Inhibit Fibril Formation Research Articles

Aromaticity of Phenylalanine Residues Is Essential for Amyloid Formation by Alzheimer's Amyloid β-Peptide.

The abnormal aggregation of amyloid β-peptide (Aβ) is central to the pathogenesis of Alzheimer's disease, the major form of dementia. Aromatic π-π interactions have been suggested to play a crucial role in the aggregation of not only Aβ, but also other amyloidogenic proteins. In this study, each or all phenylalanine (Phe) residues at the 4th, 19th, and 20th positions of Aβ-(1-40) were substituted by hydrophobic cyclohexylalanine (Cha), which is sterically similar to Phe, but lacks π-electrons, to reveal effects of interactions involving π-electrons on the aggregation of Aβ both in aqueous solution and GM1-containing membranes. We found that each Cha substitution significantly inhibited fibril formation by Aβ, indicating a pivotal role of aromatic interactions. Furthermore, the Aβ analog with three Cha residues effectively retarded the fibrillation of the wild-type Aβ.

Effect of chromium(III) gallate complex on stabilization of collagen

To improve the stability of the collagen, here we studied the interaction of chromium(III) polyphenolic complex, [Cr(GA)2] (GA: Gallic acid) with collagen using various spectroscopic techniques. Circular dichroism studies show that the [Cr(GA)2] and gallic acid did not induce any structural perturbations on the triple helix of the collagen. Both differential scanning calorimetric(DSC) data and micro-shrinkage temperature studies showed that [Cr(GA)2] stabilized the collagen by 6±1°C compared to gallic acid. Hydrodynamic studies revealed that the viscosity of collagen drastically reduced in the presence of [Cr(GA)2] while gallic acid did not. Fibrillation assay displayed a significant delay in fibril formation with Cr(III) complex compared to gallic acid treated collagen. The inhibition of fibril formation was further confirmed by microscopic data in which collagen fibres are seen with GA while [Cr(GA)2] treated collagen exhibit a thin microfibrils. From AFM studies, the d-periodicity of collagen was found to be decreased with [Cr(GA)2] while increased with gallic acid. The present study deliberate the advantage of metal complex containing polyphenolic ligand as crosslinking agent due to its synergistic effect of both metal center as well as polyphenolic groups in the stabilization of collagen structure.

Protocatechuic Acid: Inhibition of Fibril Formation, Destabilization of Preformed Fibrils of Amyloid-β and α-Synuclein, and Neuroprotection.

Protocatechuic acid (PCA) is the major metabolite of the anthocyanin known as cyanidin 3-glucoside. It is found in plasma and tissues, such as the brain, heart, liver, and kidneys, following consumption of a rich source of this flavonoid. The abnormal pathological assembly of amyloid-β (Aβ) and α-synuclein (αS) is an underlying mechanism involved in the formation of amyloid plaques and Lewy bodies in the brain, which are responsible for neuropathology symptoms in Alzheimer's (AD) and Parkinson's diseases (PD), respectively. This research was performed to evaluate the protective effects of PCA, by establishing its potential role in inhibiting aggregation and fibril destabilization of Aβ and αS proteins. It has been found that PCA inhibits the aggregation of Aβ and αS and destabilizes their preformed fibrils. These results were confirmed by TEM images, electrophoresis, and immunoblotting experiments. Furthermore, PCA prevents the death of PC12 cells triggered by Aβ- and αS-induced toxicity.

Small Heat-shock Proteins Prevent α-Synuclein Aggregation via Transient Interactions and Their Efficacy Is Affected by the Rate of Aggregation

The aggregation of α-synuclein (α-syn) into amyloid fibrils is associated with neurodegenerative diseases, collectively referred to as the α-synucleinopathies. In vivo, molecular chaperones, such as the small heat-shock proteins (sHsps), normally act to prevent protein aggregation; however, it remains to be determined how aggregation-prone α-syn evades sHsp chaperone action leading to its disease-associated deposition. This work examines the molecular mechanism by which two canonical sHsps, αB-crystallin (αB-c) and Hsp27, interact with aggregation-prone α-syn to prevent its aggregation in vitro. Both sHsps are very effective inhibitors of α-syn aggregation, but no stable complex between the sHsps and α-syn was detected, indicating that the sHsps inhibit α-syn aggregation via transient interactions. Moreover, the ability of these sHsps to prevent α-syn aggregation was dependent on the kinetics of aggregation; the faster the rate of aggregation (shorter the lag phase), the less effective the sHsps were at inhibiting fibril formation of α-syn. Thus, these findings indicate that the rate at which α-syn aggregates in cells may be a significant factor in how it evades sHsp chaperone action in the α-synucleinopathies.

Modulation of Amyloid β-Protein (Aβ) Assembly by Homologous C-Terminal Fragments as a Strategy for Inhibiting Aβ Toxicity.

Self-assembly of amyloid β-protein (Aβ) into neurotoxic oligomers and fibrillar aggregates is a key process thought to be the proximal event leading to development of Alzheimer's disease (AD). Therefore, numerous attempts have been made to develop reagents that disrupt this process and prevent the formation of the toxic oligomers and aggregates. An attractive strategy for developing such reagents is to use peptides derived from Aβ based on the assumption that such peptides would bind to full-length Aβ, interfere with binding of additional full-length molecules, and thereby prevent formation of the toxic species. Guided by this rationale, most of the studies in the last two decades have focused on preventing formation of the core cross-β structure of Aβ amyloid fibrils using β-sheet-breaker peptides derived from the central hydrophobic cluster of Aβ. Though this approach is effective in inhibiting fibril formation, it is generally inefficient in preventing Aβ oligomerization. An alternative approach is to use peptides derived from the C-terminus of Aβ, which mediates both oligomerization and fibrillogenesis. This approach has been explored by several groups, including our own, and led to the discovery of several lead peptides with moderate to high inhibitory activity. Interestingly, the mechanisms of these inhibitory effects have been found to be diverse, and only in a small percentage of cases involved interference with β-sheet formation. Here, we review the strategy of using C-terminal fragments of Aβ as modulators of Aβ assembly and discuss the relevant challenges, therapeutic potential, and mechanisms of action of such fragments.

Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases.

Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer’s, Parkinson’s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

Peptide backbone modification in the bend region of amyloid-β inhibits fibrillogenesis but not oligomer formation.

Current evidence suggests that oligomers of the amyloid-β (Aβ) peptide are involved in the cellular toxicity of Alzheimer's disease, yet their biophysical characterization remains difficult because of lack of experimental control over the aggregation process under relevant physiologic conditions. Here, we show that modification of the Aβ peptide backbone at Gly29 allows for the formation of oligomers but inhibits fibril formation at physiologic temperature and pH. Our results suggest that the putative bend region in Aβ is important for higher-order aggregate formation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Epigallocatechin Gallate Remodels Fibrils of Lattice Corneal Dystrophy Protein, Facilitating Proteolytic Degradation and Preventing Formation of Membrane-Permeabilizing Species

Lattice corneal dystrophy is associated with painful recurrent corneal erosions and amyloid corneal opacities induced by transforming growth factor β-induced protein (TGFBIp) that impairs vision. The exact mechanism of amyloid fibril formation in corneal dystrophy is unknown but has been associated with destabilizing mutations in the fourth fasciclin 1 (Fas1-4) domain of TGFBIp. The green tea compound epigallocatechin gallate (EGCG) has been found to inhibit fibril formation of various amyloidogenic proteins in vitro. In this study, we investigated the effect of EGCG as a potential treatment in lattice corneal dystrophy (LCD) using Fas1-4 with the naturally occurring LCD-inducing A546T mutation. A fewfold molar excess of EGCG was found to inhibit fibril formation in vitro by directing Fas1-4 A546T into stable EGCG-bound protein oligomers. Incubation with 2 molar equiv of EGCG led to a 4-fold reduction in the aggregates' membrane disruptive potential, potentially indicative of significantly lower cytotoxicity with regard to corneal erosions. EGCG did not induce oligomer formation by wild-type Fas1-4, indicating that treatment with EGCG would not interfere with the native function of the wild-type protein. Addition of EGCG to 10-day-old fibrils reduced fibril content in a dose-dependent manner. Proteinase K was found to reduce the light scattering of nontreated fibrils by 31% but reduced that of fibrils treated with 8 molar equiv of EGCG by 85%. This suggests that EGCG remodeling of fibril structure can facilitate aggregate removal by endogenous proteases and thus alleviate the protein deposits' light scattering symptoms.

Synthesis and structural analysis of halogen substituted fibril formation inhibitors of Human Transthyretin (TTR)

Transthyretin (TTR), a β-sheet-rich tetrameric protein, in equilibrium with an unstable amyloidogenic monomeric form is responsible for extracellular deposition of amyloid fibrils, is associated with the onset of neurodegenerative diseases, such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. One of the therapeutic strategies is to use small molecules to stabilize the TTR tetramer and thus curb amyloid fibril formation. Here, we report the synthesis, the in vitro evaluation of several halogen substituted 9-fluorenyl- and di-benzophenon-based ligands and their three-dimensional crystallographic analysis in complex with TTR. The synthesized compounds bind TTR and stabilize the tetramer with different potency. Of these compounds, 2c is the best inhibitor. The dual binding mode prevalent in the absence of substitutions on the fluorenyl ring, is disfavored by (2,7-dichloro-fluoren-9-ylideneaminooxy)-acetic acid (1b), (2,7-dibromo-fluoren-9-ylideneaminooxy)-acetic acid (1c) and (E/Z)-((3,4-dichloro-phenyl)-methyleneaminooxy)-acetic acid (2c), all with halogen substitutions.

Conformational Dynamics of the Human Islet Amyloid Polypeptide in a Membrane Environment: Toward the Aggregation Prone Form.

Human islet amyloid polypeptide (hIAPP) is a 37-residue peptide hormone, which upon misfolding changes from the physiologically active monomer into pathological amyloid fibril aggregates in the pancreas of type 2 diabetes mellitus patients. During this process, the insulin-producing pancreatic β-cells are damaged; however, the underlying mechanism of this mode of cytotoxicity remains elusive. It is known that anionic lipids accelerate amyloid fibril formation, implicating the importance of the cellular membrane in the process, and that a pH close to the level in the β-cell secretory granules (pH 5.5) inhibits amyloid fibril formation. Using all-atom molecular dynamics simulations, we have investigated the membrane-associated monomer state of α-helical hIAPP, analyzed specific interactions of hIAPP with a mixed anionic-zwitterionic lipid membrane and examined the influence of pH on the structure and dynamics of hIAPP and its interaction with the membrane. We find that hIAPP primarily interacts with the membrane by forming favorable interactions between anionic lipids and the positively charged residues in the N-terminal part of the peptide. Rationalizing experimental findings, the simulations show that the N-terminal part of the peptide interacts with the membrane in the lipid headgroup region. At neutral pH, the C-terminal part of the peptide, which contains the residues that initiate fibril formation, displays a highly dynamic, unfolded state, which interacts with the membrane significantly less than the N-terminal part. Such an unfolded form can be proposed to contribute to the acceleration of fibril formation. At low pH, protonation of His18 mediates a stronger interaction of the C-terminal part with the membrane, resulting in the immobilization of the C-terminal part on the membrane surface that might constitute a mechanism by which low pH inhibits fibril formation.

Hydrodynamic Effects on Amyloid-β Aggregation

The self-assembly of misfolded amyloid-β (Aβ) proteins into insoluble fibrils is strongly linked to the pathogenesis of Alzheimer's disease (AD). The development of new drugs capable to inhibit fibril formation requires the understanding of the mechanism leading to the formation, in the early stages, of metastable oligomers which are the main neurotoxic species.Our investigation enlighten the role of hydrodynamic interactions (HI) in the kinetics of β-amyloidogenesis, interactions which are essential for modelling such processes occurring in highly crowed environments like cells [1].Our approach is based on a new multi-scale and multi-physics method that couples Lattice Boltzmann and Molecular Dynamics (LBMD) techniques[2,3]. In our scheme solvent mediated interactions are included naturally. For the molecular system we adopt the high resolution coarse-grained model OPEP (Optimized Potential for Efficient Protein structure prediction)[4], developed in our laboratory. For the first time, we have performed quasi all-atoms simulations for very large systems containing thousands of amyloid peptides and approaching experimental concentration. After the correct tuning of the key parameter of our coupling in order to obtain the experimental diffusivity of Aβ monomer and small oligomers, we have demonstrated that HI speed up the aggregation process. In our poster we will present a detailed characterisation of the fluctuating clusters in term of their size and the structural organisation of the peptides.

A new crystal form of human transthyretin obtained with a curcumin derived ligand.

Transthyretin (TTR), a 54kDa homotetrameric protein that transports thyroxine (T4), has been associated with clinical cases of TTR amyloidosis for its tendency to aggregate to form fibrils. Many ligands with a potential to inhibit fibril formation have been studied by X-ray crystallography in complex with TTR. Unfortunately, the ligand is often found in ambiguous electron density that is difficult to interpret. The ligand validation statistics suggest over-interpretation, even for the most active compounds like diflunisal. The primary technical reason is its position on a crystallographic 2-fold axis in the most common crystal form. Further investigations with the use of polyethylene glycol (PEG) to crystallize TTR complexes have resulted in a new trigonal polymorph with two tetramers in the asymmetric unit. The ligand used to obtain this new polymorph, 4-hydroxychalcone, is related to curcumin. Here we evaluate this crystal form to understand the contribution it may bring to the study of TTR ligands complexes, which are often asymmetric.

BRICHOS binds to a designed amyloid-forming β-protein and reduces proteasomal inhibition and aggresome formation.

The BRICHOS domain is associated with proliferative, degenerative and amyloid diseases, and it has been shown to inhibit fibril formation and toxicity of the Alzheimer's disease-associated amyloid β-peptide. ProSP-C (prosurfactant protein C) BRICHOS binds to stretches of hydrophobic amino acid residues, which are unfolded or in β-strand conformation, suggesting that it may have broad anti-amyloid activity. We have studied the effect of the proSP-C BRICHOS domain on the designed amyloidogenic β-sheet proteins β17 and β23. β17 expressed in the secretory pathway of HEK (human embryonic kidney)-293 cells forms intracellular inclusions, whereas β23 is rapidly degraded. Co-expression of BRICHOS leads to a reduction in β17 inclusion size and increased levels of soluble β17 and β23. Furthermore, BRICHOS interacts with the β-proteins intracellularly, reduces their ubiquitination and decreases aggresome formation and proteasomal inhibition. Collectively, these data suggest that BRICHOS is capable of delaying the aggregation process and toxicity of amyloidogenic proteins in a generic manner.

Binding of ACE-inhibitors to in vitro and patient-derived amyloid-β fibril models.

Currently, no drugs exist that can prevent or reverse Alzheimer's disease, a neurodegenerative disease associated with the presence, in the brain, of plaques that are composed of β-amyloid (Aβ) peptides. Recent studies suggest that angiotensin-converting enzyme (ACE) inhibitors, a set of drugs used to treat hypertension, may inhibit amyloid formation in vitro. In the present study, we investigate through computer simulations the binding of ACE inhibitors to patient-derived Aβ fibrils and contrast it with that of ACE inhibitors binding to in vitro generated fibrils. The binding affinities of the ACE inhibitors are compared with that of Congo red, a dye that is used to identify amyloid structures and that is known to be a weak inhibitor of Aβ aggregation. We find that ACE inhibitors have a lower binding affinity to the patient-derived fibrils than to in vitro generated ones. For patient-derived fibrils, their binding affinities are even lower than that of Congo red. Our observations raise doubts on the hypothesis that these drugs inhibit fibril formation in Alzheimer patients by interacting directly with the amyloids.

Looking for a generic inhibitor of amyloid-like fibril formation among flavone derivatives.

A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrils in vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid.

The membrane-active amphibian peptide caerin 1.8 inhibits fibril formation of amyloid β1-42

The amphibian host-defense peptide caerin 1.8 [1GLFKVLGSV10AKHLLPHVVP20VIAEKL(NH2)] inhibits fibril formation of amyloid β 1−42 [1DAEFRHDSG10YEVHHQKLVF20FAEDVGSNKG30AIIGLMVGGV40VIA] [Aβ42] (the major precursor of the extracellular fibrillar deposits of Alzheimer's disease). Some truncated forms of caerin 1.8 also inhibit fibril formation of Aβ42. For example, caerin 1.8 (1–13) [1GLFKVLGSV10AKHL(NH2) and caerin 1.8 (22–25) [KVLGSV10AKHLLPHVVP20VIAEKL(NH2)] show 85% and 75% respectively of the inhibition activity of the parent caerin 1.8. The synthetic peptide KLVFFKKKKKK is a known inhibitor of Aβ42 fibril formation, and was used as a standard in this study. Caerin 1.8 is the more effective fibril inhibitor. IC50 values (±15%) are caerin 1.8 (75μM) and KLVFFKKKKKK (370μM). MALDI mass spectrometry shows the presence of a small peak corresponding to a protonated 1:1 adduct [caerin 1.8/Aβ42]H+. Molecular dynamics simulation suggests that both hydrogen bonding and hydrophobic interactions between Aβ42 and caerin 1.8 facilitate the formation of a 1:1 complex in water. Fibril formation from Aβ42 has been proposed to be based around the 16KLVF20F region of Aβ42; this region in the 1:1 complex is partially blocked from attachment of a further molecule of Aβ42.

Stabilization of Insulin by Adsorption on a Hydrophobic Silane Self-Assembled Monolayer.

The interaction between many proteins and hydrophobic functionalized surfaces is known to induce β-sheet and amyloid fibril formation. In particular, insulin has served as a model peptide to understand such fibrillation, but the early stages of insulin misfolding and the influence of the surface have not been followed in detail under the acidic conditions relevant to the synthesis and purification of insulin. Here we compare the adsorption of human insulin on a hydrophobic (-CH3-terminated) silane self-assembled monolayer to a hydrophilic (-NH3(+)-terminated) layer. We monitor the secondary structure of insulin with Fourier transform infrared attenuated total reflection and side-chain orientation with sum frequency spectroscopy. Adsorbed insulin retains a close-to-native secondary structure on both hydrophobic and hydrophilic surfaces for extended periods at room temperature and converts to a β-sheet-rich structure only at elevated temperature. We propose that the known acid stabilization of human insulin and the protection of the aggregation-prone hydrophobic domains on the insulin monomer by adsorption on the hydrophobic surface work together to inhibit fibril formation at room temperature.

X-ray crystal structure and activity of fluorenyl-based compounds as transthyretin fibrillogenesis inhibitors

Transthyretin (TTR) is a 54 kDa homotetrameric protein that transports thyroxine (T4) and retinol (vitamin A), through its association with retinol binding protein (RBP). Under unknown conditions, it aggregates to form fibrils associated with TTR amyloidosis. Ligands able to inhibit fibril formation have been studied by X-ray crystallography. The use of polyethylene glycol (PEG) instead of ammonium sulphate or citrate has been evaluated as an alternative to obtain new TTR complexes with (R)-3-(9-fluoren-9-ylideneaminooxy)-2-methyl-N-(methylsulfonyl) propionamide (48R(1)) and 2-(9H-fluoren-9-ylideneaminooxy) acetic acid (ES8(2)). The previously described fluorenyl based inhibitors (S)-3-((9H-fluoren-9-ylideneamino)oxy)-2-methylpropanoic acid (6BD) and 3-((9H-fluoren-9-ylideneamino)oxy)propanoic acid (7BD) have been re-evaluated with the changed crystallization method. The new TTR complexes with compounds of the same family show that the 9-fluorenyl motif can occupy alternative hydrophobic binding sites. This augments the potential use of this scaffold to yield a large variety of differently substituted mono-aryl compounds able to inhibit TTR fibril formation.

Aqueous, Unfolded OmpA Forms Amyloid-Like Fibrils upon Self-Association.

Unfolded outer membrane beta-barrel proteins have been shown to self-associate in the absence of lipid bilayers. We previously investigated the formation of high molecular weight species by OmpA, with both the transmembrane domain alone and the full-length protein, and discovered that the oligomeric form contains non-native β-sheet structure. We have further probed the conformation of self-associated OmpA by monitoring binding to Thioflavin T, a dye that is known to bind the cross-β a structure inherent in amyloid fibrils, and by observing the species by electron microscopy. The significant increase in fluorescence indicative of Thioflavin T binding and the appearance of fibrillar species by electron microscopy verify that the protein forms amyloid-like fibril structures upon oligomerization. These results are also consistent with our previous kinetic analysis of OmpA self-association that revealed a nucleated growth polymerization mechanism, which is frequently observed in amyloid formation. The discovery of OmpA’s ability to form amyloid-like fibrils provides a new model protein with which to study fibrillization, and implicates periplasmic chaperone proteins as capable of inhibiting fibril formation.

Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis.

Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors.