Silibinin: a novel inhibitor of Aβ aggregation

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid β peptide (Aβ) into extracellular fibrillar deposits known as amyloid plaque. Inhibition of Aβ aggregation is therefore viewed as a potential method to halt or slow the progression of AD. It is reported that silibinin (silybin), a flavonoid derived from the herb milk thistle (Silybum marianum), attenuates cognitive deficits induced by Aβ25-35 peptide and methamphetamine. However, it remains unclear whether silibinin interacts with Aβ peptide directly and decreases Aβ peptide-induced neurotoxicity. In the present study, we identified, through employing a ThT assay and electron microscopic imaging that silibinin also appears to act as a novel inhibitor of Aβ aggregation and this effect showed dose-dependency. We also show that silibinin prevented SH-SY5Y cells from injuries caused by Aβ(1-42)-induced oxidative stress by decreasing H(2)O(2) production in Aβ(1-42)-stressed neurons. Taken together, these results indicate that silibinin may be a novel therapeutic agent for the treatment of AD.     

Characterization of Aβ aggregation mechanism probed by congo red

β-Amyloid peptide (1) (Aβ) aggregates are toxic to neuron and the main cause of Alzheimer's disease (AD). The role of congo red (CR) on Aβ aggregation is controversial in aqueous solution. Both prevention and promotion of Aβ aggregation have been proposed, suggesting that CR may interact with Aβ of different structural conformations resulting in different effects on Aβ aggregation behavior. CR with these characteristics can be applied to probe the molecular mechanism of Aβ aggregation. Therefore, in the present study, we used CR as a probe to study the Aβ aggregation behavior in sodium dodecyl sulfate (SDS) condition. Our results show that Aβ(40) adopts two short helices at Q15-S26 and K28-L34 in the SDS environment. CR can interact with the helical form of Aβ(40), and the main interaction site is located at the first helical and hydrophobic core region, residues 17-25, which is assigned as a discordant helix region. Furthermore, CR may prevent Aβ(40) undergoing α-helix to β-strand conversion, and therefore aggregation through stabilizing the helical conformation of discordant helix in SDS environment, suggesting that the discordant helix plays a key role on the conformational stabilization of Aβ. Our present study implies that any factors or molecules that can stabilize the discordant helical conformation may also prevent the Aβ aggregation in membrane associated state. This leads to a new therapeutic strategy for the development of lead compounds to AD.     

Characterization of Aβ aggregation mechanism probed by congo red

β-Amyloid peptide ^{1 1}These authors contributed equally to this work. Communicated by Ramaswamy H. Sarma (Aβ) aggregates are toxic to neuron and the main cause of Alzheimer’s disease (AD). The role of congo red (CR) on Aβ aggregation is controversial in aqueous solution. Both prevention and promotion of Aβ aggregation have been proposed, suggesting that CR may interact with Aβ of different structural conformations resulting in different effects on Aβ aggregation behavior. CR with these characteristics can be applied to probe the molecular mechanism of Aβ aggregation. Therefore, in the present study, we used CR as a probe to study the Aβ aggregation behavior in sodium dodecyl sulfate (SDS) condition. Our results show that Aβ₄₀ adopts two short helices at Q15-S26 and K28-L34 in the SDS environment. CR can interact with the helical form of Aβ₄₀, and the main interaction site is located at the first helical and hydrophobic core region, residues 17–25, which is assigned as a discordant helix region. Furthermore, CR may prevent Aβ₄₀ undergoing α-helix to β-strand conversion, and therefore aggregation through stabilizing the helical conformation of discordant helix in SDS environment, suggesting that the discordant helix plays a key role on the conformational stabilization of Aβ. Our present study implies that any factors or molecules that can stabilize the discordant helical conformation may also prevent the Aβ aggregation in membrane associated state. This leads to a new therapeutic strategy for the development of lead compounds to AD.     

Computational selection of inhibitors of Aβ aggregation and neuronal toxicity

Alzheimer’s disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-β protein (Aβ). Disease symptoms can be alleviated, in vitro and in vivo, by ‘β-sheet breaker’ pentapeptides that reduce plaque load. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related β-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Aβ. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features and a conformation similar to the active peptides were selected, ranked by docking and biochemical parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Aβ aggregation at 2–3 μM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Aβ on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD.     

The co-effect of copper and lipid vesicles on Aβ aggregation

Both metal ions and lipid membranes have a wide distribution in amyloid plaques and play significant roles in AD pathogenesis. Although influences of different metal ions or lipid vesicles on the aggregation of Aβ peptides have been extensively studied, their combined effects are less understood. In this study, we reported a unique effect of copper ion on Aβ aggregation in the presence of lipid vesicles, different from other divalent metal ions. Cu²⁺ in a super stoichiometric amount leads to the rapid formation of β-sheet rich structure, containing abundant low molecular weight (LMW) oligomers. We demonstrated that oligomerization of Aβ40 induced by Cu²⁺ binding was an essential prerequisite for the rapid conformation transition. Overall, the finding provided a new view on the complex triple system of Aβ, copper ion and lipid vesicles, which might help understanding of Aβ pathologies.     

Mechanism of fiber assembly: treatment of Aβ peptide aggregation with a coarse-grained united-residue force field

The growth mechanism of β-amyloid (Aβ) peptide fibrils was studied by a physics-based coarse-grained united-residue model and molecular dynamics (MD) simulations. To identify the mechanism of monomer addition to an Aβ_1-40 fibril, we placed an unstructured monomer at a distance of 20 Å from a fibril template and allowed it to interact freely with the latter. The monomer was not biased towards fibril conformation by either the force field or the MD algorithm. With the use of a coarse-grained model with replica-exchange molecular dynamics, a longer timescale was accessible, making it possible to observe how the monomers probe different binding modes during their search for the fibril conformation. Although different assembly pathways were seen, they all follow a dock-lock mechanism with two distinct locking stages, consistent with experimental data on fibril elongation. Whereas these experiments have not been able to characterize the conformations populating the different stages, we have been able to describe these different stages explicitly by following free monomers as they dock onto a fibril template and to adopt the fibril conformation (i.e., we describe fibril elongation step by step at the molecular level). During the first stage of the assembly (“docking”), the monomer tries different conformations. After docking, the monomer is locked into the fibril through two different locking stages. In the first stage, the monomer forms hydrogen bonds with the fibril template along one of the strands in a two-stranded β-hairpin; in the second stage, hydrogen bonds are formed along the second strand, locking the monomer into the fibril structure. The data reveal a free-energy barrier separating the two locking stages. The importance of hydrophobic interactions and hydrogen bonds in the stability of the Aβ fibril structure was examined by carrying out additional canonical MD simulations of oligomers with different numbers of chains (4-16 chains), with the fibril structure as the initial conformation. The data confirm that the structures are stabilized largely by hydrophobic interactions and show that intermolecular hydrogen bonds are highly stable and contribute to the stability of the oligomers as well.     

Characterization of insulin-degrading enzyme-mediated cleavage of Aβ in distinct aggregation states

To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met₃₅–Val₃₆ as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity.     

Characterization of early stage intermediates in the nucleation phase of Aβ aggregation

Alzheimer's disease (AD) is a common form of dementia, which is characterized by the presence of extracellular amyloid plaques comprising the amyloid β peptide (Aβ). Although the mechanism underlying AD pathogenesis remains elusive, accumulating evidence suggests that the process of amyloid fibril formation is a surface-mediated event, which plays an important role in AD onset and progression. In this study, the mechanism of Aβ aggregation on hydrophobic surfaces was investigated with dual polarization interferometry (DPI), which provides real-time information on early stages of the aggregation process. Aggregation was monitored on a hydrophobic C18 surface and a polar silicon oxynitride surface. The DPI results showed a characteristic Aβ aggregation pattern involving a decrease in the density of Aβ at the surface followed by an increase in the thickness on the hydrophobic C18 chip. Most importantly, the DPI measurements provided unique information on the early stages of Aβ aggregation, which is characterized by the presence of initially slow nucleus formation process followed by exponential fibril elongation. The dimensions of the putative nucleus corresponded to a thickness of ～5 nm for both Aβ40 and Aβ42, which may represent about 10-15 molecules. The results thus support the nucleation-dependent polymerization model as indicated by the presence of a nucleation phase followed by an exponential growth phase. These results are the first reported measurements of the real-time changes in Aβ molecular structure during the early stages of amyloid formation at the nanometer level.     

Alzheimer Aβ peptide interactions with lipid membranes

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

Aβ polymerization through interaction with membrane gangliosides

Clarification of the molecular and cellular mechanisms underlying the assembly of amyloid β-protein (Aβ) into insoluble fibrils in the brain has been one of the biggest challenges in the research on Alzheimer disease (AD). We previously identified a novel Aβ species, which was characterized by its tight binding to GM1 ganglioside (GM1), in the brain showing early pathological changes of AD. The ganglioside-bound Aβ (GAβ) possessed unique characteristics, including its altered immunoreactivity, which suggests its distinct conformation from native Aβ, and its strong potency to accelerate Aβ assembly into fibrils. On the basis of these characteristics, it was hypothesized that Aβ adopts an altered conformation following interaction with GM1, leading to the generation of GAβ, and then GAβ acts as an endogenous seed for Alzheimer amyloid in the brain. To date, various in vitro and in vivo studies on GAβ have revealed how Aβ binds to gangliosides, i.e., what are the favorable physicochemical and neurobiological conditions for generating GAβ, and what is the pathological significance of ganglioside-induced Aβ assembly in the development of AD. Interestingly, GAβ is favorably generated in the unique ganglioside-enriched (clustered), raft-like microdomains; moreover, amyloid fibrils formed in the presence of gangliosides are neurotoxic. Furthermore, the conformational change of Aβ in the presence of ganglioside has been characterized by an NMR study. In this review, we focus on the recent progress of GAβ studies and highlight the possibility that ganglioside binding is the initial and common step in the development of a part of human misfolding-type amyloidoses, including AD.     

Mutations that replace aromatic side chains promote aggregation of the Alzheimer's Aβ peptide

The aggregation of polypeptides into amyloid fibrils is associated with a number of human diseases. Because these fibrils--or intermediates on the aggregation pathway--play important roles in the etiology of disease, considerable effort has been expended to understand which features of the amino acid sequence promote aggregation. One feature suspected to direct aggregation is the π-stacking of aromatic residues. Such π-stacking interactions have also been proposed as the targets for various aromatic compounds that are known to inhibit aggregation. In the case of Alzheimer's disease, the aromatic side chains Phe19 and Phe20 in the wild-type amyloid beta (Aβ) peptide have been implicated. To explicitly test whether the aromaticity of these side chains plays a role in aggregation, we replaced these two phenylalanine side chains with leucines or isoleucines. These residues have similar sizes and hydrophobicities as Phe but are not capable of π-stacking. Thioflavin-T fluorescence and electron microscopy demonstrate that replacement of residues 19 and 20 by Leu or Ile did not prevent aggregation, but rather enhanced amyloid formation. Further experiments showed that aromatic inhibitors of aggregation are as effective against Ile- and Leu-substituted versions of Aβ42 as they are against wild-type Aβ. These results suggest that aromatic π-stacking interactions are not critical for Aβ aggregation or for the inhibition of Aβ aggregation.     

A kinetic aggregation assay allowing selective and sensitive amyloid-β quantification in cells and tissues

The process of amyloid-β (Aβ) fibril formation is genetically and pathologically linked to Alzheimer's disease (AD). Thus, a selective and sensitive method for quantifying Aβ fibrils in complex biological samples allows a variety of hypotheses to be tested. Herein, we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding-competent Aβ aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate, and in mouse brain homogenate. Sonicated, proteinase K-treated Aβ fibril-containing tissue homogenates or cell culture media were added to an initially monomeric Aβ(1-40) reporter peptide to seed an in vitro nucleated aggregation reaction. The reduction in the half-time (t(50)) of the amyloid growth phase is proportional to the quantity of seeding-competent Aβ aggregates present in the biological sample. An ion-exchange resin amyloid isolation strategy from complex biological samples is demonstrated as an alternative for improving the sensitivity and linearity of the kinetic aggregation assay.     

A new class of aggregation inhibitor of amyloid-β peptide based on an O-acyl isopeptide

Inhibition of amyloid β peptide (Aβ) aggregation is a potential therapeutic approach to treat Alzheimer’s disease. We report that an O-acyl isopeptide of Aβ1–42 (1) containing an ester bond at the Gly²⁵-Ser²⁶ moiety inhibits Aβ1–42 fibril formation at equimolar ratio. Inhibitory activity was retained by an N-Me-β-Ala²⁶ derivative (2), in which the ester of 1 was replaced with N-methyl amide to improve chemical stability at physiological pH. Inhibition was verified by fluorescence anisotropy, Western blot, and atomic force microscopy. This report suggests a new class of Aβ aggregation inhibitor based on modification of Aβ1–42 at Gly²⁵-Ser²⁶.     

In vitro ADMET and physicochemical investigations of poly-N-methylated peptides designed to inhibit Aβ aggregation

N-Methylation is a common strategy for improving oral bioavailability of peptide-based lead structures. Herein, we present a detailed study on how the degree of N-methylation affects the absorption–distribution–metabolism–excretion–toxicity (ADMET) properties such as solubility, membrane transport, proteolytic stability, and general cell toxicity of the investigated peptides. As representative structures we chose hexapeptides 1–8. These peptides, corresponding to N-methylated analogues of residues 16–21 and 32–37 of the Aβ-peptide, pathological hallmark of Alzheimer’s disease (AD), have previously been shown to inhibit aggregation of Aβ fibrils in vitro. This study suggests that poly-N-methylated peptides are non-toxic and have enhanced proteolytic stability over their non-methylated analogues. Furthermore, solubility in aqueous solution is seen to increase with increased degree of N-methylation, while membrane transport was found to be low for all investigated hexapeptides. The present results, together with those reported in the literature, suggest that poly-N-methylated peptides, especially shorter or equal to six residues, can be suitable candidates for drug design.     

Mice with dysfunctional TGF-β signaling develop altered intestinal microbiome and colorectal cancer resistant to 5FU

Emerging data show a rise in colorectal cancer (CRC) incidence in young men and women that is often chemoresistant. One potential risk factor is an alteration in the microbiome. Here, we investigated the role of TGF-β signaling on the intestinal microbiome and the efficacy of chemotherapy for CRC induced by azoxymethane and dextran sodium sulfate in mice. We used two genotypes of TGF-β-signaling-deficient mice (Smad4^+/? and Smad4^+/?Sptbn1^+/?), which developed CRC with similar phenotypes and had similar alterations in the intestinal microbiome. Using these mice, we evaluated the intestinal microbiome and determined the effect of dysfunctional TGF-β signaling on the response to the chemotherapeutic agent 5-Fluoro-uracil (5FU) after induction of CRC. Using shotgun metagenomic sequencing, we determined gut microbiota composition in mice with CRC and found reduced amounts of beneficial species of Bacteroides and Parabacteroides in the mutants compared to the wild-type (WT) mice. Furthermore, the mutant mice with CRC were resistant to 5FU. Whereas the abundances of E. boltae, B.dorei, Lachnoclostridium sp., and Mordavella sp. were significantly reduced in mice with CRC, these species only recovered to basal amounts after 5FU treatment in WT mice, suggesting that the alterations in the intestinal microbiome resulting from compromised TGF-β signaling impaired the response to 5FU. These findings could have implications for inhibiting the TGF-β pathway in the treatment of CRC or other cancers.     

Molecular Interactions of Alzheimer's Biomarker FDDNP with Aβ Peptide

All-atom explicit solvent model and replica exchange molecular dynamics were used to investigate binding of Alzheimer''s biomarker FDDNP to the Aβ_10–40 monomer. At low and high concentrations, FDDNP binds with high affinity to two sites in the Aβ_10–40 monomer located near the central hydrophobic cluster and in the C-terminal. Analysis of ligand- Aβ_10–40 interactions at both concentrations identifies hydrophobic effect as a main binding factor. However, with the increase in ligand concentration the interactions between FDDNP molecules also become important due to strong FDDNP self-aggregation propensity and few specific binding locations. As a result, FDDNP ligands partially penetrate the core of the Aβ_10–40 monomer, forming large self-aggregated clusters. Ligand self-aggregation does not affect hydrophobic interactions as a main binding factor or the location of binding sites in Aβ_10–40. Using the Aβ_10–40 conformational ensemble in ligand-free water as reference, we show that FDDNP induces minor changes in the Aβ_10–40 secondary structure at two ligand concentrations studied. At the same time, FDDNP significantly alters the peptide tertiary fold in a concentration-dependent manner by redistributing long-range, side-chain interactions. We argue that because FDDNP does not change Aβ_10–40 secondary structure, its antiaggregation effect is likely to be weak. Our study raises the possibility that FDDNP may serve as a biomarker of not only Aβ fibril species, but of monomers as well.     

Molecular interactions of Alzheimer's Aβ protofilaments with lipid membranes

Amyloid fibrils and peptide oligomers play central roles in the pathology of Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, and prion-related disease. Here, we investigate the molecular interactions between preformed amyloid β (Aβ) molecular protofilaments and lipid bilayer membranes, in the presence of explicit water molecules, using computational models and all-atom molecular dynamics. These interactions play an important role in the stability and function of both Aβ fibrils and the adjacent cellular membrane. Taking advantage of the symmetry-related and directional properties of the protofilaments, we build models that cover several relative protofilament-membrane orientations. Our molecular dynamics simulations reveal the relative contributions of different structural elements to the dynamics and stability of Aβ protofilament segments near membranes, and the first steps in the mechanism of fibril-membrane interactions. During this process, we observe a significant alteration of the side-chain contact pattern in protofilaments, although a fraction of the characteristic β-sheet content is preserved. As a major driving force, we identify the electrostatic interactions between Aβ charged side chains, including E22, D23, and K28, and lipid headgroups. Together with hydrogen bonding with atoms from lipid headgroups, these interactions can facilitate the penetration of hydrophobic C-terminal amino acids through the lipid headgroup region, which can finally lead both to further loss of the initial fibril structure and to local membrane-thinning effects. Our results may guide new experiments that could test the extent to which the structural features of water-formed amyloid fibrils are preserved, lost, or reshaped by membrane-mediated interactions.     

Inhibition of aggregation of amyloid-β through covalent modification with benzylpenicillin; potential relevance to Alzheimer's disease

The pathogenesis of Alzheimer's disease (AD) is correlated with the misfolding and aggregation of amyloid-beta protein (Aβ). Here we report that the antibiotic benzylpenicillin (BP) can specifically bind to Aβ, modulate the process of aggregation and supress its cytotoxic effect, initially via a reversible binding interaction, followed by covalent bonding between specific functional groups (nucleophiles) within the Aβ peptide and the beta-lactam ring. Mass spectrometry and computational docking supported covalent modification of Aβ by BP. BP was found to inhibit aggregation of Aβ as revealed by the Thioflavin T (ThT) fluorescence assay and atomic force microscopy (AFM). In addition, BP treatment was found to have a cytoprotective activity against Aβ-induced cell cytotoxicity as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell toxicity assay. The specific interaction of BP with Aβ suggests the possibility of structure-based drug design, leading to the identification of new drug candidates against AD. Moreover, good pharmacokinetics of beta-lactam antibiotics and safety on long-time use make them valuable candidates for drug repurposing towards neurological disorders such as AD.