Small Molecule Microarrays Enable the Discovery of Compounds That Bind the Alzheimer’s Aβ Peptide and Reduce its Cytotoxicity

Article (PDF Available)inJournal of the American Chemical Society 132(47):17015-22 · November 2010with37 Reads
DOI: 10.1021/ja107552s · Source: PubMed
The amyloid-β (Aβ) aggregation pathway is a key target in efforts to discover therapeutics that prevent or delay the onset of Alzheimer's disease. Efforts at rational drug design, however, are hampered by uncertainties about the precise nature of the toxic aggregate. In contrast, high-throughput screening of compound libraries does not require a detailed understanding of the structure of the toxic species, and can provide an unbiased method for the discovery of small molecules that may lead to effective therapeutics. Here, we show that small molecule microarrays (SMMs) represent a particularly promising tool for identifying compounds that bind the Aβ peptide. Microarray slides with thousands of compounds immobilized on their surface were screened for binding to fluorescently labeled Aβ. Seventy-nine compounds were identified by the SMM screen, and then assayed for their ability to inhibit the Aβ-induced killing of PC12 cells. Further experiments focused on exploring the mechanism of rescue for one of these compounds: Electron microscopy and Congo red binding showed that the compound enhances fibril formation, and suggest that it may rescue cells by accelerating Aβ aggregation past an early toxic oligomer. These findings demonstrate that the SMM screen for binding to Aβ is effective at identifying compounds that reduce Aβ toxicity, and can reveal potential therapeutic leads without the biases inherent in methods that focus on inhibitors of aggregation.

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    • "An important distinction of this approach from previous anti-Aβ aggregation screens303132 is that we can detect drugs that inhibit Aβ 42 oligomer formation but do not inhibit the formation of large Aβ 42 amyloid. This is important because such large aggregates are now thought to be helpful because they likely capture some of the more toxic Aβ 42 oligomers , rendering them less toxic [33, 34] Here, we show that the mechanism of the PICALM, human AD risk factor, is likely to reduce the level of Aβ 42 oligomers in cells. This strongly supports the hypothesis that oligomerization of Aβ 42 is a major cause of AD toxicity. "
    [Show abstract] [Hide abstract] ABSTRACT: The formation of small Aβ42 oligomers has been implicated as a toxic species in Alzheimer disease (AD). In strong support of this hypothesis we found that overexpression of Yap1802, the yeast ortholog of the human AD risk factor, phosphatidylinositol binding clathrin assembly protein (PICALM), reduced oligomerization of Aβ42 fused to a reporter in yeast. Thus we used the Aβ42-reporter system to identify drugs that could be developed into therapies that prevent or arrest AD. From a screen of 1,200 FDA approved drugs and drug-like small compounds we identified 7 drugs that reduce Aβ42 oligomerization in yeast: 3 antipsychotics (bromperidol, haloperidol and azaperone), 2 anesthetics (pramoxine HCl and dyclonine HCl), tamoxifen citrate, and minocycline HCl. Also, all 7 drugs caused Aβ42 to be less toxic to PC12 cells and to relieve toxicity of another yeast AD model in which Aβ42 aggregates targeted to the secretory pathway are toxic. Our results identify drugs that inhibit Aβ42 oligomers from forming in yeast. It remains to be determined if these drugs inhibit Aβ42 oligomerization in mammals and could be developed as a therapeutic treatment for AD.
    Full-text · Article · Feb 2016
    • "X-ray crystallography has also been employed but has the distinct disadvantage of not being able to monitor binding in aqueous solution because samples need to be crystallized [2,33–35]. Several array-based methods have been reported in the literature during recent years, immobilizing either a fragment of the parent peptide [36] [37] or a small molecule ligand [38] [39] on a surface and then detecting the resulting binding that occurs. Although innovative, these approaches require the use of shortened AA fragments of the full-length target protein and, thus, do not allow for assessment of ligand binding sites using the full 3D protein/peptide structure. "
    [Show abstract] [Hide abstract] ABSTRACT: Numerous aromatic small molecule modulators of amyloid-beta peptide (Aβ) monomer aggregation and neurotoxicity have been identified with the ultimate goal of Alzheimer's disease (AD) treatment. Determining binding sites of these modulators on Aβ monomer is an important topic in the mechanistic understanding of AD pathology and drug development. However, Aβ monomer binding sites have been reported for only a very limited number of Aβ modulators. In this article, we present a convenient method for determining aggregation-modulating polycyclic aromatic small molecule ligand binding sites on Aβ monomer using immunostaining with a panel of Aβ sequence-specific antibodies. To validate our technique, we first examined one modulating aromatic ligand, Congo Red, with known binding sites, which yielded consistent results with previous findings. Then, using the same technique, binding sites on Aβ of four known Aβ monomer aggregation modulators, Erythrosin B, Eosin Y, Phloxine B, and Rose Bengal, were determined. The identified ligand binding sites were also confirmed by a separate fluorescence quenching-based assay using a panel of overlapping Aβ sub-fragments. The technique described here greatly increases researchers' ability to determine the Aβ monomer binding site(s) of aggregation-modulating aromatic small molecule ligands and to screen for new ligands that bind specific regions on Aβ. Copyright © 2014 Elsevier Inc. All rights reserved.
    Full-text · Article · Nov 2014
    • "More sophisticated NMR methodologies such as ligand-based approaches may thus be required for these studies. These findings and recent theoretical [19] and experimental [65] observations on the Ab peptide associated with Alzheimer's disease suggest that IDPs in general may contain small-molecule binding sites in a subset of their conformational ensemble. Thus our fragment mapping and docking studies suggest that locallypersistent binding sites are present even within a diverse population of conformations of aSyn, similarly to the case of the Ab peptide [19]. "
    [Show abstract] [Hide abstract] ABSTRACT: The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aβ peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions.
    Full-text · Article · Feb 2014
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