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.     

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.     

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.     

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.     

The Molecular Structure of Alzheimer β‐Amyloid Fibrils Formed in the Presence of Phospholipid Vesicles

β‐amyloid (Aβ) fibrils are the major species involved in Alzheimer’s disease (AD). An atomic‐resolution molecular structure of Aβ40 fibrils formed in the presence of lipid vesicles was obtained by using magic angle spinning (MAS) solid‐state NMR spectroscopy. The fibril structures formed in the presence of the lipid vesicles are remarkably different from those formed in solution. These results provide insights into the molecular mechanism of Aβ aggregation in the presence of lipid vesicles.     

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.     

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.     

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.     

Distinct Spacing Between Anionic Groups: An Essential Chemical Determinant for Achieving Thiophene‐Based Ligands to Distinguish β‐Amyloid or Tau Polymorphic Aggregates

The accumulation of protein aggregates is associated with many devastating neurodegenerative diseases and the existence of distinct aggregated morphotypes has been suggested to explain the heterogeneous phenotype reported for these diseases. Thus, the development of molecular probes able to distinguish such morphotypes is essential. We report an anionic tetrameric oligothiophene compound that can be utilized for spectral assignment of different morphotypes of β‐amyloid or tau aggregates present in transgenic mice at distinct ages. The ability of the ligand to spectrally distinguish between the aggregated morphotypes was reduced when the spacing between the anionic substituents along the conjugated thiophene backbone was altered, which verified that specific molecular interactions between the ligand and the protein aggregate are necessary to detect aggregate polymorphism. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between different morphotypes of protein aggregates.     

Maintenance of Amyloid β Peptide Homeostasis by Artificial Chaperones Based on Mixed‐Shell Polymeric Micelles

The disruption of Aβ homeostasis, which results in the accumulation of neurotoxic amyloids, is the fundamental cause of Alzheimer’s disease (AD). Molecular chaperones play a critical role in controlling undesired protein misfolding and maintaining intricate proteostasis in vivo. Inspired by a natural molecular chaperone, an artificial chaperone consisting of mixed‐shell polymeric micelles (MSPMs) has been devised with tunable surface properties, serving as a suppressor of AD. Taking advantage of biocompatibility, selectivity toward aberrant proteins, and long blood circulation, these MSPM‐based chaperones can maintain Aβ homeostasis by a combination of inhibiting Aβ fibrillation and facilitating Aβ aggregate clearance and simultaneously reducing Aβ‐mediated neurotoxicity. The balance of hydrophilic/hydrophobic moieties on the surface of MSPMs is important for their enhanced therapeutic effect.     

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.     

Quantitative Aspects of Electrochemical Detection of Amyloid‐β Aggregation

The tyrosine based electrochemical analysis of synthetic amyloid‐β (Aβ) peptide – an analog of natural peptide implicated in Alzheimer's disease pathogenesis – was applied for a quantitative estimation of peptide aggregation in vitro. The analysis was carried out by square wave voltammetry (SWV) on carbon screen printed electrodes (SPE). The electrooxidation peak current (I_p) for Aβ42 peptide in different aggregation states was directly compared with the size and structure of Aβ42 aggregates occurring in the analyzed sample. Dynamic light scattering (DLS) and thioflavin T (ThT) based fluorescence assay were employed to estimate the size and structure of Aβ42 aggregates. The I_p was found to decrease in a linear fashion when the average diameter of aggregates and the relative ThT fluorescence in Aβ42 solutions exceeded 35 nm and 3, respectively, while being nearly constant below these values. It was suggested that the electrooxidation current is mostly generated by peptide monomers and that a depletion of the monomer pool due to inclusion of Aβ42 molecules in aggregates is responsible for the decrease of electrooxidation current. The direct electrochemistry is emerging as a method complementary to methods based on aggregates’ detection and commonly employed for monitoring Aβ aggregation. The work further enlarges the basis for application of the cost‐effective and rapid electrochemical techniques, such as SWV on carbon SPE, to in vitro studies of Aβ aggregation.     

Synthesis of a Targeted Library of Heparan Sulfate Hexa‐ to Dodecasaccharides as Inhibitors of β‐Secretase: Potential Therapeutics for Alzheimer’s Disease

Heparan sulfates (HS) are a class of sulfated polysaccharides that function as dynamic biological regulators of the functions of diverse proteins. The structural basis of these interactions, however, remains elusive, and chemical synthesis of defined structures represents a challenging but powerful approach for unravelling the structure–activity relationships of their complex sulfation patterns. HS has been shown to function as an inhibitor of the β‐site cleaving enzyme β‐secretase (BACE1), a protease responsible for generating the toxic Aβ peptides that accumulate in Alzheimer’s disease (AD), with 6‐O‐sulfation identified as a key requirement. Here, we demonstrate a novel generic synthetic approach to HS oligosaccharides applied to production of a library of 16 hexa‐ to dodecasaccharides targeted at BACE1 inhibition. Screening of this library provided new insights into structure–activity relationships for optimal BACE1 inhibition, and yielded a number of potent non‐anticoagulant BACE1 inhibitors with potential for development as leads for treatment of AD through lowering of Aβ peptide levels.