Identification of Physiological and Toxic Conformations in Aβ42 Aggregates

Aggregation of the 42‐residue amyloid β‐protein (Aβ42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Despite numerous structural studies on Aβ aggregates, the relationship between tertiary structure and toxicity remains unclear. Our proline scanning and solid‐state NMR studies suggested that aggregates both of wild‐type Aβ42 and of E22K‐Aβ42 (one of the mutants related to cerebral amyloid angiopathy) contain two conformers: a major one with a turn at positions 25 and 26, and a minor one with a turn at positions 22 and 23. To identify the toxic conformer, the derivative Aβ42‐lactam(22K–23E), in which the side chains at positions 22 and 23 were covalently linked, was synthesized as a minor conformer surrogate, along with Aβ42‐lactam(25K–26E) as a major conformer surrogate. The Aβ42‐lactam(22K–23E) showed stronger aggregation, neurotoxicity, radical generation, and oligomerization than wild‐type Aβ42, whereas in Aβ42‐lactam(25K–26E) were weak. The transition from the physiological conformation with a turn at positions 25 and 26 to the toxic conformation with a turn at positions 22 and 23 might be a key event in the pathogenesis of AD.     

Rational Design,Binding Studies,and Crystal‐Structure Evaluation of the First Ligand Targeting the Dimerization Interface of the 14‐3‐3ζ Adapter Protein

14‐3‐3 Proteins play a central role in signalling pathways in cells: they interact as gatekeeper proteins with a huge number of binding partners. Their function as hub for intracellular communication can explain why these adapter proteins are associated with a wide range of diseases. How they control the various cellular mechanisms is still unclear, but it is assumed that the dimeric nature of the 14‐3‐3 proteins plays a key role in their activity. Here, we present, to the best of our knowledge, the first example of a small molecule binding to the 14‐3‐3ζ dimerisation interface. This compound was designed by rational in silico optimisation of a peptidic ligand identified from biochemical screening of a peptidic library, and the binding was characterised by UV/Vis spectroscopy, microscale thermophoresis, multiscale simulations, and X‐ray crystallography.     

Biophysical Studies of the Amyloid β‐Peptide: Interactions with Metal Ions and Small Molecules

Alzheimer's disease is the most common of the protein misfolding (“amyloid”) diseases. The deposits in the brains of afflicted patients contain as a major fraction an aggregated insoluble form of the so‐called amyloid β‐peptides (Aβ peptides): fragments of the amyloid precursor protein of 39–43 residues in length. This review focuses on biophysical studies of the Aβ peptides: that is, of the aggregation pathways and intermediates observed during aggregation, of the molecular structures observed along these pathways, and of the interactions of Aβ with Cu and Zn ions and with small molecules that modify the aggregation pathways. Particular emphasis is placed on studies based on high‐resolution and solid‐state NMR methods. Theoretical studies relating to the interactions are also included. An emerging picture is that of Aβ peptides in aqueous solution undergoing hydrophobic collapse together with identical partners. There then follows a relatively slow process leading to more ordered secondary and tertiary (quaternary) structures in the growing aggregates. These aggregates eventually assemble into elongated fibrils visible by electron microscopy. Small molecules or metal ions that interfere with the aggregation processes give rise to a variety of aggregation products that may be studied in vitro and considered in relation to observations in cell cultures or in vivo. Although the heterogeneous nature of the processes makes detailed structural studies difficult, knowledge and understanding of the underlying physical chemistry might provide a basis for future therapeutic strategies against the disease. A final part of the review deals with the interactions that may occur between the Aβ peptides and the prion protein, where the latter is involved in other protein misfolding diseases.     

Retro‐Enantio N‐Methylated Peptides as β‐Amyloid Aggregation Inhibitors

An emerging and attractive target for the treatment of Alzheimer's disease is to inhibit the aggregation of β‐amyloid protein (Aβ). We applied the retro‐enantio concept to design an N‐methylated peptidic inhibitor of the Aβ42 aggregation process. This inhibitor, inrD, as well as the corresponding all‐L (inL) and all‐D (inD) analogues were assayed for inhibition of Aβ42 aggregation. They were also screened in neuroblastoma cell cultures to assess their capacity to inhibit Aβ42 cytotoxicity and evaluated for proteolytic stability. The results reveal that inrD and inD inhibit Aβ42 aggregation more effectively than inL, that inrD decreases Aβ42 cytotoxicity to a greater extent than inL and inD, and that as expected, both inD and inrD are stable to proteases. Based on these results, we propose that the retro‐enantio approach should be considered in future designs of peptide inhibitors of protein aggregation.     

Probing the Mint2 Protein–Protein Interaction Network Relevant to the Pathophysiology of Alzheimer's Disease

The intracellular adaptor protein Mint2 binds amyloid precursor protein (APP) and presenilin‐1, which are both central constituents of the amyloidogenic pathway associated with Alzheimer's disease (AD). Additional interaction partners have also been suggested for Mint2; several of them are also pertinent to AD pathogenesis. However, no comparative mapping of the Mint2 protein–protein interaction network is available. Here we provide a systematic characterization of seven interaction partners and address their specificities towards the different binding domains of Mint2, which reveal domain‐specific and ‐nonspecific interaction partners. Moreover, we show that the last two C‐terminal amino acids of Mint2 are both important for the intramolecular interaction with the PDZ1 domain and for the stability of Mint2.     

In vitro and in vivo Staining Characteristics of Small,Fluorescent, Aβ42‐Binding D‐Enantiomeric Peptides in Transgenic AD Mouse Models

Plaque visualisation : We identified three different D ‐enantiomeric peptides that bind to Alzheimer's amyloid β (Aβ1‐42). As there is currently no definitive pre‐mortem diagnosis for Alzheimer's disease, we investigated the peptides' suitability as molecular probes for in vivo imaging in transgenic mouse models.

     

Modulation of Amyloid‐β Aggregation by Histidine‐Coordinating Cobalt(III) Schiff Base Complexes

Oligomers of the Aβ42 peptide are significant neurotoxins linked to Alzheimer's disease (AD). Histidine (His) residues present at the N terminus of Aβ42 are believed to influence toxicity by either serving as metal–ion binding sites (which promote oligomerization and oxidative damage) or facilitating synaptic binding. Transition metal complexes that bind to these residues and modulate Aβ toxicity have emerged as therapeutic candidates. Cobalt(III) Schiff base complexes (Co–sb) were evaluated for their ability to interact with Aβ peptides. HPLC‐MS, NMR, fluorescence, and DFT studies demonstrated that Co–sb complexes could interact with the His residues in a truncated Aβ16 peptide representing the Aβ42 N terminus. Coordination of Co–sb complexes altered the structure of Aβ42 peptides and promoted the formation of large soluble oligomers. Interestingly, this structural perturbation of Aβ correlated to reduced synaptic binding to hippocampal neurons. These results demonstrate the promise of Co–sb complexes in anti‐AD therapeutic approaches.     

Trans-Chalcone Plus Baicalein Synergistically Reduce Intracellular Amyloid Beta (Aβ42) and Protect from Aβ42 Induced Oxidative Damage in Yeast Models of Alzheimer’s Disease

Finding an effective therapeutic to prevent or cure AD has been difficult due to the complexity of the brain and limited experimental models. This study utilized unmodified and genetically modified Saccharomyces cerevisiae as model organisms to find potential natural bioactive compounds capable of reducing intracellular amyloid beta 42 (Aβ₄₂) and associated oxidative damage. Eleven natural bioactive compounds including mangiferin, quercetin, rutin, resveratrol, epigallocatechin gallate (EGCG), urolithin A, oleuropein, rosmarinic acid, salvianolic acid B, baicalein and trans-chalcone were screened for their ability to reduce intracellular green fluorescent protein tagged Aβ₄₂ (GFP-Aβ₄₂) levels. The two most effective compounds from the screens were combined in varying concentrations of each to study the combined capacity to reduce GFP-Aβ₄₂. The most effective combinations were examined for their effect on growth rate, turnover of native Aβ₄₂ and reactive oxygen species (ROS). The bioactive compounds except mangiferin and urolithin A significantly reduced intracellular GFP-Aβ₄₂ levels. Baicalein and trans-chalcone were the most effective compounds among those that were screened. The combination of baicalein and trans-chalcone synergistically reduced GFP-Aβ₄₂ levels. A combination of 15 μM trans-chalcone and 8 μM baicalein was found to be the most synergistic combination. The combination of the two compounds significantly reduced ROS and Aβ₄₂ levels in yeast cells expressing native Aβ₄₂ without affecting growth of the cells. These findings suggest that the combination of baicalein and trans-chalcone could be a promising multifactorial therapeutic strategy to cure or prevent AD. However, further studies are recommended to look for similar cytoprotective activity in humans and to find an optimal dosage.     

Small‐Molecule Proteomimetic Inhibitors of the HIF‐1α–p300 Protein–Protein Interaction

The therapeutically relevant hypoxia inducible factor HIF‐1α–p300 protein–protein interaction can be orthosterically inhibited with α‐helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C‐terminal helix of the HIF‐1α C‐TAD (C‐terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side‐chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF‐1α–p300 PPI inhibitors and the first examples of small‐molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF‐1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42‐residue HIF‐1α C‐TAD from which it is derived.     

Spatiotemporal Control of Amyloid‐Like Aβ Plaque Formation Using a Multichannel Organic Electronic Device

We herein report on an iontronic device to drive and control Aβ1‐40 and Aβ1‐42 fibril formation. This system allows kinetic control of Aβ aggregation by regulation of H⁺ flows. The formed aggregates show both nanometer‐sized fibril structure and microscopic growth, thus mimicking senile plaques, at the H⁺‐outlet. Mechanistically we observed initial accumulation of Aβ1‐40 likely driven by electrophoretic migration which preceded nucleation of amyloid structures in the accumulated peptide cluster.

     

A Cyclic KLVFF‐Derived Peptide Aggregation Inhibitor Induces the Formation of Less‐Toxic Off‐Pathway Amyloid‐β Oligomers

Inhibition of amyloid‐β (Aβ) aggregation could be a target of drug development for the treatment of currently incurable Alzheimer's disease. We previously reported that a head‐to‐tail cyclic peptide of KLVFF (cyclic‐KLVFF), a pentapeptide fragment corresponding to the Aβ16–20 region (which plays a critical role in the generating Aβ fibrils), possesses potent inhibitory activity against Aβ aggregation. Here we found that the inhibitory activity of cyclic‐KLVFF was significantly improved by incorporating an additional phenyl group at the β‐position of the Phe4 side chain (inhibitor 3 ). Biophysical and biochemical analyses revealed the rapid formation of 3 ‐embedded oligomer species when Aβ1–42 was mixed with 3 . The oligomer species is an “off‐pathway” species with low affinity for cross‐β‐sheet‐specific dye thioflavin T and oligomer‐specific A11 antibodies. The oligomer species had a sub‐nanometer height and little capability of aggregation to amyloid fibrils. Importantly, the toxicity of the oligomer species was significantly lower than that of native Aβ oligomers. These insights will be useful for further refinement of cyclic‐KLVFF‐based aggregation inhibitors.     

A Novel Cu(II)-Binding Peptide Identified by Phage Display Inhibits Cu2+-Mediated Aβ Aggregation

Copper (Cu) has been implicated in the progression of Alzheimer’s disease (AD), and aggregation of Cu and amyloid β peptide (Aβ) are considered key pathological features of AD. Metal chelators are considered to be potential therapeutic agents for AD because of their capacity to reduce metal ion-induced Aβ aggregation through the regulation of metal ion distribution. Here, we used phage display technology to screen, synthesize, and evaluate a novel Cu(II)-binding peptide that specifically blocked Cu-triggered Aβ aggregation. The Cu(II)-binding peptide (S-A-Q-I-A-P-H, PCu) identified from the phage display heptapeptide library was used to explore the mechanism of PCu inhibition of Cu²⁺-mediated Aβ aggregation and Aβ production. In vitro experiments revealed that PCu directly inhibited Cu²⁺-mediated Aβ aggregation and regulated copper levels to reduce biological toxicity. Furthermore, PCu reduced the production of Aβ by inhibiting Cu²⁺-induced BACE1 expression and improving Cu(II)-mediated cell oxidative damage. Cell culture experiments further demonstrated that PCu had relatively low toxicity. This Cu(II)-binding peptide that we have identified using phage display technology provides a potential therapeutic approach to prevent or treat AD.     

Copper and Zinc Binding to Amyloid‐β: Coordination,Dynamics, Aggregation,Reactivity and Metal‐Ion Transfer

The metal ions copper, zinc and iron have been shown to be involved in Alzheimer's disease (AD). Cu, Zn and Fe ions are proposed to be implicated in two key steps of AD pathology: 1) aggregation of the peptide amyloid‐β (Aβ), and 2) production of reactive oxygen species (ROS) induced by Aβ. There is compelling evidence that Cu and Zn bind directly to Aβ in AD. This formation of Cu/Zn–Aβ complexes is thought to be aberrant as they have been detected only in AD, but not under healthy conditions. In this context, the understanding of how these metal ions interact with Aβ, their influence on structure and oligomerization become an important issue for AD. Moreover, the mechanism of ROS production by Cu–Aβ in relation to its aggregations state, as well as the metal‐transfer reaction from and to Aβ are crucial in order to understand why Aβ oligomers are highly toxic and why Aβ seems to bind Cu and Zn only in AD.     

Characterization of the Stereochemical Structures of 2H‐Thiazolo[3,2‐a]pyrimidine Compounds and Their Binding Affinities for Anti‐apoptotic Bcl‐2 Family Proteins

In a previous study we reported a class of compounds with a 2H‐thiazolo[3,2‐a]pyrimidine core structure as general inhibitors of anti‐apoptotic Bcl‐2 family proteins. However, the absolute stereochemical configuration of one carbon atom on the core structure remained unsolved, and its potential impact on the binding affinities of compounds in this class was unknown. In this study, we obtained pure R and S enantiomers of four selected compounds by HPLC separation and chiral synthesis. The absolute configurations of these enantiomers were determined by comparing their circular dichroism spectra to that of an appropriate reference compound. In addition, a crystal structure of one selected compound revealed the exocyclic double bond in these compounds to be in the Z configuration. The binding affinities of all four pairs of enantiomers to Bcl‐x_L, Bcl‐2, and Mcl‐1 proteins were measured in a fluorescence‐polarization‐based binding assay, yielding inhibition constants (K_i values) ranging from 0.24 to 2.20 μM . Interestingly, our results indicate that most R and S enantiomers exhibit similar binding affinities for the three tested proteins. A binding mode for this compound class was derived by molecular docking and molecular dynamics simulations to provide a reasonable interpretation of this observation.     

A Highly Potent and Cellularly Active β‐Peptidic Inhibitor of the p53/hDM2 Interaction

New and improved : The incorporation of a 6‐chlorotryptophan (6‐Cl‐Trp) into a β‐peptide (M)‐3₁₄ helix leads to a high‐affinity hDM2 inhibitor, as demonstrated by fluorescence fluctuation analysis at single molecule resolution. When conjugated to penetratin, the newly derived hDM2 binder specifically inhibits tumour cell growth in vitro.