Attenuation of the Aggregation and Neurotoxicity of Amyloid‐β Peptides by Catalytic Photooxygenation

Alzheimer’s disease (AD), a progressive severe neurodegenerative disorder, is currently incurable, despite intensive efforts worldwide. Herein, we demonstrate that catalytic oxygenation of amyloid‐β peptides (Aβ) might be an effective approach to treat AD. Aβ1–42 was oxygenated under physiologically‐relevant conditions (pH 7.4, 37 °C) using a riboflavin catalyst and visible light irradiation, with modifications at the Tyr¹⁰, His¹³, His¹⁴, and Met³⁵ residues. The oxygenated Aβ1–42 exhibited considerably lower aggregation potency and neurotoxicity compared with native Aβ. Photooxygenation of Aβ can be performed even in the presence of cells, by using a selective flavin catalyst attached to an Aβ‐binding peptide; the Aβ cytotoxicity was attenuated in this case as well. Furthermore, oxygenated Aβ1–42 inhibited the aggregation and cytotoxicity of native Aβ.     

A Hexameric Peptide Barrel as Building Block of Amyloid‐β Protofibrils

Oligomeric and protofibrillar aggregates formed by the amyloid‐β peptide (Aβ) are believed to be involved in the pathology of Alzheimer’s disease. Central to Alzheimer pathology is also the fact that the longer Aβ₄₂ peptide is more prone to aggregation than the more prevalent Aβ₄₀. Detailed structural studies of Aβ oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aβ that forms stable protofibrils and here we use solid‐state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aβ protomers into hexameric barrel‐like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C‐terminal hydrophobic regions of Aβ, and hairpin loops extend from the core. The model accounts for why Aβ₄₂ forms oligomers and protofibrils more easily than Aβ₄₀.     

A Method for Evaluating the Level of Soluble β‐Amyloid(1–40/1–42) in Alzheimer’s Disease Based on the Binding of Gelsolin to β‐Amyloid Peptides

In the present work, a new electrochemical strategy for the sensitive and specific detection of soluble β‐amyloid Aβ_{(1–40/1–42)} peptides in a rat model of Alzheimer’s disease (AD) is described. In contrast to previous antibody‐based methods, β‐amyloid_{(1–40/1–42)} was quantified based on its binding to gelsolin, a secretory protein present in the cerebrospinal fluid (CSF) and plasma. The level of soluble β‐amyloid peptides in the CSF and various brain regions were found with this method to be lower in rats with AD than in normal rats.     

Modification of a Small β‐Barrel Protein,To Give Pseudo‐Amyloid Structures,Inhibits Amyloid β‐Peptide Aggregation

Aggregation of amyloid β‐peptide (Aβ) is closely related to the pathogenesis of Alzheimer’s disease (AD). Although much effort has been devoted to the construction of molecules that inhibit the aggregation of Aβ1‐42, high doses are needed for the inhibition of Aβ aggregation in many cases. Previously, we reported that designed green fluorescent protein (GFP) analogues that gives pseudo‐Aβ β‐sheet structures can work as an aggregation inhibitor against Aβ. To further test this design strategy, we constructed protein analogues that mimic Aβ β‐sheet structures of amyloids by using insulin‐like growth factor 2 receptor domain 11 (IGF2R‐d11) as a scaffold. A designed protein, named IG11KK, which has a parallel configuration of Aβ‐like β sheets, can bind more preferentially to oligomeric Aβ1‐42 than the monomer. Moreover, IG11KK suppressed the aggregation of Aβ1‐42 efficiently, even though lower concentrations of IG11KK than Aβ were used. The aggregation kinetics of Aβ in the presence of the designed proteins revealed that IG11KK can work as an inhibitor not only for the early to middle stages, but also in the latter stage of Aβ aggregation owing to its favorable binding to oligomeric structures of Aβ. The design strategy using β‐barrel proteins such as IGF2R‐d11 and GFP is useful in generating excellent inhibitors of protein misfolding and amyloid formation.     

Multi‐Frequency,Multi‐Technique Pulsed EPR Investigation of the Copper Binding Site of Murine Amyloid β Peptide

Copper‐amyloid peptides are proposed to be the cause of Alzheimer’s disease, presumably by oxidative stress. However, mice do not produce amyloid plaques and thus do not suffer from Alzheimer’s disease. Although much effort has been focused on the structural characterization of the copper‐ human amyloid peptides, little is known regarding the copper‐binding mode in murine amyloid peptides. Thus, we investigated the structure of copper‐murine amyloid peptides through multi‐frequency, multi‐technique pulsed EPR spectroscopy in conjunction with specific isotope labeling. Based on our pulsed EPR results, we found that Ala2, Glu3, His6, and His14 are directly coordinated with the copper ion in murine amyloid β peptides at pH 8.5. This is the first detailed structural characterization of the copper‐binding mode in murine amyloid β peptides. This work may advance the knowledge required for developing inhibitors of Alzheimer’s disease.     

Gallotannins and Tannic Acid: First Chemical Syntheses and In Vitro Inhibitory Activity on Alzheimer’s Amyloid β‐Peptide Aggregation

The screening of natural products in the search for new lead compounds against Alzheimer’s disease has unveiled several plant polyphenols that are capable of inhibiting the formation of toxic β‐amyloid fibrils. Gallic acid based gallotannins are among these polyphenols, but their antifibrillogenic activity has thus far been examined using “tannic acid”, a commercial mixture of gallotannins and other galloylated glucopyranoses. The first total syntheses of two true gallotannins, a hexagalloylglucopyranose and a decagalloylated compound whose structure is commonly used to depict “tannic acid”, are now described. These depsidic gallotannins and simpler galloylated glucose derivatives all inhibit amyloid β‐peptide (Aβ) aggregation in vitro, and monogalloylated α‐glucogallin and a natural β‐hexagalloylglucose are shown to be the strongest inhibitors.     

Randomization of Amyloid‐β‐Peptide(1‐42) Conformation by Sulfonated and Sulfated Nanoparticles Reduces Aggregation and Cytotoxicity

The amyloid‐β peptide (Aβ) plays a central role in the mechanism of Alzheimer's disease, being the main constituent of the plaque deposits found in AD brains. Aβ amyloid formation and deposition are due to a conformational switching to a β‐enriched secondary structure. Our strategy to inhibit Aβ aggregation involves the re‐conversion of Aβ conformation by adsorption to nanoparticles. NPs were synthesized by sulfonation and sulfation of polystyrene, leading to microgels and latexes. Both polymeric nanostructures affect the conformation of Aβ inducing an unordered state. Oligomerization was delayed and cytotoxicity reduced. The proper balance between hydrophilic moieties and hydrophobic chains seems to be an essential feature of effective NPs.

     

Rational Design and Identification of a Non‐Peptidic Aggregation Inhibitor of Amyloid‐β Based on a Pharmacophore Motif Obtained from cyclo[‐Lys‐Leu‐Val‐Phe‐Phe‐]

Inhibition of pathogenic protein aggregation may be an important and straightforward therapeutic strategy for curing amyloid diseases. Small‐molecule aggregation inhibitors of Alzheimer’s amyloid‐β (Aβ) are extremely scarce, however, and are mainly restricted to dye‐ and polyphenol‐type compounds that lack drug‐likeness. Based on the structure‐activity relationship of cyclic Aβ16–20 (cyclo‐[KLVFF]), we identified unique pharmacophore motifs comprising side‐chains of Leu², Val³, Phe⁴, and Phe⁵ residues without involvement of the backbone amide bonds to inhibit Aβ aggregation. This finding allowed us to design non‐peptidic, small‐molecule aggregation inhibitors that possess potent activity. These molecules are the first successful non‐peptidic, small‐molecule aggregation inhibitors of amyloids based on rational molecular design.     

Molecular Simulations of Human and Mouse Aβ1–16 at Different pH Values: Structural Characteristics toward Understanding Cu2+‐Coordinated Amyloid Beta Spheres

As the main sequence responsible for metal ion coordination in the amyloid beta (Aβ) peptide, Aβ_1–16 plays a key role in the understanding of the aggregation of Aβ induced by Cu²⁺ ions. There is no consensus on the nature of the coordination sphere of the Cu²⁺–Aβ complex so far due to the amorphous conformation of the Aβ_1–16 peptide itself and the pH dependence of Cu²⁺–Aβ coordination. The simulation reported here reveals that human Aβ_1–16 monomer has a U‐shape morphology, which is preserved at any pH. This morphology accommodates Cu²⁺ ions with several binding sites and is also the basis for establishing a center‐distance statistical method (CDSM). Based on this CDSM, specific histidine residues for a Cu²⁺‐coordinated sphere are identified and the corresponding accurate pH range is established, indicating that the CDSM can be used as a reference to predict the potential coordination sites of metal ions in other amorphous peptides. By contrast, mouse Aβ_1–16 monomer has a more open and random morphology than human Aβ_1–16 due to the differences of three sequence positions. These mutations not only reduce the number of binding sites required by a stable Cu²⁺‐binding sphere but also diminish the capacity to generate salt bridges compared to the human peptide. These observations offer insights into the roles of three residues that differ in the mouse Aβ_1–16 and perhaps into the reasons mice seldom develop Alzheimer's disease.     

Natural Compounds against Alzheimer’s Disease: Molecular Recognition of Aβ1–42 Peptide by Salvia sclareoides Extract and its Major Component,Rosmarinic Acid,as Investigated by NMR

Amyloid peptides, Aβ1–40 and Aβ1–42, represent major molecular targets to develop potential drugs and diagnostic tools for Alzheimer’s Disease (AD). In fact, oligomeric and fibrillar aggregates generated by these peptides are amongst the principal components of amyloid plaques found post mortem in patients suffering from AD. Rosmarinic acid has been demonstrated to be effective in preventing the aggregation of amyloid peptides in vitro and to delay the progression of the disease in animal models. Nevertheless, no information is available about its molecular mechanism of action. Herein, we report the NMR characterization of the interaction of Salvia sclareoides extract and that of its major component, rosmarinic acid, with Aβ1–42 peptide, whose oligomers have been described as the most toxic Aβ species in vivo. Our data shed light on the structural determinants of rosmarinic acid–Aβ1–42 oligomers interaction, thus allowing the elucidation of its mechanism of action. They also provide important information for the rational design of new compounds with higher affinity for Aβ peptides to generate new anti‐amyloidogenic molecules and/or molecular tools for the specific targeting of amyloid aggregates in vivo. In addition, we identified methyl caffeate, another natural compound present in different plants and human diet, as a good ligand of Aβ1–42 oligomers, which also shows anti‐amyloidogenic activity. Finally, we demonstrated the possibility to exploit STD‐NMR and trNOESY experiments to screen extracts from natural sources for the presence of Aβ peptide ligands.     

Natural Compounds against Neurodegenerative Diseases: Molecular Characterization of the Interaction of Catechins from Green Tea with Aβ1–42, PrP106–126, and Ataxin‐3 Oligomers

By combining NMR spectroscopy, transmission electron microscopy, and circular dichroism we have identified the structural determinants involved in the interaction of green tea catechins with Aβ1–42, PrP106–126, and ataxin‐3 oligomers. The data allow the elucidation of their mechanism of action, showing that the flavan‐3‐ol unit of catechins is essential for interaction. At the same time, the gallate moiety, when present, seems to increase the affinity for the target proteins. These results provide important information for the rational design of new compounds with anti‐amyloidogenic activity and/or molecular tools for the specific targeting of amyloid aggregates in vivo.     

Solid‐phase Total Synthesis of (−)‐Apratoxin A and Its Analogues and Their Biological Evaluation

Two approaches for the solid‐phase total synthesis of apratoxin A and its derivatives were accomplished. In synthetic route A, the peptide was prepared by the sequential coupling of the corresponding amino acids on trityl chloride SynPhase Lanterns. After cleavage from the polymer‐support, macrolactamization of 10 , followed by thiazoline formation, provided apratoxin A. This approach, however, resulted in low yield because the chemoselectivity was not sufficient for the formation of the thiazoline ring though its analogue 33 was obtained. However, in synthetic route B, a cyclization precursor was prepared by solid‐phase peptide synthesis by using amino acids 13 – 15 and 18 . The final macrolactamization was performed in solution to provide apratoxin A in high overall yield. This method was then successfully applied to the synthesis of apratoxin analogues. The cytotoxic activity of the synthetic derivatives was then evaluated. The epimer 34 was as potent as apratoxin A, and O‐methyl tyrosine can be replaced by 7‐azidoheptyl tyrosine without loss of activity. The 1,3‐dipolar cycloaddition of 38 with phenylacetylene was performed in the presence of a copper catalyst without affecting the thiazoline ring.     

Spectroscopic Evidence for Polymorphic Aggregates Formed by Amyloid‐β Fragments

Understanding the structure of amyloid‐β (Aβ) aggregates is a key step towards elucidating the pathology of Alzheimer’s disease. In this work, three fragments of the Aβ_1–42 protein, Aβ_1–25 (DAEFRHDSGYEVHHQKLVFFAEDVG), Aβ_25–35 (GSNKGAIIGLM), and Aβ_33–42 (GLMVGGVVIA), were synthesized, and their aggregated structures were examined by linear infrared spectroscopy in the amide‐I (mainly the C?O stretching) region. The structures of the formed aggregates were found to be both sequence and pH dependent. The results suggest that instead of forming matured fibrils, as in the case of full‐length Aβ_1–42, both Aβ_1–25 and Aβ_33–42 form a mixture of threadlike β‐sheet fibril, soluble β‐sheet oligomer, and random coil structures. The β‐sheet conformations were found to be mainly antiparallel for the former and both parallel and antiparallel for the latter. However, the Aβ_25–35 fragment was found to form assembled fibrils containing predominantly parallel β‐sheets. The conformation and morphology of the aggregates were also confirmed by circular dichroism measurements and transmission electron microscopy. Factors influencing the structures of the aggregates formed by the Aβ fragments were discussed.     

Real‐time Amyloid Aggregation Monitoring with a Photonic Crystal‐based Approach

We propose the application of a new label‐free optical technique based on photonic nanostructures to real‐time monitor the amyloid‐beta 1‐42 (Aβ(1‐42)) fibrillization, including the early stages of the aggregation process, which are related to the onset of the Alzheimer’s Disease (AD). The aggregation of Aβ peptides into amyloid fibrils has commonly been associated with neuronal death, which culminates in the clinical features of the incurable degenerative AD. Recent studies revealed that cell toxicity is determined by the formation of soluble oligomeric forms of Aβ peptides in the early stages of aggregation. At this phase, classical amyloid detection techniques lack in sensitivity. Upon a chemical passivation of the sensing surface by means of polyethylene glycol, the proposed approach allows an accurate, real‐time monitoring of the refractive index variation of the solution, wherein Aβ(1‐42) peptides are aggregating. This measurement is directly related to the aggregation state of the peptide throughout oligomerization and subsequent fibrillization. Our findings open new perspectives in the understanding of the dynamics of amyloid formation, and validate this approach as a new and powerful method to screen aggregation at early stages.