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.     

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.     

Dye aggregation and interaction of dyes with a water‐soluble polymer in ink‐jet ink for textiles

Detailed understanding of the interaction between dyes and additives and the aggregation behaviour of the dye molecules in aqueous solutions is required to develop ink‐jet ink for textiles. In the present study, the aggregation behaviour of three acid dyes (CI Acid Red 88, CI Acid Red 13 and CI Acid Red 27) containing different number of sulphonate groups in aqueous solutions was investigated by means of visible absorption spectroscopy. As a result, the higher the solubility of the dyes in water (the larger the number of sulphonate groups in the dyes), the lower are the aggregation constants of the dyes. For all the dyes, the aggregation constants decreased with increasing temperature, indicating the exothermic process of the dye aggregation. The thermodynamic process for CI Acid Red 88 with one sulphonate group is less enthalpic and more entropic than that for CI Acid Red 13, which contains two sulphonate groups. CI Acid Red 27, which includes three sulphonate groups, hardly forms any aggregates. To elucidate the effects of the polymer additive, the binding constants of the dyes with the water‐soluble polymer, poly(vinylpyrrolidone) and the aggregation constants of the dyes in aqueous polymer solutions were also estimated. In addition, the binding constants were influenced by the number of sulphonate groups in the acid dyes: the larger number of sulphonate groups diminished the binding constants. The aggregation constants in the presence of poly(vinylpyrrolidone) were smaller than those in its absence at every temperature for all dyes. This suggests that poly(vinylpyrrolidone) has disaggregation effects. Furthermore, poly(vinylpyrrolidone) makes the aggregation process less enthalpic with a greater entropic change. Thus, the aggregation process of the dyes in the polymer solutions is thermodynamically different from that in water, reflecting the interactions between the dyes and the polymer.     

Lactose‐Functionalized Dendrimers Arbitrate the Interaction of Galectin‐3/MUC1 Mediated Cancer Cellular Aggregation

By using lactose‐functionalized poly(amidoamine) dendrimers as a tunable multivalent platform, we studied cancer cell aggregation in three different cell lines (A549, DU‐145, and HT‐1080) with galectin‐3. We found that small lactose‐functionalized G(2)‐dendrimer 1 inhibited galectin‐3‐induced aggregation of the cancer cells. In contrast, dendrimer 4 (a larger, generation 6 dendrimer with 100 carbohydrate end groups) caused cancer cells to aggregate through a galectin‐3 pathway. This study indicates that inhibition of cellular aggregation occurred because 1 provided competitive binding sites for galectin‐3 (compared to its putative cancer cell ligand, TF‐antigen on MUC1). Dendrimer 4 , in contrast, provided an excess of ligands for galectin‐3 binding; this caused crosslinking and aggregation of cells to be increased.     

Effect of 1-Ethyl-3-methylimidazolium Tetrafluoroborate and Acetate Ionic Liquids on Stability and Amyloid Aggregation of Lysozyme

Amyloid fibrils draw attention as potential novel biomaterials due to their high stability, strength, elasticity or resistance against degradation. Therefore, the controlled and fast fibrillization process is of great interest, which raises the demand for effective tools capable of regulating amyloid fibrillization. Ionic liquids (ILs) were identified as effective modulators of amyloid aggregation. The present work is focused on the study of the effect of 1-ethyl-3-methyl imidazolium-based ILs with kosmotropic anion acetate (EMIM-ac) and chaotropic cation tetrafluoroborate (EMIM-BF₄) on the kinetics of lysozyme amyloid aggregation and morphology of formed fibrils using fluorescence and CD spectroscopy, differential scanning calorimetry, AFM with statistical image analysis and docking calculations. We have found that both ILs decrease the thermal stability of lysozyme and significantly accelerate amyloid fibrillization in a dose-dependent manner at concentrations of 0.5%, 1% and 5% (v/v) in conditions and time-frames when no fibrils are formed in ILs-free solvent. The effect of EMIM-BF₄ is more prominent than EMIM-ac due to the different specific interactions of the anionic part with the protein surface. Although both ILs induced formation of amyloid fibrils with typical needle-like morphology, a higher variability of fibril morphology consisting of a different number of intertwining protofilaments was identified for EMIM-BF_4.     

Melatonin Inhibits hIAPP Oligomerization by Preventing β-Sheet and Hydrogen Bond Formation of the Amyloidogenic Region Revealed by Replica-Exchange Molecular Dynamics Simulation

The pathogenesis of type 2 diabetes (T2D) is highly related to the abnormal self-assembly of the human islet amyloid polypeptide (hIAPP) into amyloid aggregates. To inhibit hIAPP aggregation is considered a promising therapeutic strategy for T2D treatment. Melatonin (Mel) was reported to effectively impede the accumulation of hIAPP aggregates and dissolve preformed fibrils. However, the underlying mechanism at the atomic level remains elusive. Here, we performed replica-exchange molecular dynamics (REMD) simulations to investigate the inhibitory effect of Mel on hIAPP oligomerization by using hIAPP_20–29 octamer as templates. The conformational ensemble shows that Mel molecules can significantly prevent the β-sheet and backbone hydrogen bond formation of hIAPP_20–29 octamer and remodel hIAPP oligomers and transform them into less compact conformations with more disordered contents. The interaction analysis shows that the binding behavior of Mel is dominated by hydrogen bonding with a peptide backbone and strengthened by aromatic stacking and CH–π interactions with peptide sidechains. The strong hIAPP–Mel interaction disrupts the hIAPP_20–29 association, which is supposed to inhibit amyloid aggregation and cytotoxicity. We also performed conventional MD simulations to investigate the influence and binding affinity of Mel on the preformed hIAPP_1–37 fibrillar octamer. Mel was found to preferentially bind to the amyloidogenic region hIAPP_20–29, whereas it has a slight influence on the structural stability of the preformed fibrils. Our findings illustrate a possible pathway by which Mel alleviates diabetes symptoms from the perspective of Mel inhibiting amyloid deposits. This work reveals the inhibitory mechanism of Mel against hIAPP_20–29 oligomerization, which provides useful clues for the development of efficient anti-amyloid agents.     

Simultaneous IR‐Spectroscopic Observation of α‐Synuclein,Lipids, and Solvent Reveals an Alternative Membrane‐Induced Oligomerization Pathway

The intrinsically disordered protein α‐synuclein (αS), a known pathogenic factor for Parkinson's disease, can adopt defined secondary structures when interacting with membranes or during fibrillation. The αS–lipid interaction and the implications of this process for aggregation and damage to membranes are still poorly understood. Therefore, we established a label‐free infrared (IR) spectroscopic approach to allow simultaneous monitoring of αS conformation and membrane integrity. IR showed its unique sensitivity for identifying distinct β‐structured aggregates. A comparative study of wild‐type αS and the naturally occurring splicing variant αS Δexon3 yielded new insights into the membrane's capability for altering aggregation pathways.     

Nanoscale Surface Topography Modulates hIAPP Aggregation Pathways at Solid–Liquid Interfaces

The effects that solid–liquid interfaces exert on the aggregation of proteins and peptides are of high relevance for various fields of basic and applied research, ranging from molecular biology and biomedicine to nanotechnology. While the influence of surface chemistry has received a lot of attention in this context, the role of surface topography has mostly been neglected so far. In this work, therefore, we investigate the aggregation of the type 2 diabetes-associated peptide hormone hIAPP in contact with flat and nanopatterned silicon oxide surfaces. The nanopatterned surfaces are produced by ion beam irradiation, resulting in well-defined anisotropic ripple patterns with heights and periodicities of about 1.5 and 30 nm, respectively. Using time-lapse atomic force microscopy, the morphology of the hIAPP aggregates is characterized quantitatively. Aggregation results in both amorphous aggregates and amyloid fibrils, with the presence of the nanopatterns leading to retarded fibrillization and stronger amorphous aggregation. This is attributed to structural differences in the amorphous aggregates formed at the nanopatterned surface, which result in a lower propensity for nucleating amyloid fibrillization. Our results demonstrate that nanoscale surface topography may modulate peptide and protein aggregation pathways in complex and intricate ways.     

Terpendole E and its Derivative Inhibit STLC‐ and GSK‐1‐Resistant Eg5

Terpendole E is first natural product found to inhibit mitotic kinesin Eg5, but its inhibitory mechanism remains to be revealed. Here, we report the effects of terpendole E and 11ketopaspaline (a new natural terpendole E analogue) on the Eg5–microtubule interaction and in several Eg5 mutants. 11‐Ketopaspaline is a shunt product from terpendole E, and it shows potent inhibitory activity against the microtubule‐stimulated ATPase activity of Eg5. Unlike other Eg5 inhibitors, such as S‐trityl‐L ‐cysteine (STLC) and GSK‐1, both terpendole E and 11‐ketopaspaline only partially inhibited Eg5–microtubule interaction. Furthermore, terpendole E and 11‐ketopaspaline inhibited several Eg5 mutants that are resistant to STLC (Eg5^D130A, Eg5^L214A) or GSK‐1 (Eg5^I299F, Eg5^A356T), but with the same extent of inhibition against wild‐type Eg5. Because Eg5^D130A and Eg5^L214A show cross‐resistance to most known Eg5 inhibitors, which bind the L5 loop, these results suggest that terpendole E and its analogues have a different binding site and/or inhibitory mechanism to those for L5 loop‐binding type Eg5 inhibitors.     

Sensitivity analysis of a proposed model mechanism for newly created glucose‐6‐oxidases

Parametric sensitivity analysis of a proposed model mechanism can serve as a valuable diagnostic for studying the fundamental characteristics of an enzymatic reaction. As the first step toward understanding the kinetics of a series of newly engineered glucose‐6‐oxidases, the activity of which has never been found in nature, we have proposed a mechanistic kinetic model and performed a parametric sensitivity analysis. The mechanism of our model consists of two enzyme conformations, namely “less active” and “more active,” and is shown to be consistent with experimental observations of prolonged periods of enzyme induction. The extended lag phase phenomenon was found to be well described mechanistically by a slow rate of interconversion between the two enzyme conformations (relative to the rate of product formation), and this prediction was further supported by our experimental results. The proposed enzymatic model will serve as a blueprint with which to better understand the mechanistic behavior of newly generated glucose‐6‐oxidases. © 2011 American Institute of Chemical Engineers AIChE J, 58: 2303–2308, 2012     

Not Oligomers but Amyloids are Cytotoxic in the Membrane‐Mediated Amyloidogenesis of Amyloid‐β Peptides

The formation of neurotoxic aggregates by amyloid‐β peptide (Aβ) is considered to be a key step in the onset of Alzheimer's disease. It is widely accepted that oligomers are more neurotoxic than amyloid fibrils in the aqueous‐phase aggregation of Aβ. Membrane‐mediated amyloidogenesis is also relevant to the pathology, although the relationship between the aggregate size and cytotoxicity has remained elusive. Here, aggregation processes of Aβ on living cells and cytotoxic events were monitored by fluorescence techniques. Aβ formed amyloids after forming oligomers composed of ≈10 Aβ molecules. The formation of amyloids was necessary to activate apoptotic caspase‐3 and reduce the ability of the cell to proliferate; this indicated that amyloid formation is a key event in Aβ‐induced cytotoxicity.     

Multitarget Therapeutic Leads for Alzheimer’s Disease: Quinolizidinyl Derivatives of Bi‐ and Tricyclic Systems as Dual Inhibitors of Cholinesterases and β‐Amyloid (Aβ) Aggregation

Multitarget therapeutic leads for Alzheimer’s disease were designed on the models of compounds capable of maintaining or restoring cell protein homeostasis and of inhibiting β‐amyloid (Aβ) oligomerization. Thirty‐seven thioxanthen‐9‐one, xanthen‐9‐one, naphto‐ and anthraquinone derivatives were tested for the direct inhibition of Aβ(1–40) aggregation and for the inhibition of electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBChE). These compounds are characterized by basic side chains, mainly quinolizidinylalkyl moieties, linked to various bi‐ and tri‐cyclic (hetero)aromatic systems. With very few exceptions, these compounds displayed inhibitory activity on both AChE and BChE and on the spontaneous aggregation of β‐amyloid. In most cases, IC₅₀ values were in the low micromolar and sub‐micromolar range, but some compounds even reached nanomolar potency. The time course of amyloid aggregation in the presence of the most active derivative (IC₅₀=0.84 μM ) revealed that these compounds might act as destabilizers of mature fibrils rather than mere inhibitors of fibrillization. Many compounds inhibited one or both cholinesterases and Aβ aggregation with similar potency, a fundamental requisite for the possible development of therapeutics exhibiting a multitarget mechanism of action. The described compounds thus represent interesting leads for the development of multitarget AD therapeutics.     

Aggregation behavior of 3,6‐O‐carboxymethylated chitin in aqueous solutions

The aggregation behavior of 3,6‐O‐carboxymethylated chitin (3,6‐O‐CM‐chitin) in aqueous solutions was investigated by viscometry, gel permeation chromatography (GPC), and GPC combined with laser light scattering (GPC‐LLS) techniques. 3,6‐O‐CM‐chitin has a strong tendency to form aggregates in NaCl aqueous solutions with the apparent aggregation number (N_ap) of about 27. There were three kinds of aggregates corresponding to different cohesive energies, the aggregates with low cohesive energy were first dissociated at 60°C, the aggregates with middle cohesive energy were then dissociated at 80 to 90°C, and the aggregates with high cohesive energy were difficult to be disrupted by heating. Decreasing polysaccharide concentration (c_p) or increasing NaCl concentration (c_s) reduced the content of the aggregates. At the critical c_p of 2.5 × 10^?5 g/mL, the aggregates were dissociated into single chains completely. The change of aggregation and disaggregation of 3,6‐O‐CM‐chitin in water–cadoxen mixtures occurred from 0.1 to 0.4 of v_cad, and were irreversible. Intermolecular hydrogen bonding can be ascribed as main driving force for aggregation. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1838–1843, 2002     

Fluorine‐Mediated Editing of a G‐Quadruplex Folding Pathway

A (3+1)‐hybrid‐type G‐quadruplex was substituted within its central tetrad by a single 2′‐fluoro‐modified guanosine. Driven by the anti‐favoring nucleoside analogue, a novel quadruplex fold with inversion of a single G‐tract and conversion of a propeller loop into a lateral loop emerges. In addition, scalar couplings across hydrogen bonds demonstrate the formation of intra‐ and inter‐residual F ??? H8?C8 pseudo‐hydrogen bonds within the modified quadruplexes. Alternative folding can be rationalized by the impact of fluorine on intermediate species on the basis of a kinetic partitioning mechanism. Apparently, chemical or other environmental perturbations are able to redirect folding of a quadruplex, possibly modulating its regulatory role in physiological processes.     

Aggregation‐Enhanced Emission of Fluorescent‐Gemini Surfactants with High Photostability for Cell‐Membrane Imaging

In situ studies of the aggregation behavior of traditional surfactants at the liquid interface using spectroscopic methods are often significantly affected by the large volume of fluorescent groups, such as pyrene. Fluorescent‐Gemini surfactants provide an ideal solution since the fluorescent block can be designed as a spacer or a tail. In this work, we report the synthesis of a new fluorescent‐Gemini surfactant with a rigid spacer (referred to as 8‐TBT‐8). The aggregation behavior and application in cell‐membrane imaging were investigated. The unique aggregation behavior in an organic solvent and aqueous solution was studied using spectroscopy. UV–vis and photoluminescence spectra of 8‐TBT‐8 revealed that this new fluorescent surfactant forms H aggregates in organic solution to give blue emission, whereas it forms J aggregates in aqueous solution to give green fluorescence under UV light. In addition, the fluorescence intensity of 8‐TBT‐8 increases abruptly at concentrations higher than the critical micellization concentration. Good photostability and a unique structure make the synthesized Gemini surfactant very suitable for membrane imaging.     

Amphipathic polymers with stimuli‐responsive microdomains for water remediation: Binding studies with p‐cresol

Amphipathic, stimuli‐responsive water‐soluble polymers have been investigated as potential remediation agents for micellar enhanced ultrafiltration (MEUF). The systems represent divergent architectural types, a triblock ABA copolymer of PEO‐PPO‐PEO, an n‐octylamide modified poly(sodium maleate‐alt‐ethyl vinyl ether), and the transport protein, bovine serum albumin. Each type exhibits stimuli‐dependent microphase separation or domain formation in response to temperature, pH, and/or ionic strength changes. Segmental associations result in hydrophobic clusters resembling those present in small molecule surfactant micelles. The effects of such segmental aggregation on sequestration of a model hydrophobic foulant, p‐cresol, have been investigated using equilibrium dialysis. The favorable molar binding values, the large hydrodynamic dimensions of the stable polymer aggregates, and potential reversibility of foulant loading could have commercial utility in high flow rate, multiple‐pass remediation processes. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2290–2300, 1999     

Creating a Water‐Soluble Resveratrol‐Based Antioxidant by Site‐Selective Enzymatic Glucosylation

The phytochemical resveratrol (trans‐3,5,4′‐trihydroxystilbene) has drawn great interest as health‐promoting food ingredient and potential therapeutic agent. However, resveratrol shows vanishingly low water solubility; this limits its uptake and complicates the development of effective therapeutic forms. Glycosylation should be useful to enhance resveratrol solubility, with the caveat that unselective attachment of sugars could destroy the molecule's antioxidant activity. UGT71A15 (a uridine 5′‐diphosphate α‐D ‐glucose‐dependent glucosyltransferase from apple) was used to synthesize resveratrol 3,5‐β‐D ‐diglucoside; this was about 1700‐fold more water‐soluble than the unglucosylated molecule (～0.18 mM ), yet retained most of the antioxidant activity. Resveratrol 3‐β‐D ‐glucoside, which is the naturally abundant form of resveratrol, was a practical substrate for perfect site‐selective conversion into the target diglucoside in quantitative yield (g L^?1 concentration).     

C3‐Symmetrical π‐Scaffolds: Useful Building Blocks to Construct Helical Supramolecular Polymers

In this review, we highlight some relevant examples of C₃‐symmetrical molecules that have been reported to form supramolecular polymers and helical aggregates. In particular, the number and type of non‐covalent forces are key to bias the supramolecular polymerization leading from a simple isodesmic or cooperative mechanism to a more complex self‐assembly process, i. e. pathway complexity. Furthermore, the attachment of stereogenic centres at the peripheral side chains of the C₃‐systems provokes efficient transfer and amplification of chirality phenomena when directional and specific non‐covalent interactions operate. Interestingly, the incorporation of hydrophilic side chains induces the formation of organized aggregates in aqueous media with potential biomedical applications. Overall, the examples shown in this review on C₃‐symmetrical scaffolds illustrate the relevance of this molecular shape in the development of functional supramolecular structures.     

Potent Tau Aggregation Inhibitor D-Peptides Selected against Tau-Repeat 2 Using Mirror Image Phage Display

Alzheimer's disease and other Tauopathies are associated with neurofibrillary tangles composed of Tau protein, as well as toxic Tau oligomers. Therefore, inhibitors of pathological Tau aggregation are potentially useful candidates for future therapies targeting Tauopathies. Two hexapeptides within Tau, designated PHF6* (275-VQIINK-280) and PHF6 (306-VQIVYK-311), are known to promote Tau aggregation. Recently, the PHF6* segment has been described as the more potent driver of Tau aggregation. We therefore employed mirror-image phage display with a large peptide library to identify PHF6* fibril binding peptides consisting of D-enantiomeric amino acids. The suitability of D-enantiomeric peptides for in vivo applications, which are protease stable and less immunogenic than L-peptides, has already been demonstrated. The identified D-enantiomeric peptide MMD3 and its retro-inverso form, designated MMD3rev, inhibited in vitro fibrillization of the PHF6* peptide, the repeat domain of Tau as well as full-length Tau. Dynamic light scattering, pelleting assays and atomic force microscopy demonstrated that MMD3 prevents the formation of tau β-sheet-rich fibrils by diverting Tau into large amorphous aggregates. NMR data suggest that the D-enantiomeric peptides bound to Tau monomers with rather low affinity, but ELISA (enzyme-linked immunosorbent assay) data demonstrated binding to PHF6* and full length Tau fibrils. In addition, molecular insight into the binding mode of MMD3 to PHF6* fibrils were gained by in silico modelling. The identified PHF6*-targeting peptides were able to penetrate cells. The study establishes PHF6* fibril binding peptides consisting of D-enantiomeric amino acids as potential molecules for therapeutic and diagnostic applications in AD research.