The Necessity of Having a Tetradentate Ligand to Extract Copper(II) Ions from Amyloids

The accumulation of redox-active metal ions, in particular copper, in amyloid plaques is considered to the cause of the intensive oxidation damage to the brain of patients with Alzheimers disease (AD). Drug candidates based on a bis(8-aminoquinoline) tetradentate ligand are able to efficiently extract Cu²⁺ from copper-loaded amyloids (Cu–Aβ). Contrarily, in the presence of a bidentate hydroxyquinoline, such as clioquinol, the copper is not released from Aβ, but remains sequestrated within a Aβ–Cu–clioquinol ternary complex that has been characterized by mass spectrometry. Facile extraction of copper(II) at a low amyloid/ligand ratio is essential for the re-introduction of copper in regular metal circulation in the brain. As, upon reduction, the Cu⁺ is easily released from the bis(8-aminoquinoline) ligand unable to accommodate Cu^I, it should be taken by proteins with an affinity for copper. So, the tetradentate bis(8-aminoquinoline) described here might act as a regulator of copper homeostasis.     

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.     

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 Single Biosensor for Evaluating the Levels of Copper Ion and L‐Cysteine in a Live Rat Brain with Alzheimer's Disease

Copper ion (Cu²⁺) and L ‐cysteine (CySH) are closely correlated with physiological and pathological events of Alzheimer’s Disease (AD), however the detailed mechanism is still unclear, mainly owing to a lack of accurate analytical methods in live brains. Herein, we report a single biosensor for electrochemical ratiometric detection of Cu²⁺ and CySH in live rat brains with AD. N,N‐di‐(2‐picoly)ethylenediamine (DPEA) is first synthesized for specific recognition of Cu²⁺ to form a DPEA–Cu²⁺ complex. This complex shows high selectivity for CySH owing to the release of Cu²⁺ from the complex through CySH binding to Cu²⁺ center. In parallel, 5′‐MB‐GGCGCGATTTTTTTTTTTTT‐SH‐3′ (HS‐DNA‐MB, MB=Methylene Blue) is designed as an inner‐reference for providing a built‐in correction to improve the accuracy. As a result, combined with the amplified effect of Au nanoleaves, our single ratiometric biosensor can be successfully applied in real‐time detection of Cu²⁺ and CySH in the live rat brains with AD. To our knowledge, this is the first report on the accurate concentrations of Cu²⁺ and CySH in live rat brains with AD.     

Copper‐Catalyzed One‐Pot Trifluoromethylation/Aryl Migration/Carbonyl Formation with Homopropargylic Alcohols

A novel copper‐catalyzed one‐pot functionalization of homopropargylic alcohols that involves trifluoromethylation, aryl migration, and formation of a carbonyl moiety has been developed. This reaction constitutes the first direct conversion of homopropargylic alcohols into CF₃‐containing 3‐butenal or 3‐buten‐1‐one derivatives in a regioselective manner. Mechanistic studies indicate that the 1,4‐aryl migration proceeds through a radical pathway.     

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.     

Enzyme‐Mediated Site‐Specific Bioconjugation of Metal Complexes to Proteins: Sortase‐Mediated Coupling of Copper‐64 to a Single‐Chain Antibody

The enzyme‐mediated site‐specific bioconjugation of a radioactive metal complex to a single‐chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single‐chain variable fragment. The antibody fragment scFv_anti‐LIBS targets ligand‐induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron‐emitting isotope ⁶⁴Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site‐specific enzyme‐mediated conjugation and should be broadly applicable to other metal complexes and proteins.     

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.     

Cyclodextrins 3‐Functionalized with 8‐Hydroxyquinolines: Copper‐Binding Ability and Inhibition of Synuclein Aggregation

Neurodegenerative diseases such as Parkinson's and Alzheimer's diseases are multifactorial disorders related to protein aggregation, metal dyshomeostasis, and oxidative stress. To advance understanding in this area and to contribute to therapeutic development, many efforts have been directed at devising suitable agents that can target metal ions associated with relevant biomolecules such as α‐synuclein. This paper presents a new cyclodextrin–8‐hydroxyquinoline conjugate and discusses the properties of four cyclodextrins 3‐functionalized with 8‐hydroxyquinoline as copper(II) chelators and inhibitors of copper‐induced synuclein aggregation. The encouraging results establish the potential of cyclodextrin–8‐hydroxyquinoline conjugates as chelators for the control of copper toxicity.     

Free Superoxide is an Intermediate in the Production of H2O2 by Copper(I)‐Aβ Peptide and O2

Oxidative stress is considered as an important factor and an early event in the etiology of Alzheimer's disease (AD). Cu bound to the peptide amyloid‐β (Aβ) is found in AD brains, and Cu‐Aβ could contribute to this oxidative stress, as it is able to produce in vitro H₂O₂ and HO^. in the presence of oxygen and biological reducing agents such as ascorbate. The mechanism of Cu‐Aβ‐catalyzed H₂O₂ production is however not known, although it was proposed that H₂O₂ is directly formed from O₂ via a 2‐electron process. Here, we implement an electrochemical setup and use the specificity of superoxide dismutase‐1 (SOD1) to show, for the first time, that H₂O₂ production by Cu‐Aβ in the presence of ascorbate occurs mainly via a free O₂^.? intermediate. This finding radically changes the view on the catalytic mechanism of H₂O₂ production by Cu‐Aβ, and opens the possibility that Cu‐Aβ‐catalyzed O₂^.? contributes to oxidative stress in AD, and hence may be of interest.     

A Hot‐Segment‐Based Approach for the Design of Cross‐Amyloid Interaction Surface Mimics as Inhibitors of Amyloid Self‐Assembly

The design of inhibitors of protein–protein interactions mediating amyloid self‐assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self‐assembly. Here we present a hot‐segment‐linking approach to design a series of mimics of the IAPP cross‐amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self‐ and cross‐seeded IAPP self‐assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer’s disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self‐assembly and pathogenic interactions of other proteins as well.     

Methenamine as an Efficient Ligand for Copper‐catalyzed Coupling of Phenols with Aryl Halides

Employing methenamine as a new supporting ligand, the copper‐catalyzed coupling reactions of aryl bromides or aryl iodides with various phenols successfully proceeded in good yields under mild conditions. A series of diaryl ethers were obtained with excellent yields in the presence of K₃PO₄ and copper(I) salt.     

Interaction of PiB‐Derivative Metal Complexes with Beta‐Amyloid Peptides: Selective Recognition of the Aggregated Forms

Metal complexes are increasingly explored as imaging probes in amyloid peptide related pathologies. We report the first detailed study on the mechanism of interaction between a metal complex and both the monomer and the aggregated form of Aβ_1–40 peptide. We have studied lanthanide(III) chelates of two PiB‐derivative ligands (PiB=Pittsburgh compound B), L¹ and L², differing in the length of the spacer between the metal‐complexing DO3A macrocycle (DO3A= 1,4,7,10‐tetraazacyclododecane‐1,4,7‐triacetic acid) and the peptide‐recognition PiB moiety. Surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR spectroscopy revealed that they both bind to aggregated Aβ_1–40 (K_D=67–160 μM ), primarily through the benzothiazole unit. HSQC NMR spectroscopy on the ¹⁵N‐labeled, monomer Aβ_1–40 peptide indicates nonsignificant interaction with monomeric Aβ. Time‐dependent circular dichroism (CD), dynamic light scattering (DLS), and TEM investigations of the secondary structure and of the aggregation of Aβ_1–40 in the presence of increasing amounts of the metal complexes provide coherent data showing that, despite their structural similarity, the two complexes affect Aβ fibril formation distinctly. Whereas GdL¹, at higher concentrations, stabilizes β‐sheets, GdL² prevents aggregation by promoting α‐helical structures. These results give insight into the behavior of amyloid‐targeted metal complexes in general and contribute to a more rational design of metal‐based diagnostic and therapeutic agents for amyloid‐ associated pathologies.