Roni Scherzer-Attali

Orally administrated cinnamon extract reduces β-amyloid oligomerization and corrects cognitive impairment in Alzheimer's disease animal models.

An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of β-amyloid polypeptide (Aβ) play a key role in Alzheimers disease (AD) pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aβ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aβ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt), which markedly inhibits the formation of toxic Aβ oligomers and prevents the toxicity of Aβ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aβ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aβ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aβ species formation in AD through the utilization of a compound that is currently in use in human diet.

Complete phenotypic recovery of an Alzheimer's disease model by a quinone-tryptophan hybrid aggregation inhibitor.

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimers disease-associated β-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Aβ oligomerization and fibrillization, as well as the cytotoxic effect of Aβ oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimers disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Aβ while immuno-staining of the 3rd instar larval brains showed a significant reduction in Aβ accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Aβ. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimers disease.

Generic inhibition of amyloidogenic proteins by two naphthoquinone–tryptophan hybrid molecules

Amyloid formation is associated with several human diseases including Alzheimers disease (AD), Parkinsons disease, Type 2 Diabetes, and so forth, no disease modifying therapeutics are available for them. Because of the structural similarities between the amyloid species characterizing these diseases, (despite the lack of amino acid homology) it is believed that there might be a common mechanism of toxicity for these conditions. Thus, inhibition of amyloid formation could be a promising disease‐modifying therapeutic strategy for them. Aromatic residues have been identified as crucial in formation and stabilization of amyloid structures. This finding was corroborated by high‐resolution structural studies, theoretical analysis, and molecular dynamics simulations. Amongst the aromatic entities, tryptophan was found to possess the most amyloidogenic potential. We therefore postulate that targeting aromatic recognition interfaces by tryptophan could be a useful approach for inhibiting the formation of amyloids. Quinones are known as inhibitors of cellular metabolic pathways, to have anti‐ cancer, anti‐viral and anti‐bacterial properties and were shown to inhibit aggregation of several amyloidogenic proteins in vitro. We have previously described two quinone‐tryptophan hybrids which are capable of inhibiting amyloid‐beta, the protein associated with AD pathology, both in vitro and in vivo. Here we tested their generic properties and their ability to inhibit other amyloidogenic proteins including α‐synuclein, islet amyloid polypeptide, lysozyme, calcitonin, and insulin. Both compounds showed efficient inhibition of all five proteins examined both by ThT fluorescence analysis and by electron microscope imaging. If verified in vivo, these small molecules could serve as leads for developing generic anti‐amyloid drugs. Proteins 2012;

Naphthoquinone-tyrptophan reduces neurotoxic Aβ*56 levels and improves cognition in Alzheimer's disease animal model.

An increasing body of evidence indicates a role for oligomers of the amyloid-β peptide (Aβ) in the neurotoxicity of this peptide and the pathology of Alzheimers disease (AD). Several neurotoxic oligomeric forms of Aβ have been noted ranging from the larger Amyloid β-Derived Diffusible Ligands (ADDLs) to smaller trimers and dimers of Aβ. More recently a dodecameric form of Aβ with a 56 kDa molecular weight, denoted Aβ*56, was shown to cause memory impairment in AD model mice. Here, we present for the first time a potential therapeutic strategy for AD that targets the early stages in the formation of neurotoxic Aβ*56 oligomers using a modified quinone-Tryptophan small molecule N-(3-chloro-1,4-dihydro-1,4-dioxo-2-naphthalenyl)-L-Tryptophan (Cl-NQTrp). Using NMR spectroscopy we show that this compound binds the aromatic recognition core of Aβ and prevents the formation of oligomers. We assessed the effect of Cl-NQTrp in vivo in transgenic flies expressing Aβ(1-42) in their nervous system. When these flies were fed with Cl-NQTrp a marked alleviation of their Aβ-engendered reduced life span and defective locomotion was observed. Finally, intraperitoneal injection of Cl-NQTrp into an aggressive AD mouse model reduced the level of the Aβ*56 species in their brain and reversed their cognitive defects. Further experiments should assess whether this is a direct effect of the drug in the brain or an indirect peripheral effect. This is the first demonstration that targeted reduction of Aβ*56 results in amelioration of AD symptoms. This second generation of tryptophan-modified naphthoquinones could therefore serve as potent disease modifying therapeutic for AD.

Monitoring and Targeting the Initial Dimerization Stage of Amyloid Self‐Assembly

Amyloid deposits are pathological hallmark of a large group of human degenerative disorders of unrelated etiologies. While accumulating evidence suggests that early oligomers may account for tissue degeneration, most detection tools do not allow the monitoring of early association events. Here we exploit bimolecular fluorescence complementation analysis to detect and quantify the dimerization of three major amyloidogenic polypeptides; islet amyloid polypeptide, β-amyloid and α-synuclein. The constructed systems provided direct visualization of protein-protein interactions in which only assembled dimers display strong fluorescent signal. Potential inhibitors that interfere with the initial intermolecular interactions of islet amyloid polypeptide were further identified using this system. Moreover, the identified compounds were able to inhibit the aggregation and cytotoxicity of islet amyloid polypeptide, demonstrating the importance of targeting amyloid dimer formation for future drug development.

Naphthoquinone-Tryptophan Hybrid Inhibits Aggregation of the Tau-Derived Peptide PHF6 and Reduces Neurotoxicity.

Tauopathies, such as Alzheimers disease (AD), are a group of disorders characterized neuropathologically by intracellular toxic accumulations of abnormal protein aggregates formed by misfolding of the microtubule-associated protein tau. Since protein self-assembly appears to be an initial key step in the pathology of this group of diseases, intervening in this process can be both a prophylactic measure and a means for modifying the course of the disease for therapeutic purposes. We and others have shown that aromatic small molecules can be effective inhibitors of aggregation of various protein assemblies, by binding to the aromatic core in aggregation-prone motifs and preventing their self-assembly. Specifically, we have designed a series of small aromatic naphthoquinone-tryptophan hybrid molecules as candidate aggregation inhibitors of β -sheet based assembly and demonstrated their efficacy toward inhibiting aggregation of the amyloid-β peptide, another culprit of AD, as well as of various other aggregative proteins involved in other protein misfolding diseases. Here we tested whether a leading naphthoquinone-tryptophan hybrid molecule, namely NQTrp, can be repurposed as an inhibitor of the aggregation of the tau protein in vitro and in vivo. We show that the molecule inhibits the in vitro assembly of PHF6, the aggregation-prone fragment of tau protein, reduces hyperphosphorylated tau deposits and ameliorates tauopathy-related behavioral defect in an established transgenic Drosophila model expressing human tau. We suggest that NQTrp, or optimized versions of it, could act as novel disease modifying drugs for AD and other tauopathies.

Methylations of Tryptophan-Modified Naphthoquinone Affect Its Inhibitory Potential toward Aβ Aggregation

Aggregation of amyloid beta (Aβ) is the hallmark of Alzheimers disease (AD). Small molecules inhibiting Aβ can be valuable therapeutics for AD. We have previously reported that 1,4-naphthoquinon-2-yl-l-tryptophan (NQTrp), reduces aggregation and oligomerization of Aβ in vitro and in vivo. In silico analysis further showed that certain functional groups of NQTrp, not in the aromatic rings, are also involved in binding and inhibiting Aβ. To better understand the exact mode of action and identify the groups crucial for NQTrp inhibitory activity, we conducted structure-activity analysis. Four derivatives of NQTrp were studied in silico: a D-isomer, two single-methylated and one double-methylated derivative. In silico results showed that the NQTrp groups involved in hydrogen bonds are the anilinic NH (i.e., the NH linker between the quinone and tryptophan moieties), the quinonic carbonyls, and the carboxylic acid. These predictions were supported by in vitro results. Our results should aid in designing improved small-molecule inhibitors of Aβ aggregation for treating AD.

Quantitative structure–activity relationship analysis of β-amyloid aggregation inhibitors

Inhibiting the aggregation process of the β-amyloid peptide is a promising strategy in treating Alzheimer’s disease. In this work, we have collected a dataset of 80 small molecules with known inhibition levels and utilized them to develop two comprehensive quantitative structure–activity relationship models: a Bayesian model and a decision tree model. These models have exhibited high predictive accuracy: 87% of the training and test sets using the Bayesian model and 89 and 93% of the training and test sets, respectively, by the decision tree model. Subsequently these models were used to predict the activities of several new potential β-amyloid aggregation inhibitors and these predictions were indeed validated by in vitro experiments. Key chemical features correlated with the inhibition ability were identified. These include the electro-topological state of carbonyl groups, AlogP and the number of hydrogen bond donor groups. The results demonstrate the feasibility of the developed models as tools for rapid screening, which could help in the design of novel potential drug candidates for Alzheimer’s disease.

Cl-NQTrp Alleviates Tauopathy Symptoms in a Model Organism through the Inhibition of Tau Aggregation-Engendered Toxicity

Alzheimers disease (AD) is the most abundant tauopathy and is characterized by Aβ-derived plaques and tau-derived tangles, resulting from the unfolding of the corresponding monomeric subunits into ordered β-sheet oligomers and fibrils. Intervening in the toxic aggregation process is a promising therapeutic approach, but, to date, a disease-modifying therapy is neither available for AD nor for other tauopathies. Along these lines, we have previously demonstrated that a small naphthoquinone-tryptophan hybrid, termed NQTrp, is an effective modulator of tauopathy in vitro and in vivo. However, NQTrp is difficult to synthesize and is not very stable. Therefore, we tested whether a more stable and easier-to-synthesize modified version of NQTrp, containing a Cl ion, namely Cl-NQTrp, is also an effective inhibitor of tau aggregation in vitro and in vivo. Cl-NQTrp was previously shown to efficiently inhibit the aggregation of various amyloidogenic proteins and peptides. We demonstrate that Cl-NQTrp inhibits the in vitro assembly of PHF6, the aggregation-prone fragment of tau, and alleviates tauopathy symptoms in a transgenic Drosophila model through the inhibition of tau aggregation-engendered toxicity. These results suggest that Cl-NQTrp could be a unique potential therapeutic for AD since it targets aggregation of both Aβ and tau.