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.     

Controlling Amyloid‐β Peptide(1–42) Oligomerization and Toxicity by Fluorinated Nanoparticles

The amyloid‐β peptide (Aβ) is a major fibrillar component of neuritic plaques in Alzheimer's disease brains and is related to the pathogenesis of the disease. Soluble oligomers that precede fibril formation have been proposed as the main neurotoxic species that contributes to neurodegeneration and dementia. We hypothesize that oligomerization and cytotoxicity can be repressed by nanoparticles (NPs) that induce conformational changes in Aβ42. We show here that fluorinated and hydrogenated NPs with different abilities to change Aβ42 conformation influence oligomerization as assessed by atomic force microscopy, immunoblot and SDS‐PAGE. Fluorinated NPs, which promote an increase in α‐helical content, exert an antioligomeric effect, whereas hydrogenated analogues do not and lead to aggregation. Cytotoxicity assays confirmed our hypothesis by indicating that the conformational conversion of Aβ42 into an α‐helical‐enriched secondary structure also has antiapoptotic activity, thereby increasing the viability of cells treated with oligomeric species.     

Single‐Molecule Imaging Reveals that Small Amyloid‐β1–42 Oligomers Interact with the Cellular Prion Protein (PrPC)

Oligomers of the amyloid‐β peptide (Aβ) play a central role in the pathogenesis of Alzheimer’s disease and have been suggested to induce neurotoxicity by binding to a plethora of cell‐surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aβ42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single‐molecule imaging to characterize Aβ42 oligomers (oAβ42) and to confirm the controversial interaction of oAβ42 with the cellular prion protein (PrP^C) on live neuronal cells. Our results show that, at nanomolar concentrations, oAβ42 interacts with PrP^C and that the species bound to PrP^C are predominantly small oligomers (dimers and trimers). Single‐molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor‐mediated oAβ‐induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways.     

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.     

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.     

Perspectives on Inhibiting β‐Amyloid Aggregation through Structure‐Based Drug Design

Targeting β‐amyloid (Aβ) remains the most desired strategy in Alzheimer’s disease (AD) drug discovery research. Many peptides that specifically target Aβ aggregates are known, encompassing efforts from both industrial and academic research settings. However, in clinical terms, not much success has been gained with peptide research; in turn, small drug‐like molecules are already globally recognized as showing promise as an alternate approach. Aβ aggregation inhibitors are the most important part of the multifunctional drug design regimen for treating AD. Unfortunately, rational drug design approaches with small molecules are still in the initial stages. Herein we highlight, update, and elaborate on the structural anatomy of Aβ and known Aβ aggregation inhibitors in hopes of helping to optimize their use in structure‐based drug design approaches toward inhibitors with greater specificity. Furthermore, we present the first review of efforts to target a previously uncharacterized region of acetylcholinesterase: the N‐terminal 7–20 sub‐region, which was experimentally elucidated to participate in Aβ aggregation and deposition.     

Transthyretin Mimetics as Anti‐β‐Amyloid Agents: A Comparison of Peptide and Protein Approaches

β‐Amyloid (Aβ) aggregation is causally linked to neuronal pathology in Alzheimer's disease; therefore, several small molecules, antibodies, and peptides have been tested as anti‐Aβ agents. We developed two compounds based on the Aβ‐binding domain of transthyretin (TTR): a cyclic peptide cG8 and an engineered protein mTTR, and compared them for therapeutically relevant properties. Both mTTR and cG8 inhibit fibrillogenesis of Aβ, with mTTR inhibiting at a lower concentration than cG8. Both inhibit aggregation of amylin but not of α‐synuclein. They both bind more Aβ aggregates than monomer, and neither disaggregates preformed fibrils. cG8 retained more of its activity in the presence of biological materials and was more resistant to proteolysis than mTTR. We examined the effect of mTTR or cG8 on Aβ binding to human neurons. When mTTR was co‐incubated with Aβ under oligomer‐forming conditions, Aβ morphology was drastically changed and Aβ‐cell deposition significantly decreased. In contrast, cG8 did not affect morphology but decreased the amount of Aβ deposited. These results provide guidance for further evolution of TTR‐mimetic anti‐amyloid agents.     

Alanine and Lysine Scans of the LL‐37‐Derived Peptide Fragment KR‐12 Reveal Key Residues for Antimicrobial Activity

The human host defence peptide LL‐37 is a broad‐spectrum antibiotic with immunomodulatory functions. Residues 18–29 in LL‐37 have previously been identified as a minimal peptide (KR‐12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR‐12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad‐spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR‐12 is a tuneable template for antibiotic development.     

A Novel Competitive Class of α‐Glucosidase Inhibitors: (E)‐1‐Phenyl‐3‐(4‐Styrylphenyl)Urea Derivatives

Competitive glycosidase inhibitors are generally sugar mimics that are costly and tedious to obtain because they require challenging and elongated chemical synthesis, which must be stereo‐ and regiocontrolled. Here, we show that readily accessible achiral (E)‐1‐phenyl‐3‐(4‐strylphenyl)ureas are potent competitive α‐glucosidase inhibitors. A systematic synthesis study shows that the 1‐phenyl moiety on the urea is critical for ensuring competitive inhibition, and substituents on both terminal phenyl groups contribute to inhibition potency. The most potent inhibitor, compound 12 (IC₅₀=8.4 μM , K_i=3.2 μM ), manifested a simple slow‐binding inhibition profile for α‐glucosidase with the kinetic parameters k₃=0.005256 μM ^?1 min^?1, k₄=0.003024 min^?1, and ${K{{{\rm app}\hfill \atop {\rm i}\hfill}}}$ =0.5753 μM .     

Controlled Production of Amyloid β Peptide from a Photo‐Triggered,Water‐Soluble Precursor “Click Peptide“

In biological experiments, poor solubility and uncontrolled assembly of amyloid β peptide (Aβ) 1–42 pose significant obstacles to establish an experiment system that clarifies the function of Aβ1–42 in Alzheimer's disease (AD). Herein, as an experimental tool to overcome these problems, we developed a water‐soluble photo‐“click peptide” with a coumarin‐derived photocleavable protective group that is based on an O‐acyl isopeptide method. The click peptide had nearly 100‐fold higher water solubility than Aβ1–42 and did not self‐assemble, as the isomerized structure in its peptide backbone drastically changed the conformation that was derived from Aβ1–42. Moreover, the click peptide afforded Aβ1–42 quickly under physiological conditions (pH 7.4, 37 °C) by photoirradiation followed by an O–N intramolecular acyl migration. Because the in situ production of intact Aβ1–42 from the click peptide could improve the difficulties in handling Aβ1–42 caused by its poor solubility and highly aggregative nature, this click peptide strategy would provide a reliable experiment system for investigating the pathological function of Aβ1–42 in AD.     

A One‐Pot “Triple‐C” Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides

A broadly applicable one‐pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)‐catalyzed reaction (CEPS/CLIPS/CuAAC; “triple‐C”) locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head‐to‐tail by using the peptiligase variant omniligase‐1 (CEPS). Subsequent ligation–cyclization with tetravalent (T4_1/2) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF‐T4_1/2) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero‐bifunctional variants (UF/FU‐T4_1/2), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the “bifunctional tetracyclic peptide” inhibitor concept.     

Aggregation‐Prone Amyloid‐β⋅CuII Species Formed on the Millisecond Timescale under Mildly Acidic Conditions

Metal ions and their interaction with the amyloid beta (Aβ) peptide might be key elements in the development of Alzheimer's disease. In this work the effect of Cu^II on the aggregation of Aβ is explored on a timescale from milliseconds to days, both at physiological pH and under mildly acidic conditions, by using stopped‐flow kinetic measurements (fluorescence and light‐scattering), ¹H NMR relaxation and ThT fluorescence. A minimal reaction model that relates the initial Cu^II binding and Aβ folding with downstream aggregation is presented. We demonstrate that a highly aggregation prone Aβ ? Cu^II species is formed on the sub‐second timescale at mildly acidic pH. This observation might be central to the molecular origin of the known detrimental effect of acidosis in Alzheimer's disease.     

α‐Bromoacrylamido N‐Substituted Isatin Derivatives as Potent Inducers of Apoptosis in Human Myeloid Leukemia Cells

A novel series of α‐bromoacryloyl N‐substituted isatin analogues were found to inhibit the growth and viability of human myeloid leukemia HL‐60 and U‐937 cells as well as human lymphoid leukemia MOLT‐3 cells. Cell death induced by these molecules was preceded by a rapid release of cytochrome c from mitochondria into the cytosol and subsequent caspase activation involving caspase‐3, to cleave poly(ADP‐ribose) polymerase (PARP). These findings suggest that these compounds present antiproliferative activity which may be mediated by apoptosis caused by cytochrome c release and caspase activation in human leukemia cells.