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A Novel Inhibitor of Amyloid β (Aβ) Peptide Aggregation

FROM HIGH THROUGHPUT SCREENING TO EFFICACY IN AN ANIMAL MODEL OF ALZHEIMER DISEASE*
  • Angela Fortner McKoy
    Affiliations
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544
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  • Jermont Chen
    Affiliations
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544
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  • Trudi Schupbach
    Affiliations
    Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
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  • Michael H. Hecht
    Correspondence
    To whom correspondence should be addressed. Tel.: 609-258-2901
    Affiliations
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant 5R21AG028462 (to M. H. H.) and the NCI Initiative for Chemical Genetics, National Institutes of Health, under Contract N01-CO-12400 and has been performed with the assistance of the Chemical Biology Platform of the Broad Institute of Harvard and MIT. This work was also supported by Award IIRG-08-89944 from the Alzheimer Association (to M. H. H.) and by the Howard Hughes Medical Institute (to T. S.).
    This article contains supplemental Figs. 1–4 and Movie 1.
Open AccessPublished:September 19, 2012DOI:https://doi.org/10.1074/jbc.M112.348037
      Compelling evidence indicates that aggregation of the amyloid β (Aβ) peptide is a major underlying molecular culprit in Alzheimer disease. Specifically, soluble oligomers of the 42-residue peptide (Aβ42) lead to a series of events that cause cellular dysfunction and neuronal death. Therefore, inhibiting Aβ42 aggregation may be an effective strategy for the prevention and/or treatment of disease. We describe the implementation of a high throughput screen for inhibitors of Aβ42 aggregation on a collection of 65,000 small molecules. Among several novel inhibitors isolated by the screen, compound D737 was most effective in inhibiting Aβ42 aggregation and reducing Aβ42-induced toxicity in cell culture. The protective activity of D737 was most significant in reducing the toxicity of high molecular weight oligomers of Aβ42. The ability of D737 to prevent Aβ42 aggregation protects against cellular dysfunction and reduces the production/accumulation of reactive oxygen species. Most importantly, treatment with D737 increases the life span and locomotive ability of flies in a Drosophila melanogaster model of Alzheimer disease.

      Introduction

      Alzheimer disease (AD)
      The abbreviations used are: AD
      Alzheimer disease
      amyloid β
      LMW
      low molecular weight
      HMW
      high molecular weight
      HTS
      high throughput screen
      DMSO
      dimethyl sulfoxide
      ThT
      thioflavin T
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      ROS
      reactive oxygen species
      DCF
      dichlorofluorescein.
      is a progressive and fatal brain disorder affecting as many as 6 million Americans, yet there are currently no effective treatments targeting the underlying molecular cause of this debilitating neurodegenerative disease (
      • Alexandrescu A.
      Amyloid accomplices and enforcers.
      ,
      • Clippingdale A.B.
      • Wade J.D.
      • Barrow C.J.
      The amyloid β peptide and its role in Alzheimer disease.
      ,
      • Selkoe D.J.
      • Podlisny M.B.
      Deciphering the genetic basis of Alzheimer disease.
      ,
      • Selkoe D.J
      • Schenk D.
      Alzheimer disease: molecular understanding predicts amyloid-based therapeutics.
      ,
      • Hardy J.A.
      • Higgins G.A.
      Alzheimer disease: the amyloid cascade hypothesis.
      ). Pathologically, the AD brain at its end stage is characterized by atrophy of the hippocampus and cerebral cortex, as well as accumulation of extracellular proteinaceous insoluble plaques composed of amyloid, a fibrillar form of protein. These amyloid plaques contain ordered assemblies of the amyloid β (Aβ) peptide (specifically the 42-residue alloform, Aβ42) (
      • Selkoe D.
      Alzheimer disease: genes, proteins, and therapy.
      ,
      • Citron M.
      Alzheimer disease: strategies for disease modification.
      ,
      • Tiraboschi P.
      • Hansen L.A.
      • Thal L.J.
      • Corey-Bloom J.
      The importance of neuritic plaques and tangles to the development and evolution of AD.
      ,
      • Masters C.L.
      • Simms G.
      • Weinman N.A.
      • Multhaup G.
      • McDonald B.L.
      • Beyreuther K.
      Amyloid plaque core protein in Alzheimer disease and Down syndrome.
      ,
      • Hardy J.
      • Allsop D.
      Amyloid deposition as the central event in the etiology of Alzheimer disease.
      ,
      • Hardy J.
      • Selkoe D.
      The amyloid hypothesis of Alzheimer disease: progress and problems on the road to therapeutics.
      ,
      • Walsh D.M.
      • Selkoe D.J.
      Aβ oligomers–a decade of discovery.
      ).
      Several pathogenic species that precede plaque formation have been isolated from AD brains. These include soluble low molecular weight (LMW) oligomers (dimers to dodecamers), Aβ-derived diffusible ligands or high molecular weight (HMW) oligomers (globular structures <5 nm in diameter), protofibrils (circular prefibrils 5–100 nm in diameter), and fibrils. The exact pathway of Aβ42 aggregation is not fully understood, and several competing pathways may occur simultaneously. A general view of the aggregation of soluble monomers into insoluble fibrils involves the following features: (i) nucleation dominated by a short lag phase, (ii) formation of LMW oligomers (dimers to dodecamers), (iii) assembly of LMW oligomers into larger β-sheet-rich oligomeric states, and (iv) growth of the β-sheet-rich oligomers into higher order aggregates of heterogeneous size and morphology. (v) Ultimately, the higher order oligomeric intermediates disappear with the formation of fibrils (
      • Hardy J.
      • Selkoe D.
      The amyloid hypothesis of Alzheimer disease: progress and problems on the road to therapeutics.
      ,
      • Walsh D.M.
      • Selkoe D.J.
      Aβ oligomers–a decade of discovery.
      ,
      • Walsh D.M.
      • Selkoe D.J.
      Oligomers on the brain: the emerging role of soluble protein aggregates in neurodegeneration.
      ,
      • Glabe C.
      Structural classification of toxic amyloid oligomers.
      ).
      Although fibrils were once thought to be the molecular culprit in AD, recent studies show a more decisive correlation between the levels of soluble Aβ oligomers and the extent of synaptic loss and cognitive impairment (
      • Glabe C.
      Structural classification of toxic amyloid oligomers.
      ). The link between Aβ aggregation, cellular dysfunction, and AD suggests that inhibition of Aβ oligomerization may ultimately lead to therapeutics that prevent and/or treat AD (
      • Tiraboschi P.
      • Hansen L.A.
      • Thal L.J.
      • Corey-Bloom J.
      The importance of neuritic plaques and tangles to the development and evolution of AD.
      ,
      • Shi C.
      • Zhao L.
      • Zhu B.
      • Li Q.
      • Yew D.T.
      • Yao Z.
      • Xu J.
      Protective effects of Ginkgo biloba extract (EGb761) and its constituents quercetin and ginkgolide B against β-amyloid peptide-induced toxicity in SH-SY5Y cells.
      ,
      • Necula M.
      • Kayed R.
      • Milton S.
      • Glabe C.
      Small molecule inhibitors of aggregation indicate that amyloid β oligomerization and fibrillization pathways are independent and distinct.
      ,
      • Kayed R.
      • Head E.
      • Thompson J.L.
      • McIntire T.M.
      • Milton S.C.
      • Cotman C.W.
      • Glabe C.G.
      Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.
      ).
      Here, we describe the isolation and characterization of aggregation inhibitors discovered by implementing a high throughput screen (HTS) based on the fluorescence of Aβ42-GFP fusions (
      • Kim W.
      • Kim Y.
      • Min J.
      • Kim D.J.
      • Chang Y.T.
      • Hecht M.H.
      A high throughput screen for compounds that inhibit aggregation of the Alzheimer peptide.
      ,
      • Waldo G.S.
      • Standish B.M.
      • Berendzen J.
      • Terwilliger T.C.
      Rapid protein-folding assay using green fluorescent protein.
      ). A compound, hereafter called D737, was the most potent aggregation inhibitor identified by the screen. We show that D737 inhibited formation of specific aggregates of Aβ42 and protected against Aβ42-induced cytotoxicity. Specifically, D737 was most effective in reducing the toxicity of the HMW oligomers. Most importantly, D737 increased the life span and locomotive ability of flies in a Drosophila model of AD.

      DISCUSSION

      The high resolution structure of the exact molecular species responsible for Aβ42 toxicity is not known. Therefore, structure-based drug design is not possible, and high throughput screening remains the most powerful way to identify inhibitors of Aβ42 aggregation that may lead to therapeutics for AD. Given the growing body of evidence suggesting that soluble oligomers play a major role in neurodegeneration, it is important to utilize screens capable of identifying compounds that inhibit the formation of oligomers. Here, we have show that an inexpensive high throughput screen is effective in identifying compounds that not only inhibit Aβ aggregation but also reduce toxicity in vitro and in vivo. In particular, compound D737, isolated from this screen, inhibits Aβ42 aggregation, reduces the cytotoxicity of Aβ42, and diminishes the cellular accumulation of ROS. Most importantly, D737 leads to significant improvements in both life span and locomotive ability in a transgenic D. melanogaster model of AD.

      Acknowledgments

      We thank Daniel Segal (Tel Aviv University) for generously providing transgenic flies and for hosting A. F. M. in his laboratory. We also thank Stephanie Norton, Frank An, Jason Burbank, and Lynn VerPlank for assistance in scaling the GFP assay to a high throughput assay, data analysis, and subsequent selection of compounds.

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