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

Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States.
Journal of the American Chemical Society (Impact Factor: 12.11). 11/2010; 132(47):17015-22. 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.

Download full-text


Available from: Michael Hecht,
  • Source
    • "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.
    Analytical Biochemistry 11/2014; 470. DOI:10.1016/j.ab.2014.10.016 · 2.22 Impact Factor
  • Source
    • "These findings and recent theoretical [19] and experimental [65] observations on the Aβ 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 locally-persistent binding sites are present even within a diverse population of conformations of αSyn, similarly to the case of the Aβ 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.
    PLoS ONE 02/2014; 9(2):e87133. DOI:10.1371/journal.pone.0087133 · 3.23 Impact Factor
  • Source
    • "Hecht and coworkers devised a small molecule microarray to identify amyloid peptide binders (Chen et al., 2010). In this design, a total of 17,905 compounds from various sources, including natural product, commercial compound collections, and DOS library, were immobilized onto slides and screened with fluorescently labeled amyloid peptides (Figure 5E). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proteins are fundamental components of all living systems and critical drivers of biological functions. The large-scale study of proteins, their structures and functions, is defined as proteomics. This systems-wide analysis leads to a more comprehensive view of the intricate signaling transduction pathways that proteins engage in and improves the overall understanding of the complex processes supporting the living systems. Over the last two decades, the development of high-throughput analytical tools, such as microarray technologies, capable of rapidly analyzing thousands of protein-functioning and protein-interacting events, has fueled the growth of this important field. Herein, we review the most recent advancements in microarray technologies, with a special focus on peptide microarray, small molecule microarray, and protein microarray. These technologies have become prominent players in proteomics and have made significant changes to the landscape of life science and biomedical research. We will elaborate on their performance, advantages, challenges, and future directions.
    Chemistry & biology 05/2013; 20(5):685-699. DOI:10.1016/j.chembiol.2013.04.009 · 6.65 Impact Factor
Show more