediates, and catalysts of the reaction. It is a complex problem, therefore, to choose the states of the aggregating proteins that should be bound by the compounds to achieve the most potent inhibition. We present here a comprehensive kinetic theory of amyloid-aggregation inhibition that reveals the fundamental thermodynamic and kinetic signatures characterizing effective inhibitors by identifying quantitative relationships between the aggregation and binding rate constants. These results provide general physical laws to guide the design and optimization of inhibitors of amyloid-fibril formation, revealing in particular the important role of on-rates in the binding of the inhibitors.

Novel fluorinated derivative of curcumin negatively regulates thioredoxin-interacting protein expression in retinal pigment epithelial and macrophage cells

Thioredoxin-interacting protein (TXNIP) has multiple disease-associated functions including inducing oxidative stress by inhibiting the anti-oxidant and thiol reducing activity of thioredoxin (TRX), reducing cellular glucose transport, and is a component of the activated inflammasome complex. Increased expression of TXNIP is encountered in diabetic conditions of high glucose. Curcumin and chemical derivatives have multiple therapeutic properties as anti-inflammatories, anti-oxidants, amyloid aggregation inhibitors and modulate a number of cellular signaling pathways. Using a fluorinated-derivative of curcumin (designated Shiga-Y6), we showed significant inhibition of TXNIP mRNA and protein expression, and induction of TRX mRNA and protein in ARPE-19 retinal pigment epithelial cells and THP-1-derived macrophages, while the non-fluorinated structural equivalent (Shiga-Y52) and native curcumin did not show these same effects. Shiga-Y6 was effective in reducing high glucose, endoplasmic reticulum stress-induced TXNIP in ARPE-19 cells, and reducing lipopolysaccharide and endoplasmic stress-induced proinflammatory gene expression in THP-1 macrophages. Moreover, TXNIP-knockdown experiments showed that the anti-inflammatory effect of Shiga-Y6 in LPS-stimulated THP-1 macrophages was TXNIP-independent.

Tryptophan-galactosylamine conjugates inhibit and disaggregate amyloid fibrils of A\u03b242 and hIAPP peptides while reducing their toxicity

Self-assembly of proteins into amyloid fibrils is a hallmark of various diseases, including Alzheimer’s disease (AD) and Type-2 diabetes Mellitus (T2DM). Aggregation of specific peptides, like Aβ42 in AD and hIAPP in T2DM, causes cellular dysfunction resulting in the respective pathology. While these amyloidogenic proteins lack sequence homology, they all contain aromatic amino acids in their hydrophobic core that play a major role in their self-assembly. Targeting these aromatic residues by small molecules may be an attractive approach for inhibiting amyloid aggregation. Here, various biochemical and biophysical techniques revealed that a panel of tryptophan-galactosylamine conjugates significantly inhibit fibril formation of Aβ42 and hIAPP, and disassemble their pre-formed fibrils in a dose-dependent manner. They are also not toxic to mammalian cells and can reduce the cytotoxicity induced by Aβ42 and hIAPP aggregates. These tryptophan-galactosylamine conjugates can therefore serve as a scaffold for the development of therapeutics towards AD and T2DM.

Nanochaperone-Based Strategies to Control Protein Aggregation Linked to Conformational Diseases.

The generation of highly organized amyloid fibrils is associated with a wide range of conformational pathologies, including primarily neurodegenerative diseases. Such disorders are characterized by misfolded proteins that lose their normal physiological roles and acquire toxicity. Recent findings suggest that proteostasis network impairment may be one of the causes leading to the accumulation and spread of amyloids. These observations are certainly contributing to a new focus in anti-amyloid drug design, whose efforts are so far being centered on single-target approaches aimed at inhibiting amyloid aggregation. Chaperones, known to maintain proteostasis, hence represent interesting targets for the development of novel therapeutics owing to their potential protective role against protein misfolding diseases. In this minireview, research on nanoparticles that can either emulate or help molecular chaperones in recognizing and/or correcting protein misfolding is discussed. The nascent concept of "nanochaperone" may indeed set future directions towards the development of cost-effective, disease-modifying drugs to treat several currently fatal disorders.

Nanochaperone‐Based Strategies to Control Protein Aggregation Linked to Conformational Diseases

AbstractThe generation of highly organized amyloid fibrils is associated with a wide range of conformational pathologies, including primarily neurodegenerative diseases. Such disorders are characterized by misfolded proteins that lose their normal physiological roles and acquire toxicity. Recent findings suggest that proteostasis network impairment may be one of the causes leading to the accumulation and spread of amyloids. These observations are certainly contributing to a new focus in anti‐amyloid drug design, whose efforts are so far being centered on single‐target approaches aimed at inhibiting amyloid aggregation. Chaperones, known to maintain proteostasis, hence represent interesting targets for the development of novel therapeutics owing to their potential protective role against protein misfolding diseases. In this minireview, research on nanoparticles that can either emulate or help molecular chaperones in recognizing and/or correcting protein misfolding is discussed. The nascent concept of “nanochaperone” may indeed set future directions towards the development of cost‐effective, disease‐modifying drugs to treat several currently fatal disorders.

New coumarin-benzotriazole based hybrid molecules as inhibitors of acetylcholinesterase and amyloid aggregation

A novel series of triazole tethered coumarin-benzotriazole hybrids based on donepezil skeleton has been designed and synthesized as multifunctional agents for the treatment of Alzheimer’s disease (AD). Among the synthesized compounds 13b showed most potent acetylcholinesterase (AChE) inhibition (IC50 = 0.059 μΜ) with mixed type inhibition scenario. Structure-activity relationship revealed that three-carbon alkyl chain connecting coumarin and triazole is well tolerable for inhibitory potential. Hybrids obtained from 4-hydroxycoumarin and 1-benzotriazole were most potent AChE inhibitors. The inhibitory potential of all compounds against butyrylcholinesterase was also evaluated but all showed negligible activity suggesting that the hybrid molecules are selective AChE inhibitors. 13b (most potent AChE inhibitor) also showed copper-induced Aβ1-42 aggregation inhibition (34.26% at 50 μΜ) and chelating properties for metal ions (Cu2+, Fe2+, and Zn2+) involved in AD pathogenesis along with DNA protective potential against degenerative actions of OH radicals. Molecular modelling studies confirm the potential of 13b in blocking both PAS and CAS of AChE. In addition, interactions of 13b with Aβ1-42 monomer are also streamlined. Therefore, hybrid 13b can act as an effective hit lead molecule for further development of selective AChE inhibitors as multifunctional anti-Alzheimer’s agents.

Catechins as Tools to Understand the Molecular Basis of Neurodegeneration.

Protein misfolding as well as the subsequent self-association and deposition of amyloid aggregates is implicated in the progression of several neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases. Modulators of amyloidogenic aggregation serve as essential tools to dissect the underlying molecular mechanisms and may offer insight on potential therapeutic solutions. These modulators include green tea catechins, which are potent inhibitors of amyloid aggregation. Although catechins often exhibit poor pharmacokinetic properties and bioavailability, they are still essential tools for identifying the drivers of amyloid aggregation and for developing other aggregation modulators through structural mimicry. As an illustration of such strategies, here we review how catechins have been used to map the toxic surfaces of oligomeric amyloid-like species and develop catechin-based phenolic compounds with enhanced anti-amyloid activity.

Preparation and study of the inhibitory effect of nano-niosomes containing essential oil from artemisia absinthium on amyloid fibril formation

Objective(s): Artemisia absinthium is an aromatic, perennial small shrub that shows multiple medical benefits, including anticancerous, neuroprotective, antifungal, hepatoprotective, antidepressant and antioxidant properties. One of the effective approaches to treat Alzheimer’s disease is targeting amyloid aggregation by antiamyloid drugs. In the current research study, an excellent grouping of niosomal, lipid nano-carriers drugs containing artemisia absinthium is advanced and characterized to inhibit amyloid aggregation.Materials and Methods: Niosomal vesicles were made employing phosphatidylcholine, span 60, cholesterol and DSPE-PEG2000 by the thin-film method. Then artemisia absinthium was loaded into the niosomes. Their physico-chemical attributes were analyzed utilizing Zeta-Sizer, FTIR, and SEM, and the amount of drug release was measured at 37° C. Finally, the inhibitory effect of artemisia absinthium that loaded niosomal vesicles on the aggregation of amyloid-β peptides was investigated using Thioflavin T fluorescence measurements and atomic force microscopy.Results: Niosomes containing artemisia absinthium have a size of 174±2.56nm, the encapsulation efficiency of 66.73%, zeta potential of -26.5±1/42 mV and polydispersity index (PDI) of 0.373±0/02. The release of the drug is controlled in this nano-carrier and FTIR and SEM investigations showed that the drug and nano-carrier did not interact and their particles had a spherical structure. In the end, the inhibitory effect of artemisia absinthium that loaded niosomal vesicles on the aggregation of amyloid-β peptides was examined and confirmed through Thioflavin T fluorescence measurements and atomic force microscopy.Conclusion: Meanwhile, the findings of the current study, confirm the appropriate physicochemical features of the system, a slow-release system, show that this nano-carrier inhibits amyloid aggregation, thus, the nano-niosomes containing essential oil from artemisia absinthium has the capability to preclude amyloid development.

Lead discovery of a guanidinyl tryptophan derivative on amyloid cascade inhibition

AbstractAmyloid cascade, one of pathogenic pathways of Alzheimer’s disease (AD), was focused as one of drug discovery targets. In this study, β-secretase (BACE1) inhibitors were designed aiming at the development of multifunctional compounds targeting amyloid pathogenic cascade. Tryptophan was used as a core structure due to its properties of the central nervous system (CNS) penetration and BACE1 inhibition activity. Three amino acid residues and guanidine were selected as linkers to connect the tryptophan core structure and the extended aromatic moieties. The distance between the aromatic systems of the core structure and the extended moieties was kept at the optimal length for amyloid-β (Aβ) peptide binding to inhibit its fibrillation and aggregation. Sixteen designed compounds were evaluated in silico. Eight hit compounds of TSR and TGN series containing serine and guanidine linkers, respectively, were identified and synthesized based on docking results. TSR2 and TGN2 were found to exert strong actions as BACE1 (IC50 24.18 µM and 22.35 µM) and amyloid aggregation inhibitors (IC50 37.06 µM and 36.12 µM). Only TGN2 demonstrated a neuroprotective effect in SH-SY5Y cells by significantly reducing Aβ-induced cell death at a concentration of 2.62 µM. These results support the validity of multifunctional approaches to inhibition of the β-amyloid cascade.

Amyloid-β oligomers are captured by the DNAJB6 chaperone: Direct detection of interactions that can prevent primary nucleation

A human molecular chaperone protein, DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6), efficiently inhibits amyloid aggregation. This inhibition depends on a unique motif with conserved serine and threonine (S/T) residues that have a high capacity for hydrogen bonding. Global analysis of kinetics data has previously shown that DNAJB6 especially inhibits the primary nucleation pathways. These observations indicated that DNAJB6 achieves this remarkably effective and sub-stoichiometric inhibition by interacting not with the monomeric unfolded conformations of the amyloid-β symbol (Aβ) peptide but with aggregated species. However, these pre-nucleation oligomeric aggregates are transient and difficult to study experimentally. Here, we employed a native MS-based approach to directly detect oligomeric forms of Aβ formed in solution. We found that WT DNAJB6 considerably reduces the signals from the various forms of Aβ (1–40) oligomers, whereas a mutational DNAJB6 variant in which the S/T residues have been substituted with alanines does not. We also detected signals that appeared to represent DNAJB6 dimers and trimers to which varying amounts of Aβ are bound. These data provide direct experimental evidence that it is the oligomeric forms of Aβ that are captured by DNAJB6 in a manner which depends on the S/T residues. We conclude that, in agreement with the previously observed decrease in primary nucleation rate, strong binding of Aβ oligomers to DNAJB6 inhibits the formation of amyloid nuclei.

A Spectroscopic Marker for Structural Transitions Associated with Amyloid-β Aggregation.

An amyloid aggregate evolves through a series of intermediates that have different secondary structures and intra- and intermolecular contacts. The structural parameters of these intermediates are important determinants of their toxicity. For example, the early oligomeric species of the amyloid-β (Aβ) peptide have been implicated as the most cytotoxic species in Alzheimer's disease but are difficult to identify because of their dynamic and transitory nature. Conventional aggregation monitors such as the fluorescent dye thioflavin T report on only the overall transition of the soluble species to the final amyloid fibrillar aggregated state. Here, we show that the fluorescent dye bis(triphenylphosphonium) tetraphenylethene (TPE-TPP) identifies at least three distinct aggregation intermediates of Aβ. Some atomic-level features of these intermediates are known from solid state nuclear magnetic resonance spectroscopy. Hence, the TPE-TPP fluorescence data may be interpreted in terms of these Aβ structural transitions. Steady state fluorescence and lifetime characteristics of TPE-TPP distinguish between the small oligomeric species (emission wavelength maximum, λmax = 465 nm; average fluorescence lifetime, τFl measured at 420 nm = 3.58 ± 0.04 ns), the intermediate species (λmax = 452 nm; τFl = 3.00 ± 0.03 ns), and the fibrils (λmax = 406 nm; τFl = 5.19 ± 0.08 ns). Thus, TPE-TPP provides a ready diagnostic for differentiating between the various, including the toxic, Aβ aggregates and potentially can be utilized to screen for amyloid aggregation inhibitors.

Nontoxic Black Phosphorus Quantum Dots Inhibit Insulin Amyloid Fibrillation at an Ultralow Concentration.

SummaryAmyloid are protein aggregates formed by cross β structures assemblies. Inhibiting amyloid aggregation or facilitating its disassembly are considered to be two major effective therapeutic strategies in diseases involving peptide or protein fibrillation such Alzheimer's disease or diabetes. Using thioflavin-T fluorescence, far-UV circular dichroism spectroscopy, and atomic force microscopy, we found nontoxic and biocompatible black phosphorus quantum dots (BPQDs) appear to have an exceptional capacity to inhibit insulin aggregation and to disassemble formed mature fibrils, even at an ultralow concentration (100 ng/mL). The inhibition of fibrillation persists at all stages of insulin aggregation and increases PC12 cells survival when exposed to amyloid fibrils. Molecular dynamics simulations suggest that BPQDs are able to stabilize the α-helix structure of insulin and obliterate the β-sheet structure to promote the fibril formation. These characteristics make BPQDs be promising candidate in preventing amyloidosis, disease treatment, as well as in the storage and processing of insulin.

Maintenance of Amyloid-beta Homeostasis by Carbenoxolone Post Aβ-42 Oligomer Injection in Rat Brain

The equilibrium between cerebral production and clearance of Aβ is maintained either by the active removal through blood–brain barrier or by the uptake and degradation through ubiquitin–proteasome system (UPS) and autophagy. The dysfunction of UPS and dysregulation of molecular chaperones such as heat shock proteins (HSPs) is well correlated with the progression of Alzheimer’s disease (AD). Therefore, the restoration of heat shock system and UPS appears to be an effective approach to maintain the Aβ homeostasis. The alteration in the Aβ homeostasis was induced by a single bilateral intrahippocampal injection of Aβ 42 oligomers (10 μl/rat) into the dorsal hippocampus of the rat brain. Cbx (carbenoxolone), a HSP inducer and Aβ 42 aggregation inhibitor, was tested (20 mg/kg body weight, i.p. for six-weeks) against the altered Aβ homeostasis in the rat brain. Aβ 42 oligomers downregulated the expression of HSPs and co-treatment with Cbx prevented this decline. Cbx was able to restore the UPS function post Aβ 42 oligomer injection. Aβ 42 oligomers induced upregulation of the expression levels of APP, BACE-1 and Tau was also normalized after the co-treatment with Cbx. A significant decrease in the thioflavin-T and Aβ positive deposits in different regions of the rat brain was observed after Cbx co-treatment. Thus, the present study projects Cbx as a potential candidate for the maintenance of Aβ homeostasis through inhibition of amyloid aggregation and restoration of the functioning of molecular chaperones and UPS system in the progression of AD.

Alzheimer's drug candidate, SAK3 enhances the proteasome activity and inhibits amyloid aggregation in Alzheimer‘s disease brain

Alzheimer's drug candidate, SAK3 enhances the proteasome activity and inhibits amyloid aggregation in Alzheimer‘s disease brain

Interrogating Amyloid Aggregates using Fluorescent Probes.

Amyloids are a broad class of proteins and peptides that can misfold and assemble into long unbranched fibrils with a cross-β conformation. These misfolding and aggregation events are associated with the onset of a variety of human diseases, among them, Alzheimer's disease, Parkinson's disease, and Huntington disease. Our understanding of amyloids has been greatly supported by fluorescent molecular probes, such as thioflavin-T, which shows an increase in fluorescence emission upon binding to fibrillar aggregates. Since the first application of thioflavin-T in amyloid studies nearly 30 years ago, many probes have emerged exhibiting a variety of responses to amyloids, such as intensity changes, shifts in fluorescence maxima, and variations in lifetimes, among many others. These probes have shed light on a variety of topics including the kinetics of amyloid aggregation, the effectiveness of amyloid aggregation inhibitors, the elucidation of binding sites in amyloid structures, and the staining of amyloids aggregates in vitro, ex vivo, and in vivo. In this Review, we discuss the design, properties, and application of photoactive probes used to study amyloid aggregation, as well as the challenges faced by current probes and techniques, and the novel approaches that are emerging to address these challenges.

Inhibitory activities of kukoamines A and B from Lycii Cortex on amyloid aggregation related to Alzheimer's disease and type 2 diabetes.

Alzheimer's diseases (AD) and type2 diabetes (T2D) are two age-related diseases characterized by amyloid fibrillogenesis. Prevention of amyloid aggregation is a promising therapeutic strategy for AD and T2D. Two spermine alkaloids, kukoaminesA and B, isolated from Lycii Cortex (LyC) were investigated for their inhibitory effect on amyloid aggregation. Both kukoaminesA and B inhibited aggregation of amyloidβ (Aβ) and human islet amyloid polypeptide (hIAPP) in a dose-dependent manner. KukoamineB showed stronger inhibitory activity than kukoamineA. These results on the inhibitory activity of kukoaminesA and B on Aβ and hIAPP indicate that the catechol moiety is essential for inhibition of amyloid aggregation.

Inhibition of Amyloid Fibrillation by Small Molecules and Nanomaterials: Strategic Development of Pharmaceuticals Against Amyloidosis.

Amyloid fibrils are a special class of self-assembled protein molecules, which exhibit various toxic effects in cells. Different physiological disorders such as Alzheimer's, Parkinson's, Huntington's diseases, etc. happen due to amyloid formation and lack of proper cellular mechanism for the removal of fibrils. Therefore, inhibition of amyloid fibrillation will find immense applications to combat the diseases associated with amyloidosis. The development of therapeutics against amyloidosis is definitely challenging and numerous strategies have been followed to find out anti-amyloidogenic molecules. Inhibition of amyloid aggregation of proteins can be achieved either by stabilizing the native conformation or by decreasing the chances of assembly formation by the unfolded/misfolded structures. Various small molecules such as naturally occurring polyphenols, flavonoids, small organic molecules, surfactants, dyes, chaperones, etc. have demonstrated their capability to interrupt the amyloid fibrillation of proteins. In addition to that, in last few years, different nanomaterials were evolved as effective therapeutic inhibitors against amyloidosis. Aromatic and hydrophobic interactions between the partially unfolded protein molecules and the inhibitors had been pointed as a general mechanism for inhibition. In this review article, we are presenting an overview on the inhibition of amyloidosis by using different small molecules (both natural and synthetic origin) as well as nanomaterials for development of pharmaceutical strategies against amyloid diseases.

Disassembly of Alzheimer’s amyloid fibrils by functional upconversion nanoparticles under near-infrared light irradiation

Alzheimer's disease (AD) is a common neurodegenerative disease characterized by cognitive and memory function impairment. Studies have shown that the overproduction and further misfolding of amyloid polypeptide (Aβ) are the main causes of AD. Therefore, how to reduce Aβ species, especially to disassemble the preformed amyloid aggregates, has become the focus of related research. Photodynamic therapy (PDT) using normal photosensitizers eg. porphyrins, is a traditional way of inhibiting amyloid aggregation or degrading the amyloid aggregates, but UV light irradiation presents a side effect for the damage of normal tissue, which limits its medical application. Upconversion nanoparticles (UCNPs) is a luminous material which can be modified by photodynamic agent to form the complex generating reactive oxygen species (ROS) with noninvasive light irradiation. It presents a good advantage in the disassembly of amyloid aggregates via ROS in noninvasive light irradiation. Herein, we prepared β-NaYF4:Yb/Er@SiO2@RB by combing UCNPs with photosensitizer to disassemble preformed Aβ aggregates under near-infrared light. The results displayed upconversion nanomaterials could degrade Aβ1–42 fibrils effectively by photo-generating ROS, which demonstrated that the functional UCNPs may have potential applications in the treatment of amyloid diseases in future.