Anat Frydman-Marom

Cognitive-Performance Recovery of Alzheimer's Disease Model Mice by Modulation of Early Soluble Amyloidal Assemblies†

A rationally designed oligomerization inhibitor interacts with early intermediate assemblies of amyloid-beta polypeptide (Abeta) through the aromatic elements and inhibits their assembly into the toxic oligomers that cause Alzheimers disease by a unique C(alpha)-methylation beta-breakage strategy. The electrostatic potential of the low-energy conformation of the dipeptide inhibitor bound to Abeta is shown.

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.

Inhibiting α-Synuclein Oligomerization by Stable Cell-Penetrating β-Synuclein Fragments Recovers Phenotype of Parkinson's Disease Model Flies

The intracellular oligomerization of α-synuclein is associated with Parkinsons disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we indentified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinsons disease.

Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies

Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy.

Structural Basis for Inhibiting β-Amyloid Oligomerization by a Non-coded β-Breaker-Substituted Endomorphin Analogue

The distribution of endomorphins (EM) 1 and 2 in the human brain inversely correlates with cerebral neurodegeneration in Alzheimers disease (AD), implying a protective role. These endogenous opioid peptides incorporate aromatic residues and a β-breaker motif, as seen in several optimized inhibitors of Aβ aggregation. The activity of native endomorphins was studied, as well as the rationally designed analogue Aib-1, which includes a remarkably efficient β-breaker, α-aminoisobutyric acid (Aib). In vitro and GFP fusion protein assays showed that Aib-1 interacted with Aβ and markedly inhibited the formation of toxic oligomer and fibril growth. Moreover, Aib-1 prevented the toxicity of Aβ toward neuronal PC12 cells and markedly rectified reduced longevity of an AD fly model. Atomistic simulations and NMR-derived solution structures revealed that Aib-1 significantly reduced the propensity of Aβ to aggregate due to multimode interactions including aromatic, hydrophobic, and polar contacts. We suggest that hindering the self-assembly process by interfering with the aromatic core of amyloidogenic peptides may pave the way toward developing therapeutic agents to treat amyloid-associated diseases.

The generic amyloid formation inhibition effect of a designed small aromatic β-breaking peptide

The development of generic inhibitors in order to control the formation of amyloid fibrils and early oligomers is still an unmet medical need. As it is hypothesized that amyloid assemblies represent a generic protein supramolecular structure of low free energy, targeting the key molecular recognition and self-assembly events may provide the route for the development of such potential therapeutic agents. We have previously demonstrated the ability of hybrid molecules composed of an aromatic moiety and the α-aminoisobutyric acid β-sheet breaker elements to act as excellent inhibitors of amyloid fibril formation. Specifically, the D-Trp-Aib was shown to be a superb inhibitor of the formation of Alzheimer’s disease β-amyloid fibrils and oligomers both in vitro and in vivo. Here, we demonstrate that the rationally designed molecule has the generic ability to inhibit amyloid fibril formation by calcitonin, α-synuclein, and the islet amyloid polypeptide. Moreover, we demonstrate the inability of two modified peptides, D-Ala-Aib and D-Trp-Ala, to inhibit and disassemble amyloid fibril formation, a fact that provides an additional evidence for the suggested structural basis of the inhibitor activity. Taken together, we believe that the use of β-breaker elements combined with aromatic moiety may present a promising approach for the development of fibrillization inhibition drug candidate.

Diphenylalanine as a Reductionist Model for the Mechanistic Characterization of β-Amyloid Modulators

The phenomenon of protein aggregation into amyloid fibrils is associated with a large number of major diseases of unrelated etiology. Unraveling the mechanism of amyloid self-assembly and identifying therapeutic directions to control this process are of utmost importance. Research in this field has been hampered by several challenges, including reproducibility, low protein purification yields, and the inherent aggregation propensity of amyloidogenic proteins, making them extremely difficult to study. Herein, on the basis of the similarity in the assembly mechanism, as well as the physical, chemical, and biological characteristics, of diphenylalanine nanostructures and aromatic amino acid containing amyloid fibrils, we report a simple, yet robust peptide-based platform that could be used for screening of small molecules potentially capable of interfering with the aggregation process and for mechanistic exploration of their mode of action. The system was validated using four small-molecule inhibitors, and the effect was examined via turbidity assay, thioflavin T fluorescence, and electron microscopy. The aggregation profile of diphenylalanine was very similar to that of β-amyloid polypeptide in the presence of the modulators. Rosmarinic acid emerged as an extremely potent inhibitor and a destabilizer of the aggregates. The effect of stoichiometric variation of rosmarinic acid on the process of destabilization was also probed using a microfluidic technique. Finally, the formation of equimolar complexes of diphenylalanine and inhibitors was detected using mass spectrometry. This approach not only provides a system for high-throughput screening of possible inhibitor molecules from larger libraries of modulators, but is also highly useful for understanding the mechanistic aspects of the interactions leading to the process of inhibition.

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.