Inhibits Aβ Fibril Formation Research Articles

The D-amino acid peptide D3 reduces amyloid fibril boosted HIV-1 infectivity

BackgroundAmyloid fibrils such as Semen-Derived Enhancer of Viral Infection (SEVI) or amyloid-β-peptide (Aβ) enhance HIV-1 attachment and entry. Inhibitors destroying or converting those fibrils into non-amyloidogenic aggregates effectively reduce viral infectivity. Thus, they seem to be suitable as therapeutic drugs expanding the current HIV-intervening repertoire of antiretroviral compounds.FindingsIn this study, we demonstrate that the small D-amino acid peptide D3, which was investigated for therapeutic studies on Alzheimer’s disease (AD), significantly reduces both SEVI and Aβ fibril boosted infectivity of HIV-1.ConclusionsSince amyloids could play an important role in the progression of AIDS dementia complex (ADC), the treatment of HIV-1 infected individuals with D3, that inhibits Aβ fibril formation and converts preformed Aβ fibrils into non-amyloidogenic and non-fibrillar aggregates, may reduce the vulnerability of the central nervous system of HIV patients for HIV associated neurological disorders.

3,3',4',5'-Tetrahydroxyflavone induces formation of large aggregates of amyloid β protein.

Amyloid β protein (Aβ) self-assembles into insoluble fibrils, and forms the senile plaques associated with Alzheimer's disease. 3,3',4',5'-Tetrahydroxyflavone, a synthetic analogue of the natural flavonoid fisetin, has been found to potently inhibit Aβ fibril formation. In the present study, we investigated how inhibition of Aβ fibril formation by this flavonoid affects Aβ conformation and neurotoxicity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of Aβ1-42 (20 µM) incubated with or without 3,3',4',5'-tetrahydroxyflavone demonstrated that 3,3',4',5'-tetrahydroxyflavone (100 µM) rapidly caused formation of atypical Aβ conformers, which appeared as a very broad, smear-like band in the high molecular weight region and were distinguishable from soluble Aβ oligomers or mature Aβ fibrils. Transmission electron microscopy (TEM) revealed that large spherical Aβ aggregates were preferentially formed in the presence of 3,3',4',5'-tetrahydroxyflavone. The SDS-resistant, smear-like band on SDS-PAGE and the large spherical aggregates in TEM both disappeared after heat treatment (100°C, 10 min). Furthermore, a neurotoxicity assay with cultured rat hippocampal neurons demonstrated that Aβ incubated with 3,3',4',5'-tetrahydroxyflavone was significantly less toxic than Aβ incubated without the flavonoid. These results suggest that the newly synthesized fisetin analogue 3,3',4',5'-tetrahydroxyflavone directly produces atypical, large Aβ aggregates and reduces Aβ toxicity.

Small Amphipathic Molecules Modulate Secondary Structure and Amyloid Fibril-forming Kinetics of Alzheimer Disease Peptide Aβ1–42

Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation.

Design, Synthesis and Evaluation of Novel 2‐(Aminoalkyl)‐isoindoline‐1,3‐dione Derivatives as Dual‐Binding Site Acetylcholinesterase Inhibitors

A new series of 2-(diethylaminoalkyl)-isoindoline-1,3-dione derivatives intended as dual binding site cholinesterase inhibitors were designed using molecular modeling and evaluated as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and the formation of the β-amyloid (Aβ) plaques. For AChE inhibitory activity, the spectrophotometric method of Ellman and the electrophoretically mediated microanalysis assay were used, giving good results. Most of the synthesized compounds had AChE inhibitory activity with IC(50) values ranging from IC(50) = 0.9 to 19.5 µM and weak Aβ anti-aggregation inhibitory activity. These results support the outcome of docking studies which tested compounds targeting both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. The most promising selective AChE inhibitors are compounds 10 (IC(50) = 1.2 µM) and 11 (IC(50) = 1.1 µM), with 6-7 methylene chains, which also inhibit Aβ fibril formation.

3,3′,4′,5,5′-Pentahydroxyflavone is a potent inhibitor of amyloid β fibril formation

The natural flavonoid fisetin (3,3′,4′,7-tetrahydroxyflavone) is neurotrophic and prevents fibril formation of amyloid β protein (Aβ). It is a promising lead compound for the development of therapeutic drugs for Alzheimer's disease. To find even more effective drugs based on the structure of fisetin, we synthesized a series of fisetin analogues lacking the 7-hydroxyl group and compared their effects on Aβ fibril formation determined by the thioflavin T fluorescence assay. 3,3′,4′-Trihydroxyflavone and 3′,4′-dihydroxyflavone inhibited Aβ fibril formation more potently than fisetin or 3′,4′,7-trihydroxyflavone, suggesting that the 7-hydroxy group is not necessary for anti-amyloidogenic activity. 3,3′,4′,5′-Tetrahydroxyflavone and 3′,4′,5′-trihydroxyflavone inhibited Aβ fibril formation far more potently than 3,3′,4′-trihydroxyflavone and 3′,4′-dihydroxyflavone, suggesting that 3′,4′,5′-trihydroxyl group of the B ring is crucial for the anti-amyloidogenic activity of flavonoids. Based on the structure–activity relationship, we synthesized 3,3′,4′,5,5′-pentahydroxyflavone, and confirmed that this compound is the most potent inhibitor of Aβ fibril formation among fisetin analogues that have been tested. Cytotoxicity assay using rat hippocampal neuron cultures demonstrated that Aβ preincubated with 3,3′,4′,5,5′-pentahydroxyflavone was significantly less toxic than Aβ preincubated with vehicle. 3,3′,4′,5,5′-Pentahydroxyflavone could be a new therapeutic drug candidate for the treatment of Alzheimer's disease.

Crocetin inhibits beta-amyloid fibrillization and stabilizes beta-amyloid oligomers

Aggregation of a peptide, beta-amyloid (Aβ), is a hallmark molecular process found in Alzheimer’s disease (AD). During Aβ aggregation, oligomeric and fibrillar Aβ are formed, and these molecular self-assembly steps are implicated in generation of toxic effects in AD. Crocetin is a natural carotenoid dicarboxyl acid displaying various pharmaceutical effects and may be co-localized with Aβ mediated by human serum albumin. In the study presented here, we examined the effects of crocetin on Aβ aggregation in three different molecular pathways. Our results demonstrate that crocetin inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils. Moreover, crocetin caused stabilization of Aβ oligomers and prevented their conversion into Aβ fibrils. Our study reveals potential pathological and pharmaceutical implication of crocetin in AD and suggests possible application of crocetin for currently limited structural studies on unstable Aβ oligomers.

Neuronal Production of Transthyretin in Human and Murine Alzheimer's Disease: Is It Protective?

Transthyretin (TTR), a systemic amyloid precursor in the human TTR amyloidoses, interacts with β-amyloid (Aβ) in vitro, inhibits Aβ fibril formation, and suppresses the Alzheimer's disease (AD) phenotype in APP23 mice bearing a human APP gene containing the Swedish autosomal dominant AD mutation. In the present study, we show that TTR is a neuronal product upregulated in AD. Immunohistochemical analysis reveals that, in contrast to brains from non-demented age-matched individuals and control mice, the majority of hippocampal neurons from human AD and all those from the APP23 mouse brains contain TTR. Quantitative PCR for TTR mRNA and Western blot analysis show that primary neurons from APP23 mice transcribe TTR mRNA, and the cells synthesize and secrete TTR protein. TTR mRNA abundance is greatly increased in cultured cortical and hippocampal embryonic neurons and cortical lysates from adult APP23 mice. Antibodies specific for TTR and Aβ pulled down TTR/Aβ complexes from cerebral cortical extracts of APP23 mice and some human AD patients but not from control brains. In complementary tissue culture experiments, recombinant human TTR suppressed the cytotoxicity of soluble Aβ aggregates added to mouse neurons and differentiated human SH-SY5Y neuroblastoma cells. The findings that production of Aβ, its precursor, or its related peptides induces neuronal TTR transcription and synthesis and the presence of Aβ/TTR complexes in vivo suggest that increased TTR production coupled with interaction between TTR and Aβ and/or its related peptides may play a role in natural resistance to human AD.

Butanol extract of Ecklonia cava prevents production and aggregation of beta-amyloid, and reduces beta-amyloid mediated neuronal death

Beta-amyloid (Aβ) is a major pathogenic peptide for Alzheimer’s disease (AD) and is generated by the processing of amyloid precursor protein (APP). The Aβ monomers aggregate into oligomeric and fibrillar forms which have been implicated as the toxic species inducing the neuronal dysfunction. Brown algae Ecklonia cava is known for its anti-oxidant and anti-inflammatory functions. Therefore, we tested the effect of E. cava extract on the production and aggregation of Aβ peptides. The butanol extract of E. cava reduced Aβ secretion from HEK293 cells expressing APP with Swedish mutation and increased soluble APPα and C-terminal fragment-α (CTFα), of which activity was similar to BACE (β-site of APP cleaving enzyme) inhibitors. Furthermore, the extract inhibited Aβ oligomerization, particularly mid-size oligomer formation, confirmed by the ultrastructural morphology. Congo red, thioflavin T assays, and electron microscopy showed that the extract inhibited Aβ fibril formation effectively. Finally, the extract protected primary cortical neurons from various Aβ-induced cell deaths, especially oligomer-induced death. Although further study is needed to test the effectiveness of the extract in vivo, our results demonstrate, for the first time, that the butanol extract of E. cava could be used as an anti-Aβ agent for AD therapeutics.

An Aqueous Extract of Withania somnifera Root Inhibits Amyloid β Fibril Formation In Vitro

The ability of an aqueous extract of W. somnifera L. Dunal (Family: Solanaceae) roots to inhibit fibril formation by the amyloid-β peptide in vitro was investigated. W. somnifera is used extensively in traditional Ayurvedic medicine as a nerve tonic with reputed memory enhancing properties. Inhibition of fibrillogenesis measured by transmission electron microscopy and ThT fluorescence assay showed that an aqueous extract of W. somnifera strongly inhibited Aβ fibril formation in a concentration-dependent manner, when compared with control samples. These results suggest that the aqueous extract of W. somnifera root has an ability to inhibit the formation of mature amyloid-β fibrils in vitro, which are known to lead to amyloid plaque formation in vivo.

Chronic Intranasal Treatment with an Anti-Aβ30-42 scFv Antibody Ameliorates Amyloid Pathology in a Transgenic Mouse Model of Alzheimer's Disease

Amyloid-beta peptide (Aβ)-directed active and passive immunization therapeutic strategies reduce brain levels of Aβ, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aβ-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aβ, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aβ-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aβ fibril formation and Aβ-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aβ from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aβ-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aβ-directed immunotherapy can reduce Aβ deposition in brain vessels in transgenic mice.

N-terminal Domain of Myelin Basic Protein Inhibits Amyloid β-Protein Fibril Assembly

Accumulation of amyloid β-protein (Aβ) into brain parenchymal plaques and the cerebral vasculature is a pathological feature of Alzheimer disease and related disorders. Aβ peptides readily form β-sheet-containing oligomers and fibrils. Previously, we reported a strong interaction between myelin basic protein (MBP) and Aβ peptides that resulted in potent inhibition of fibril assembly (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720-4727). MBP is recognized as a highly post-translationally modified protein. In the present study, we demonstrate that human MBP purified from either brain or a bacterial recombinant expression system comparably bound to Aβ and inhibited Aβ fibril assembly indicating that post-translational modifications are not required for this activity. We also show that purified mouse brain MBP and recombinantly expressed mouse MBP similarly inhibited Aβ fibril formation. Through a combination of biochemical and ultrastructural techniques, we demonstrate that the binding site for Aβ is located in the N-terminal 64 amino acids of MBP and that a stable peptide (MBP1) comprising these residues was sufficient to inhibit Aβ fibrillogenesis. Under conditions comparable with those used for Aβ, the fibrillar assembly of amylin, another amyloidogenic peptide, was not inhibited by MBP1, although MBP1 still bound to it. This observation suggests that the potent inhibitory effect of MBP on fibril formation is not general to amyloidogenic peptides. Finally, MBP1 could prevent the cytotoxic effects of Aβ in primary cortical neurons. Our findings suggest that inhibition of Aβ fibril assembly by MBP, mediated through its N-terminal domain, could play a role in influencing amyloid formation in Alzheimer disease brain and corresponding mouse models.

Sequestration of Copper from β-Amyloid Promotes Selective Lysis by Cyclen-Hybrid Cleavage Agents

Decelerated degradation of beta-amyloid (Abeta) and its interaction with synaptic copper may be pathogenic in Alzheimer disease. Recently, Co(III)-cyclen tagged to an aromatic recognition motif was shown to degrade Abeta in vitro. Here, we report that apocyclen attached to selective Abeta recognition motifs (KLVFF or curcumin) can capture copper bound to Abeta and use the Cu(II) in place of Co(III) to become proteolytically active. The resultant complexes interfere with Abeta aggregation, degrade Abeta into fragments, preventing H2O2 formation and toxicity in neuronal cell culture. Because Abeta binds Cu in amyloid plaques, apocyclen-tagged targeting molecules may be a promising approach to the selective degradation of Abeta in Alzheimer disease. The principle of copper capture could generalize to other amyloidoses where copper is implicated.

Structural requirements for the flavonoid fisetin in inhibiting fibril formation of amyloid β protein

Fisetin (3,3′,4′,7-tetrahydroxyflavone) has been found to be neuroprotective, induce neuronal differentiation, enhance memory, and inhibit the aggregation of the amyloid β protein (Aβ) that may cause the progressive neuronal loss in Alzheimer's disease. The diverse collection of biological activities of this compound may lead to a new type of therapeutic drug for Alzheimer's disease. As the first step to design even more effective drugs based upon the structure of fisetin, the present study investigated the structural requirements for the anti-amyloidogenic activity of fisetin by comparing the effects of several structurally related flavonoids on Aβ fibril formation in vitro. Aβ1-42 (20 μM) and the flavonoids were incubated for 0–48 h at 37 °C, and fibril formation was quantitatively determined by the thioflavin T fluorescence assay. Among ten flavonoids tested, fisetin, 3′,4′,7-trihydroxylflavone, 3,3′,4′-trihydroxyflavone, luteolin, quercetin and myricetin inhibited Aβ fibril formation. On the other hand, 3,3′,7-trihydroxyflavone, 5-deoxykaempferol, chrysin and kaempferol enhanced Aβ fibril formation. These results suggest that the 3′,4′-dihydroxyl group, but not the 3- or 7-hydroxyl group, is essential for the inhibitory effect of fisetin on Aβ fibril formation.

Salvianolic acid B inhibits Aβ fibril formation and disaggregates preformed fibrils and protects against Aβ-induced cytotoxicty

One of the major pathological features of Alzheimer's disease (AD) is the appearance of senile plaques characterized by extracellular aggregation of amyloid β-peptide (Aβ) fibrils. Inhibition of Aβ fibril aggregation is therefore viewed as one possible method to halt the progression of AD. Salvianolic acid B (Sal B) is an active ingredient isolated from Salvia miltiorrhiza, a Chinese herbal medicine commonly used for the treatment of cardiovascular and cerebrovascular disorders. Recent findings show that Sal B prevents Aβ-induced cytotoxicity in a rat neural cell line. To understand the mechanism of Sal B-mediated neuroprotection, its effects on the inhibition of Aβ1–40 fibril formation and destabilization of the preformed Aβ1–40 fibrils were studied. The results were obtained using Thioflavin T fluorescence assay and Aβ aggregating immunoassay. We found that Sal B can inhibit fibril aggregation (IC 50: 1.54–5.37 μM) as well as destabilize preformed Aβ fibril (IC 50: 5.00–5.19 μM) in a dose- and time-dependent manner. Sal B is a better aggregation inhibitor than ferulic acid but less active than curcumin in the inhibition of Aβ1–40 aggregation. In electron microscope study, Sal B-treated Aβ1–40 fibrils are seen in various stages of shortening or wrinkling with numerous deformed aggregates of amorphous structure. Circular dichroism data indicate that Sal B dose dependently prevents the formation of β-structured aggregates of Aβ1–40. Addition of preincubated Sal B with Aβ1–42 significantly reduces its cytotoxic effects on human neuroblastoma SH-SY5Y cells. These results suggest that Sal B has therapeutic potential in the treatment of AD, and warrant its study in animal models.

High density lipoproteins bind Aβ and apolipoprotein C-II amyloid fibrils

Disease-associated amyloid deposits contain both fibrillar and nonfibrillar components. The majority of these amyloid components originate or coexist in the bloodstream. To understand the nature of the interaction between the nonfibrillar and fibrillar components, we have developed a centrifugation method to isolate fibril binding proteins from human serum. Amyloid fibrils composed of either Abeta peptide or apolipoprotein C-II (apoC-II) cosedimented with specific serum proteins. Gel electrophoresis, mass spectrometry peptide fingerprinting, and Western analysis identified the major binding species as proteins found in HDL particles, including apoA-I, apoA-II, apoE, clusterin, and serum amyloid A. Sedimentation analysis showed that purified human HDL and recombinant apoA-I lipid particles bound directly to Abeta and apoC-II amyloid fibrils. These studies reveal a novel function of HDL that may contribute to the well-established protective effect of this lipoprotein class in heart disease.

A hybrid molecule that prohibits amyloid fibrils and alleviates neuronal toxicity induced by β-amyloid (1–42)

Inhibition of oligomeric amyloid β (Aβ) peptide or fibril formation has emerged as a major therapeutic target for developing new drugs for Alzheimer’s disease. We focused on developing inhibitors by synthesizing hybrid molecules of ferulic acid and styryl benzene, which has been known as a fibril binder. Initially, cell-based assay was carried out to evaluate the effective compound. A selected effector, 1, alleviated the Aβ-induced neuronal toxicity in differentiated SH-SY5Y human neuroblastoma cells. The effector could also inhibit Aβ fibril formation, monitored by thioflavin T fluorescence intensity assay and transmitted electron microscopic images. A strong binding affinity of 1 to non-fibrous monomer-like Aβ, which was immobilized to a surface chip, was measured using a surface plasmon resonance technique. The data suggest that the effector shifts the equilibrium of multimeric Aβ, inhibiting the pathogenic oligomer or fibril formation.

Common structural features determine the effectiveness of carvedilol, daunomycin and rolitetracycline as inhibitors of Alzheimer β-amyloid fibril formation

One of the major pathological features of Alzheimer's disease is the deposition of β-amyloid peptide (Aβ). Cellular toxicity has been shown to be associated with fibrillar forms of Aβ; preventing this fibril formation is therefore viewed as a possible method of slowing disease progression in Alzheimer's disease. With the use of a series of tetracyclic and carbazole-type compounds as inhibitors of Aβ fibril formation, we here describe a number of common structural features that seem to be associated with the inhibitory properties of these agents. Compounds such as carvedilol, rolitetracycline and daunomycin, which are shown to inhibit Aβ fibril formation, also prevent the formation of species of peptide that demonstrate biological activity in a human neuroblastoma cell line. Molecular modelling data suggest that these compounds have in common the ability to adopt a specific three-dimensional pharmacophore conformation that might be essential for binding to Aβ and preventing it from forming fibrils. Understanding such drug-peptide interactions might aid the development of disease-modifying agents.

Common structural features determine the effectiveness of carvedilol, daunomycin and rolitetracycline as inhibitors of Alzheimer β-amyloid fibril formation

One of the major pathological features of Alzheimer's disease is the deposition of beta-amyloid peptide (Abeta). Cellular toxicity has been shown to be associated with fibrillar forms of Abeta; preventing this fibril formation is therefore viewed as a possible method of slowing disease progression in Alzheimer's disease. With the use of a series of tetracyclic and carbazole-type compounds as inhibitors of Abeta fibril formation, we here describe a number of common structural features that seem to be associated with the inhibitory properties of these agents. Compounds such as carvedilol, rolitetracycline and daunomycin, which are shown to inhibit Abeta fibril formation, also prevent the formation of species of peptide that demonstrate biological activity in a human neuroblastoma cell line. Molecular modelling data suggest that these compounds have in common the ability to adopt a specific three-dimensional pharmacophore conformation that might be essential for binding to Abeta and preventing it from forming fibrils. Understanding such drug-peptide interactions might aid the development of disease-modifying agents.

Inhibition of Alzheimer's Amyloidosis by Peptides That Prevent β-Sheet Conformation

Amyloid β-peptide (Aβ) is a major fibrillar component of neuritic plaques in Alzheimer's disease (AD) brains and is related to the pathogenesis of the disease. We hypothesized that amyloid formation could be inhibited by peptides homologous to Aβ (position 17-21) with a similar degree of hydrophobicity, but with a very low propensity to adopt a β-sheet conformation by incorporating proline residues (anti-β-sheet peptides or β-sheet inhibitors). An 11-residue peptide with these characteristics binds to Aβ, inhibits Aβ fibril formation and partially disaggregates preformed fibrilsin vitro.Shorter anti-β-sheet peptides and analogs containing D-amino acids are also able to inhibit Aβ fibrillogenesis. The latter are more resistant to proteolytic degradation and may serve as a starting point to design more efficient peptides derivatives to inhibit amyloidogenesis in vivo.