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Structural Mechanism of the Interaction of Alzheimer Disease Aβ Fibrils with the Non-steroidal Anti-inflammatory Drug (NSAID) Sulindac Sulfide*

  • Elke Prade
    Affiliations
    Munich Center for Integrated Protein Science at Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany
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  • Heiko J. Bittner
    Affiliations
    Molecular Modeling, Institute of Medical Physics and Biophysics, Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
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  • Riddhiman Sarkar
    Affiliations
    Munich Center for Integrated Protein Science at Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany
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  • Juan Miguel Lopez del Amo
    Affiliations
    CIC Energigune, Albert Einstein 48, 01510 Miñano, Álava, Spain
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  • Gerhard Althoff-Ospelt
    Affiliations
    Bruker BioSpin, Silberstreifen 4, 76287 Rheinstetten, Germany
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  • Gerd Multhaup
    Footnotes
    Affiliations
    Department of Pharmacology and Therapeutics, McGill University, Montreal Quebec H3G 1Y6, Canada
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  • Peter W. Hildebrand
    Affiliations
    Molecular Modeling, Institute of Medical Physics and Biophysics, Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
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  • Bernd Reif
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Munich Center for Integrated Protein Science at Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany

    Helmholtz-Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landtstr. 1, 85764 Neuherberg, Germany
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  • Author Footnotes
    * This work was supported by the Center for Integrated Protein Science Munich, the Helmholtz-Gemeinschaft, and the Deutsche Forschungsgemeinschaft (Grant Re1435). This work was also supported by Canadian Institute of Health Research Grant MOP-133411 and infrastructure (to G. M.). The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Results, Figures S1–S8, Tables S1–S3, and References.
    1 Holder of a Canada Research Chair in Molecular Pharmacology and supported by CFI grants.
Open AccessPublished:September 28, 2015DOI:https://doi.org/10.1074/jbc.M115.675215
      Alzheimer disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation has been reported. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators; they influence Aβ populations. It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. Here we present a magic angle spinning solid-state NMR study demonstrating that the NSAID sulindac sulfide interacts specifically with Alzheimer disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic Asp23-Lys28 salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue Gly33, a residue involved in Met35 oxidation. The results presented here will assist the search for pharmacologically active molecules that can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer disease.

      Introduction

      The self-assembly of amyloidogenic proteins into fibrils and oligomers plays a pivotal role in various diseases (
      • Bucciantini M.
      • Giannoni E.
      • Chiti F.
      • Baroni F.
      • Formigli L.
      • Zurdo J.
      • Taddei N.
      • Ramponi G.
      • Dobson C.M.
      • Stefani M.
      Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.
      ). The deposition of fibrils formed by the amyloid-β peptide (Aβ)
      The abbreviations used are: Aβ
      amyloid-β
      APP
      amyloid precursor protein
      NSAID
      non-steroidal anti-inflammatory drug
      DMSO
      dimethyl sulfoxide
      MAS
      magic angle spinning
      TEDOR
      transferred echo double resonance
      REDOR
      rotational echo double resonance
      TEM
      transmission electron microscopy.
      into plaques in brain tissue is a major pathological hallmark in the progression of neurodegeneration in Alzheimer disease. Aβ peptides are generated through sequential proteolytic cleavages of the amyloid precursor protein (APP) by the β- and γ-secretases (
      • Haass C.
      Take five: BACE and the γ-secretase quartet conduct Alzheimer's amyloid β-peptide generation.
      ,
      • Kang J.
      • Lemaire H.G.
      • Unterbeck A.
      • Salbaum J.M.
      • Masters C.L.
      • Grzeschik K.H.
      • Multhaup G.
      • Beyreuther K.
      • Müller-Hill B.
      The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor.
      ). This results in the production of Aβ peptides of differing lengths (
      • Olsson F.
      • Schmidt S.
      • Althoff V.
      • Munter L.M.
      • Jin S.
      • Rosqvist S.
      • Lendahl U.
      • Multhaup G.
      • Lundkvist J.
      Characterization of intermediate steps in amyloid β (Aβ) production under near-native conditions.
      ), mainly Aβ1–40 and Aβ1–42 (
      • Roher A.E.
      • Lowenson J.D.
      • Clarke S.
      • Woods A.S.
      • Cotter R.J.
      • Gowing E.
      • Ball M.J.
      β-Amyloid-(1–42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease.
      ), and shorter variants, such as Aβ1–39 (
      • Prelli F.
      • Castaño E.
      • Glenner G.G.
      • Frangione B.
      Differences between vascular and plaque core amyloid in Alzheimer's disease.
      ). Soluble oligomers formed by Aβ1–42 represent the toxic species responsible for the decline in cognitive function associated with neurodegeneration (
      • Selkoe D.J.
      Soluble oligomers of the amyloid β-protein impair synaptic plasticity and behavior.
      ). Several studies have demonstrated that small molecules can interfere with the solubility of amyloid proteins and are, therefore, potential drug candidates (
      • Bieschke J.
      • Russ J.
      • Friedrich R.P.
      • Ehrnhoefer D.E.
      • Wobst H.
      • Neugebauer K.
      • Wanker E.E.
      EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity.
      ,
      • Bieschke J.
      • Herbst M.
      • Wiglenda T.
      • Friedrich R.P.
      • Boeddrich A.
      • Schiele F.
      • Kleckers D.
      • Lopez del Amo J.M.
      • Grüning B.A.
      • Wang Q.
      • Schmidt M.R.
      • Lurz R.
      • Anwyl R.
      • Schnoegl S.
      • Fändrich M.
      • Frank R.F.
      • Reif B.
      • Günther S.
      • Walsh D.M.
      • Wanker E.E.
      Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils.
      ). Chronic inflammation significantly enhances Alzheimer disease pathogenesis (
      • Akiyama H.
      • Barger S.
      • Barnum S.
      • Bradt B.
      • Bauer J.
      • Cole G.M.
      • Cooper N.R.
      • Eikelenboom P.
      • Emmerling M.
      • Fiebich B.L.
      • Finch C.E.
      • Frautschy S.
      • Griffin W.S.
      • Hampel H.
      • Hull M.
      • Landreth G.
      • Lue L.
      • Mrak R.
      • Mackenzie I.R.
      • McGeer P.L.
      • O'Banion M.K.
      • Pachter J.
      • Pasinetti G.
      • Plata-Salaman C.
      • Rogers J.
      • Rydel R.
      • Shen Y.
      • Streit W.
      • Strohmeyer R.
      • Tooyoma I.
      • Van Muiswinkel F.L.
      • Veerhuis R.
      • Walker D.
      • Webster S.
      • Wegrzyniak B.
      • Wenk G.
      • Wyss-Coray T.
      Inflammation and Alzheimer's disease.
      ). Fibrillar β-amyloid deposits co-localize with numerous chronic inflammatory mediators and activated microglia in the brain (
      • McGeer P.L.
      • McGeer E.G.
      The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases.
      ). The relation to inflammation suggests that non-steroidal anti-inflammatory drugs (NSAIDs) might be beneficial for the treatment of Alzheimer disease. In fact, epidemiological studies demonstrate a link between the use of anti-inflammatory drugs and the prevalence of Alzheimer disease (
      • in t' Veld B.A.
      • Ruitenberg A.
      • Hofman A.
      • Launer L.J.
      • van Duijn C.M.
      • Stijnen T.
      • Breteler M.M.
      • Stricker B.H.
      Nonsteroidal antiinflammatory drugs and the risk of Alzheimer's disease.
      ). This work aims to investigate the interaction mechanism of the NSAID sulindac sulfide (Fig. 1a) with Alzheimer peptide Aβ fibrils. In addition to sulindac sulfide (
      • Weggen S.
      • Eriksen J.L.
      • Sagi S.A.
      • Pietrzik C.U.
      • Ozols V.
      • Fauq A.
      • Golde T.E.
      • Koo E.H.
      Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid β 42 production by direct modulation of γ-secretase activity.
      ), NSAIDs, including ibuprofen, indomethacin (
      • Weggen S.
      • Eriksen J.L.
      • Sagi S.A.
      • Pietrzik C.U.
      • Ozols V.
      • Fauq A.
      • Golde T.E.
      • Koo E.H.
      Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid β 42 production by direct modulation of γ-secretase activity.
      ), and flurbiprofen (
      • Eriksen J.L.
      • Sagi S.A.
      • Smith T.E.
      • Weggen S.
      • Das P.
      • McLendon D.C.
      • Ozols V.V.
      • Jessing K.W.
      • Zavitz K.H.
      • Koo E.H.
      • Golde T.E.
      NSAIDs and enantiomers of flurbiprofen target γ-secretase and lower Aβ 42 in vivo.
      ), have been identified as γ-secretase modulators. γ-Secretase modulators interfere with APP processing and modify the relative Aβ1–42 population. In particular, sulindac sulfide decreases the relative amount of the amyloid-prone Aβ1–42, whereas the production of shorter, less amyloidogenic Aβ peptides is increased (
      • Weggen S.
      • Eriksen J.L.
      • Sagi S.A.
      • Pietrzik C.U.
      • Ozols V.
      • Fauq A.
      • Golde T.E.
      • Koo E.H.
      Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid β 42 production by direct modulation of γ-secretase activity.
      • Eriksen J.L.
      • Sagi S.A.
      • Smith T.E.
      • Weggen S.
      • Das P.
      • McLendon D.C.
      • Ozols V.V.
      • Jessing K.W.
      • Zavitz K.H.
      • Koo E.H.
      • Golde T.E.
      NSAIDs and enantiomers of flurbiprofen target γ-secretase and lower Aβ 42 in vivo.
      ,
      • Takahashi Y.
      • Hayashi I.
      • Tominari Y.
      • Rikimaru K.
      • Morohashi Y.
      • Kan T.
      • Natsugari H.
      • Fukuyama T.
      • Tomita T.
      • Iwatsubo T.
      Sulindac sulfide is a noncompetitive γ-secretase inhibitor that preferentially reduces Aβ 42 generation.
      • Wanngren J.
      • Ottervald J.
      • Parpal S.
      • Portelius E.
      • Strömberg K.
      • Borgegård T.
      • Klintenberg R.
      • Juréus A.
      • Blomqvist J.
      • Blennow K.
      • Zetterberg H.
      • Lundkvist J.
      • Rosqvist S.
      • Karlström H.
      Second generation γ-secretase modulators exhibit different modulation of Notch β and Aβ production.
      ).
      Figure thumbnail gr1
      FIGURE 1The influence of sulindac sulfide on Aβ. a, chemical structure of the NSAID sulindac sulfide. b, TEM images of 50 μm Aβ fibrils in the absence (bottom panel) and presence (top panel) of a 5-fold molar excess of sulindac sulfide. The fibrillar character is maintained, and deposits of sulindac sulfide can be observed. Scale bar = 200 nm. c, two-dimensional 13C-15N TEDOR spectra of Aβ amyloid fibrils incubated in the presence of a 5-fold molar excess of sulindac sulfide and 1% DMSO (red) and Aβ fibrils incubated with 1% DMSO (black) as a control. Corresponding 13C-13C correlation spectra are shown in . The obtained 13C line widths are in the order of 120–200 Hz (data not shown). Sequential assignments are obtained from three-dimensional NCACX and NCOCX experiments. Arrows indicate residues that experience large chemical shift changes. d, CSPs induced by sulindac sulfide on the NMR chemical shifts of Aβ fibrils. Differences in chemical shifts (Δδ (ppm)) were calculated for 13C and 15N resonances according to ΔδC = [(δCsul − δCref)2]1/2 and ΔδN = [(2 / 5 × (δNsul − δNref))2]1/2, respectively.
      Solution-state NMR structures of the APP-TM (transmembrane) dimer have been solved for the wild-type (
      • Nadezhdin K.D.
      • Bocharova O.V.
      • Bocharov E.V.
      • Arseniev A.S.
      Dimeric structure of transmembrane domain of amyloid precursor protein in micellar environment.
      ) and a familial mutant (
      • Chen W.
      • Gamache E.
      • Rosenman D.J.
      • Xie J.
      • Lopez M.M.
      • Li Y.M.
      • Wang C.
      Familial Alzheimer's mutations within APPTM increase Aβ42 production by enhancing accessibility of ϵ-cleavage site.
      ). NMR and EPR experiments have revealed a potential cholesterol-binding site within the C-terminal Cys99 sequence and highlight the significance of the GXXXG segments for binding (
      • Barrett P.J.
      • Song Y.
      • Van Horn W.D.
      • Hustedt E.J.
      • Schafer J.M.
      • Hadziselimovic A.
      • Beel A.J.
      • Sanders C.R.
      The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol.
      ). It has been suggested that NSAIDs can interact with lipids to form phospholipid complexes (
      • Manrique-Moreno M.
      • Moreno M.M.
      • Garidel P.
      • Suwalsky M.
      • Howe J.
      • Brandenburg K.
      The membrane-activity of Ibuprofen, Diclofenac, and Naproxen: a physico-chemical study with lecithin phospholipids.
      ,
      • Hüsch J.
      • Dutagaci B.
      • Glaubitz C.
      • Geppert T.
      • Schneider G.
      • Harms M.
      • Müller-Goymann C.C.
      • Fink L.
      • Schmidt M.U.
      • Setzer C.
      • Zirkel J.
      • Rebmann H.
      • Schubert-Zsilavecz M.
      • Abdel-Tawab M.
      Structural properties of so-called NSAID-phospholipid-complexes.
      • Lichtenberger L.M.
      • Zhou Y.
      • Jayaraman V.
      • Doyen J.R.
      • O'Neil R.G.
      • Dial E.J.
      • Volk D.E.
      • Gorenstein D.G.
      • Boggara M.B.
      • Krishnamoorti R.
      Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: characterization of interaction of NSAIDs with phosphatidylcholine.
      ). This may provide a general mechanism for the interaction of APP with small molecules. Reports on the interaction between sulindac sulfide and the APP transmembrane sequence are, however, contradictory. Sulindac sulfide, among other γ-secretase modulators, binds to the APP transmembrane sequence of the Cys99 motif (
      • Kukar T.L.
      • Ladd T.B.
      • Bann M.A.
      • Fraering P.C.
      • Narlawar R.
      • Maharvi G.M.
      • Healy B.
      • Chapman R.
      • Welzel A.T.
      • Price R.W.
      • Moore B.
      • Rangachari V.
      • Cusack B.
      • Eriksen J.
      • Jansen-West K.
      • Verbeeck C.
      • Yager D.
      • Eckman C.
      • Ye W.
      • Sagi S.
      • Cottrell B.A.
      • Torpey J.
      • Rosenberry T.L.
      • Fauq A.
      • Wolfe M.S.
      • Schmidt B.
      • Walsh D.M.
      • Koo E.H.
      • Golde T.E.
      Substrate-targeting γ-secretase modulators.
      ) and to Cys100 dimers in the presence of SDS micelles (
      • Botev A.
      • Munter L.M.
      • Wenzel R.
      • Richter L.
      • Althoff V.
      • Ismer J.
      • Gerling U.
      • Weise C.
      • Koksch B.
      • Hildebrand P.W.
      • Bittl R.
      • Multhaup G.
      The amyloid precursor protein C-terminal fragment C100 occurs in monomeric and dimeric stable conformations and binds γ-secretase modulators.
      ). Bacterial reporter assays show that γ-secretase modulators, including sulindac sulfide, bind to the GXXXG dimerization motif and, thereby, attenuate the dimerization of the APP transmembrane sequence (
      • Richter L.
      • Munter L.M.
      • Ness J.
      • Hildebrand P.W.
      • Dasari M.
      • Unterreitmeier S.
      • Bulic B.
      • Beyermann M.
      • Gust R.
      • Reif B.
      • Weggen S.
      • Langosch D.
      • Multhaup G.
      Amyloid β 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization.
      ), a process necessary for proteolytic cleavage (
      • Khalifa N.B.
      • Van Hees J.
      • Tasiaux B.
      • Huysseune S.
      • Smith S.O.
      • Constantinescu S.N.
      • Octave J.N.
      • Kienlen-Campard P.
      What is the role of amyloid precursor protein dimerization?.
      ). However, colloidal aggregation of sulindac sulfide in aqueous solutions can potentially induce nonspecific binding (
      • Beel A.J.
      • Barrett P.
      • Schnier P.D.
      • Hitchcock S.A.
      • Bagal D.
      • Sanders C.R.
      • Jordan J.B.
      Nonspecificity of binding of γ-secretase modulators to the amyloid precursor protein.
      ,
      • Barrett P.J.
      • Sanders C.R.
      • Kaufman S.A.
      • Michelsen K.
      • Jordan J.B.
      NSAID-based γ-secretase modulators do not bind to the amyloid-β polypeptide.
      ). Contradicting data have been reported for the influence of sulindac sulfide on the Aβ peptide itself (
      • Hirohata M.
      • Ono K.
      • Naiki H.
      • Yamada M.
      Non-steroidal anti-inflammatory drugs have anti-amyloidogenic effects for Alzheimer's β-amyloid fibrils in vitro.
      ,
      • Yesuvadian R.
      • Krishnamoorthy J.
      • Ramamoorthy A.
      • Bhunia A.
      Potent γ-secretase inhibitors/modulators interact with amyloid-β fibrils but do not inhibit fibrillation: a high-resolution NMR study.
      ).
      So far, it is not understood how sulindac sulfide interacts with amyloids. NMR is a suitable technique to study Aβ-ligand interactions for various Aβ aggregation states (
      • Prade E.
      • Lopez del Amo J.-M.
      • Reif B.
      ). Solution-state NMR can be employed to study interactions of Aβ monomers and small molecule (
      • Choi J.S.
      • Braymer J.J.
      • Nanga R.P.
      • Ramamoorthy A.
      • Lim M.H.
      Design of small molecules that target metal-Aβ species and regulate metal-induced Aβ aggregation and neurotoxicity.
      ) or peptide inhibitors (
      • Rezaei-Ghaleh N.
      • Andreetto E.
      • Yan L.M.
      • Kapurniotu A.
      • Zweckstetter M.
      Interaction between amyloid β peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide.
      ), nanoparticles (
      • Yoo S.I.
      • Yang M.
      • Brender J.R.
      • Subramanian V.
      • Sun K.
      • Joo N.E.
      • Jeong S.H.
      • Ramamoorthy A.
      • Kotov N.A.
      Inhibition of amyloid peptide fibrillation by inorganic nanoparticles: functional similarities with proteins.
      ), and various others. Besides monomers and fibrils, oligomeric intermediates formed by Aβ in solution constitute potential drug targets (
      • Vivekanandan S.
      • Brender J.R.
      • Lee S.Y.
      • Ramamoorthy A.
      A partially folded structure of amyloid-β(1–40) in an aqueous environment.
      ,
      • Ramamoorthy A.
      • Lim M.H.
      Structural characterization and inhibition of toxic amyloid-β oligomeric intermediates.
      ). However, oligomeric intermediates and insoluble fibrils are not detectable by solution-state NMR because their lines are broadened beyond detection. Solid-state NMR spectroscopy is a powerful tool that allows the study of Aβ-small molecule interactions at atomic resolution. In the past, this technique has been applied successfully for the characterization of the interaction between Aβ and the polyphenol epigallocatechin gallate (
      • Lopez del Amo J.M.
      • Fink U.
      • Dasari M.
      • Grelle G.
      • Wanker E.E.
      • Bieschke J.
      • Reif B.
      Structural properties of EGCG-induced, nontoxic Alzheimer's disease Aβ oligomers.
      ), curcumin (
      • Masuda Y.
      • Fukuchi M.
      • Yatagawa T.
      • Tada M.
      • Takeda K.
      • Irie K.
      • Akagi K.
      • Monobe Y.
      • Imazawa T.
      • Takegoshi K.
      Solid-state NMR analysis of interaction sites of curcumin and 42-residue amyloid β-protein fibrils.
      ,
      • Mithu V.S.
      • Sarkar B.
      • Bhowmik D.
      • Das A.K.
      • Chandrakesan M.
      • Maiti S.
      • Madhu P.K.
      Curcumin alters the salt bridge-containing turn region in amyloid β(1–42) aggregates.
      ), and catechol-type flavonoids (
      • Sato M.
      • Murakami K.
      • Uno M.
      • Nakagawa Y.
      • Katayama S.
      • Akagi K.
      • Masuda Y.
      • Takegoshi K.
      • Irie K.
      Site-specific inhibitory mechanism for amyloid β42 aggregation by catechol-type flavonoids targeting the Lys residues.
      ) and to study the interface of Congo red and amyloids formed by the prion domain of the HET-s protein (
      • Schütz A.K.
      • Soragni A.
      • Hornemann S.
      • Aguzzi A.
      • Ernst M.
      • Böckmann A.
      • Meier B.H.
      The amyloid-Congo red interface at atomic resolution.
      ). In this work, we investigate the interaction between sulindac sulfide and Aβ fibrils using solid-state NMR spectroscopy. On the basis of the gathered NMR data, we employ docking to derive a model for the intercalation of sulindac sulfide with Aβ fibrils.

      Results

      To probe the interaction between Aβ fibrils and sulindac sulfide (Fig. 1a), we titrated sulindac sulfide to preformed fibrils. Fibrillar Aβ is observed by TEM in the presence of the NSAID (Fig. 1b). The resulting 13C-15N (Fig. 1c) and 13C-13C (supplemental Fig. S1) correlation spectra show well dispersed peaks. The three-dimensional NCACX and NCOCX experiments allowed sequential assignment of resonances for residues Gln15-Val40 for both samples (representative strip plots are shown in supplemental Fig. S2; all assigned resonances are listed in supplemental Table S1). We detected only one set of resonances for both samples, indicating that the fibrils exist in one conformation.
      Sulindac sulfide has no significant effect on the fibrillar structure of Aβ because the spectra of both samples are relatively similar (Fig. 1c and supplemental Fig. S1). However, small but defined chemical shift perturbations (CSPs) are observed in the presence of sulindac sulfide, indicating specific interactions of the NSAID with the fibrils. This is remarkable because a non-quantitative and nonspecific binding of sulindac sulfide to Aβ fibrils would result in peak-splitting and line-broadening. Fig. 1d shows CSPs, Δδ (parts per million), upon addition of sulindac sulfide. We observe changes in chemical shift in particular for side chain resonances of Lys16, Val18-Cβ, Phe19-Cβ, Phe20-Cβ, Asn27-Cγ, and Met35-Cβ as well as for the backbone resonances of Phe19, Phe20, Ala21 and Gly33. CSPs reflect ligand binding but could as well be a consequence of local or global structural rearrangements. To unambiguously probe ligand binding, we recorded 13C-19F REDOR experiments employing the NMR-active properties of the 19F atom of sulindac sulfide. In general, only aliphatic resonances of Aβ could be detected. For comparison, a one-dimensional 13C spectrum containing assignments for all aliphatic resonances is represented (Fig. 2a). The strongest dephasing effects are observed for the Cγ resonances of Val18 or Val39. However, because of spectral overlap, these two peaks cannot be discriminated. Smaller signal attenuations are observed for Cγ of Val24 and Val36, Cβs of Ala21 or Ala30 (overlap) as well as for Cγ2 and Cδ1 of Ile32 at longer mixing times. These resonances exhibit the most severe dephasing effects. The respective residues must therefore be located close to the fluorine atom of sulindac sulfide.
      To gain further information on the potential effects of sulindac sulfide on the Aβ fibril structure, we investigated the effect on the salt bridge, which is typically formed between the side chains of residues Asp23 and Lys28 (
      • Petkova A.T.
      • Yau W.M.
      • Tycko R.
      Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils.
      ,
      • Paravastu A.K.
      • Leapman R.D.
      • Yau W.M.
      • Tycko R.
      Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils.
      ). From sequential assignments, the chemical shift of the carboxylic group of Asp23 in the presence of sulindac sulfide was assigned to 177.8 ppm and to 178.0 ppm for the reference fibrils. On the basis of one-dimensional 15N spectra, the chemical shift of Lys28-Nζ was found to be 33.9 ppm and 34.3 ppm for the two preparations, respectively. In both samples, a cross-peak between Lys28-Nζ and Asp23-Cγ was detected, implying the presence of a salt bridge in both cases (Fig. 2b).
      In the following, the CSPs in the two-dimensional 13C-13C proton-driven spin diffusion and 13C-15N TEDOR as well as the 13C-19F REDOR contacts were used as restraints to derive a model for sulindac sulfide in complex with Aβ1–40 fibrils. We used both a 2-fold (
      • Petkova A.T.
      • Yau W.M.
      • Tycko R.
      Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils.
      ) and 3-fold symmetric (
      • Paravastu A.K.
      • Leapman R.D.
      • Yau W.M.
      • Tycko R.
      Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils.
      ) Aβ1–40 fibril NMR structure as reference structures for modeling and docking experiments because they show the highest correlation with our chemical shifts (supplemental Fig. S3) compared with all structures and models analyzed (
      • Lopez del Amo J.M.
      • Schmidt M.
      • Fink U.
      • Dasari M.
      • Fändrich M.
      • Reif B.
      An asymmetric dimer as the basic subunit in Alzheimer's disease amyloid β fibrils.
      ,
      • Petkova A.T.
      • Yau W.M.
      • Tycko R.
      Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils.
      ,
      • Paravastu A.K.
      • Leapman R.D.
      • Yau W.M.
      • Tycko R.
      Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils.
      ,
      • Lu J.X.
      • Qiang W.
      • Yau W.M.
      • Schwieters C.D.
      • Meredith S.C.
      • Tycko R.
      Molecular structure of β-amyloid fibrils in Alzheimer's disease brain tissue.
      • Petkova A.T.
      • Leapman R.D.
      • Guo Z.
      • Yau W.M.
      • Mattson M.P.
      • Tycko R.
      Self-propagating, molecular-level polymorphism in Alzheimer's β-amyloid fibrils.
      ,
      • Niu Z.
      • Zhao W.
      • Zhang Z.
      • Xiao F.
      • Tang X.
      • Yang J.
      The molecular structure of Alzheimer β-amyloid fibrils formed in the presence of phospholipid vesicles.
      ,
      • Bertini I.
      • Gonnelli L.
      • Luchinat C.
      • Mao J.
      • Nesi A.
      A new structural model of Aβ40 fibrils.
      • Petkova A.T.
      • Ishii Y.
      • Balbach J.J.
      • Antzutkin O.N.
      • Leapman R.D.
      • Delaglio F.
      • Tycko R.
      A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR.
      ). The NMR structures contained 10 models each. The architecture of the Aβ1–40 fibrils obviously differs comparing the 2-fold and 3-fold-symmetric structures. In each structure, Aβ adopts a β-strand—turn—β-strand fold. Fibrils are stabilized by hydrogen bonds connecting individual β-strands along the fibril axis. The 3-fold symmetric fibril structure contains three stacks of Aβ molecules in a triangular form. The 2-fold symmetric fibril structure is built from two antiparallel stacks of Aβ molecules. To detect potential binding sites for the hydrophobic sulindac sulfide, we performed a packing density analysis for each Aβ structure. In fact, all models revealed large packing defects. On average, we found eight cavities per stack of four Aβ molecules in both structures (supplemental Figs. S4 and S5), clustering in five different regions within one stack (clusters #1-#5) (Fig. 3). To dissect polar from unpolar cavities, we applied the tool DOWSER (
      • Zhang L.
      • Hermans J.
      Hydrophilicity of cavities in proteins.
      ) to calculate positions of internal water molecules that are not resolved in the NMR structures. We found that the most hydrophobic cavities in each structure clustered in the rigid core region, with calculated water occupancies of <10% in cluster #2 and <30% in cluster #3 that are located on both sides of Phe-19 (supplemental Table S3). The 3-fold symmetric structure seemed to be more tightly packed around clusters #2 and #3, featuring fewer cavities in comparison with the 2-fold symmetric structure. At the same time, other clusters in the 3-fold symmetric structure seemed to contain a larger numbers of cavities. Clusters of polar cavities were found in the flexible turn region between residues Glu22 and Ile31 (cluster #1) and toward the termini of the peptide sequence (cluster #4). The two structures differed in their interface architecture between the termini to the adjacent loop region. Although, in the 3-fold symmetric structure, cavities of cluster #4 were stabilized through interactions with the loop, in the 2-fold symmetric structure, fewer polar cavities were found because the structure opened and cavities could become exposed. Cluster #5 (around M35) with polar cavities was located at the intersheet/contact region between termini and the loop region (3-fold symmetric structure) or at the interface of the antiparallel β-sheets (2-fold symmetric structure). In the intersheet region, where the contact interface between 3-fold and 2-fold symmetric structure was apparently different, the amount of cavities per stack (of four Aβ molecules) and their polarity did not differ. The amount of cavities and the respective water contents per cluster #1-#5 are shown in supplemental Table S3.
      Clusters #2 and #3 contain large hydrophobic cavities, which could, in principle, bury a sulindac sulfide molecule. The exact size of the individual cavities depends on the rotameric state of Phe19. The 3-fold symmetric and the 2-fold symmetric structures differ slightly in the position of Phe19 with respect to the registry of the second β-sheet containing residues Ile32 to Val36 (supplemental Fig. S6). In the 3-fold symmetric structure, Phe19 is oriented more toward Ile32, whereas, in the 2-fold symmetric structure, Phe19 is pointing toward Leu34 and is, therefore, located more central in the core region. Surprisingly, the size, polarity, and distribution of the detected cavities is very similar in all analyzed structural models even though there are slight differences between the two models.
      To test whether the hydrophobic cavities in clusters #2 and #3 are indeed suitable binding sites for sulindac sulfide, we applied an induced fit docking approach where the sulindac sulfide and side chains of Aβ were kept flexible. The size and accessibility of these cavities depends on the rotameric state of Phe19. In fact, docking into cluster #3 of the 3-fold symmetric structure allows identification of two scenarios that fit to the distance restraints obtained from the NMR analysis. In particular, we find that the distance of the sulindac sulfide 19F atom to the methyl groups of Ile32, Leu34, or Val36 is smaller than 6 Å. In pose 1, sulindac sulfide lies in the Gly33 groove and is orientated along the fibril axes, being close to Ile32 and Leu34. The aromatic side chains of Phe19 are directed toward Ala30 (Fig. 4a, top panel). In pose 2, sulindac sulfide has rotated into a position with its conjugated ring system parallel to the β-sheets and orthogonal to Phe19 so that the 19F atom approaches Leu34 and Val36. (Fig. 4a, bottom panel). Both poses suggest an aromatic π stacking interaction between the conjugated ring system of sulindac sulfide and the Phe19 side chain. Similar interactions are obtained from docking to cluster #2 of the 2-fold symmetric structure (Fig. 4b). In pose 1, sulindac sulfide is orientated along the fibril axes, with its conjugated ring system parallel to Phe19, analogous to pose 1 in the 3-fold symmetric structure. Again, the 19F atom is positioned in the Gly33 groove close to Ile32 and Leu34. In contrast to the 3-fold symmetric structure, Phe19 is positioned here on the other side of sulindac sulfide, close to Leu34, thereby shielding Val36 so that sulindac sulfide cannot change into a position to contact Val36 (Fig. 4b, top panel). An additional docking to cluster #3 yields poses 2 and 3, where the normal of the conjugated ring system of sulindac sulfide is orientated perpendicularly to the fibril axes. Thereby, sulindac sulfide extends into the terminal region (#4). The conjugated ring system is positioned parallel to the β-sheets and orthogonal to Phe19. Sulindac sulfide is either orientated in a way that the 19F atom faces Phe19, Leu34, and Val36 (Fig. 4b, center panel) or that the 19F atom is oriented toward the fibril exterior, contacting Val36 and Val39 (Fig. 4b, bottom panel).
      Figure thumbnail gr4
      FIGURE 4Induced fit docking of sulindac sulfide to Aβ. a and b, induced fit docking of sulindac sulfide to the hydrophobic cavity cluster #3 of the 3-fold symmetric Aβ1–40 fibril structure (PDB code 2LMP, model 1, poses 1 and 2) (
      • Paravastu A.K.
      • Leapman R.D.
      • Yau W.M.
      • Tycko R.
      Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils.
      ) (a) and to the hydrophobic cavity clusters #2 and #3 of the 2-fold symmetric Aβ1–40 structure (PDB code 2LMN, model 1; for cluster #2, pose 1 and cluster #3, poses 2 and 3) (
      • Petkova A.T.
      • Yau W.M.
      • Tycko R.
      Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils.
      ) (b). Sulindac sulfide is depicted as blue sticks and the protein backbones as a schematic. Residues within 6 Å of the 19F atom of sulindac sulfide are depicted as sticks in purple for the cavity cluster defining residues Ile32 and Val36 and in green for Ala30, Leu34, and Val39. Phe19 and Gly33 are colored in red.

      Discussion

      Sulindac sulfide-incubated Aβ fibrils are highly similar to control fibrils, implying that sulindac sulfide has no significant effect on fibril structure. Both the NMR chemical shift patterns (Fig. 1c and supplemental Fig. S1) and the morphology in TEM images (Fig. 1b) are maintained. Analysis of 13C chemical shifts predicts β-strands as the main secondary structural element in both fibril preparations (supplemental Fig. S7). In addition, chemical shift analysis by torsion angle likeliness obtained from shift and sequence similarities+ (TALOS+) (
      • Shen Y.
      • Delaglio F.
      • Cornilescu G.
      • Bax A.
      TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts.
      ) yields secondary structural propensities that predict the presence of two β-strands typically observed in specific regions within Aβ1–40 fibrils (
      • Lopez del Amo J.M.
      • Schmidt M.
      • Fink U.
      • Dasari M.
      • Fändrich M.
      • Reif B.
      An asymmetric dimer as the basic subunit in Alzheimer's disease amyloid β fibrils.
      ,
      • Petkova A.T.
      • Yau W.M.
      • Tycko R.
      Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils.
      ,
      • Paravastu A.K.
      • Leapman R.D.
      • Yau W.M.
      • Tycko R.
      Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils.
      ,
      • Bertini I.
      • Gonnelli L.
      • Luchinat C.
      • Mao J.
      • Nesi A.
      A new structural model of Aβ40 fibrils.
      ,
      • Tycko R.
      Molecular structure of amyloid fibrils: insights from solid-state NMR.
      ) and oligomers (
      • Chimon S.
      • Shaibat M.A.
      • Jones C.R.
      • Calero D.C.
      • Aizezi B.
      • Ishii Y.
      Evidence of fibril-like β-sheet structures in a neurotoxic amyloid intermediate of Alzheimer's β-amyloid.
      ,
      • Ahmed M.
      • Davis J.
      • Aucoin D.
      • Sato T.
      • Ahuja S.
      • Aimoto S.
      • Elliott J.I.
      • Van Nostrand W.E.
      • Smith S.O.
      Structural conversion of neurotoxic amyloid-β(1–42) oligomers to fibrils.
      ). Furthermore, we find, by TEDOR experiments, that the Asp23-Lys28 salt bridge remains intact in the presence of sulindac sulfide (Fig. 2b). Even though the overall fibrillar character is maintained, we observe defined chemical shift changes, potentially indicating local conformational changes (Fig. 1d). Previous reports have stated that small molecules such as curcumin (
      • Mithu V.S.
      • Sarkar B.
      • Bhowmik D.
      • Das A.K.
      • Chandrakesan M.
      • Maiti S.
      • Madhu P.K.
      Curcumin alters the salt bridge-containing turn region in amyloid β(1–42) aggregates.
      ) are able to disrupt the characteristic salt bridge in Aβ1–42 fibrils. Judging from EM data, curcumin has a more drastic effect on the general fibril architecture compared with sulindac sulfide because it destroys Aβ1–42 fibrils (
      • Mithu V.S.
      • Sarkar B.
      • Bhowmik D.
      • Das A.K.
      • Chandrakesan M.
      • Maiti S.
      • Madhu P.K.
      Curcumin alters the salt bridge-containing turn region in amyloid β(1–42) aggregates.
      ). We note that only one set of resonances is observed for the sulindac sulfide-incubated Aβ fibrils. No resonance splitting is observed for cross-peaks. Rather, resonances move to new positions, indicating that each peptide in a fibril interacts specifically with one or more NSAID molecules.
      Upon addition of sulindac sulfide, CSPs are observed in particular for hydrophobic residues such as Val18, Phe19 and Phe20, Gly33, and Met35 but also for the polar side chain of Lys16. The most dramatic CSPs are detected for Lys16 and Gly33 (Fig. 1d). 13C-19F REDOR experiments yield unambiguous distance restraints. The REDOR experiments show that sulindac sulfide binds in the vicinity of methyl groups of Val18 or Val39 as well as Ile31 and Ile32 Cγ2.
      To identify trends and avoid bias, we use both the 2-fold symmetric (PDB code 2LMN) (
      • Petkova A.T.
      • Yau W.M.
      • Tycko R.
      Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils.
      ) and the 3-fold symmetric (PDB code 2LMP) (
      • Paravastu A.K.
      • Leapman R.D.
      • Yau W.M.
      • Tycko R.
      Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils.
      ) Aβ1–40 fibrils as reference structures for modeling and docking experiments. The polymorphism of these structures may differ from the fibrils investigated in this study. However, they show the highest similarities of all three wild-type Aβ1–40 fibril structures currently available (
      • Petkova A.T.
      • Yau W.M.
      • Tycko R.
      Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils.
      ,
      • Paravastu A.K.
      • Leapman R.D.
      • Yau W.M.
      • Tycko R.
      Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils.
      ,
      • Lu J.X.
      • Qiang W.
      • Yau W.M.
      • Schwieters C.D.
      • Meredith S.C.
      • Tycko R.
      Molecular structure of β-amyloid fibrils in Alzheimer's disease brain tissue.
      ). Within the two Aβ fibril structural ensembles, several hydrophobic cavities are detected that are large enough to potentially host a sulindac sulfide molecule. We did not find significant deviations in the distribution, size, and polarity of the cavities between the analyzed structural ensembles and models. Therefore, changes in fibril polymorphism do not significantly affect the distribution of hydrophobic patches in amyloid fibrils. We conclude that the two employed structures provide a suitable basis for docking experiments, which is also supported by experimental NMR data, although high-resolution structures of a better defined polymorph will allow more accurate docking in the future.
      An induced fit docking reveals that the NSAID can interact with fibrils in three different ways, and all models fulfill the NMR restraints. The apparently ambiguous REDOR restraints suggest that more than one sulindac sulfide molecule might be involved in binding. In the docking poses that are in best agreement with the NMR restraints, sulindac sulfide intercalates between the two β-strands of the Aβ fibril, with the long axis of the molecule either parallel (Fig. 4, a and b, pose 1) or perpendicular (Fig. 4, a, pose 2, and b, poses 2 and 3) to the fibril axis. Therefore, residue Phe19 seems to play a crucial role in sulindac sulfide binding because its rotameric state has an influence on the size and shape of cavities in clusters #2 and #3. Furthermore, the aromatic side chain is involved in π stacking with the conjugated ring system of the NSAID.
      Pose 1 of the docking approach suggests that sulindac sulfide fits into the groove formed by Gly33 (Fig. 4, a and b, top panels). Therefore, the large CSPs may be attributed to Gly33 backbone atoms experiencing a change in chemical environment or even undergoing conformational changes to accommodate the NSAID. In theoretical studies, this glycine residue has been suggested to be involved in oxidation of Aβ1–42 because of its close proximity to the side chain of Met35 (
      • Rauk A.
      • Armstrong D.A.
      • Fairlie D.P.
      Is oxidative damage by β-amyloid and prion peptides mediated by hydrogen atom transfer from glycine α-carbon to methionine sulfur within β-sheets?.
      ,
      • Rauk A.
      • Armstrong D.A.
      Influence of β-sheet structure on the susceptibility of proteins to backbone oxidative damage: preference for αc-centered radical formation at glycine residues of antiparallel β-sheets.
      ). Furthermore, Gly33 has been reported as the key amino acid for Aβ toxicity and to be responsible for driving Aβ into neurotoxic conformations (
      • Harmeier A.
      • Wozny C.
      • Rost B.R.
      • Munter L.M.
      • Hua H.
      • Georgiev O.
      • Beyermann M.
      • Hildebrand P.W.
      • Weise C.
      • Schaffner W.
      • Schmitz D.
      • Multhaup G.
      Role of amyloid-β glycine 33 in oligomerization, toxicity, and neuronal plasticity.
      ). Both residues, Gly33 and Met35, have been hypothesized to stabilize reactive oxygen species (
      • Kanski J.
      • Varadarajan S.
      • Aksenova M.
      • Butterfield D.A.
      Role of glycine-33 and methionine-35 in Alzheimer's amyloid β-peptide 1–42-associated oxidative stress and neurotoxicity.
      ,
      • Brunelle P.
      • Rauk A.
      The radical model of Alzheimer's disease: specific recognition of Gly29 and Gly33 by Met35 in a β-sheet model of Aβ: an ONIOM study.
      ). Sulindac sulfide might, therefore, act by binding to the hydrophobic pocket in the vicinity of Gly33, and preventing oxidation of the Aβ peptide. Oxidation of Aβ1–42 reduces fibril assembly and aggregation because of the increased polarity introduced by the methionine sulfoxide (
      • Hou L.
      • Kang I.
      • Marchant R.E.
      • Zagorski M.G.
      Methionine 35 oxidation reduces fibril assembly of the amyloid aβ-(1–42) peptide of Alzheimer's disease.
      ). In accordance, we find that the Aβ-sulindac sulfide complex exists in a stable fibrillar state.
      CSPs for Lys16 may be explained by a recent docking study involving sulindac sulfide and Aβ fibrils that suggested that sulindac sulfide may bind weakly to a shallow, solvent-exposed pocket in the vicinity of Lys16 and Val18 and does not interfere with fibrillation (
      • Yesuvadian R.
      • Krishnamoorthy J.
      • Ramamoorthy A.
      • Bhunia A.
      Potent γ-secretase inhibitors/modulators interact with amyloid-β fibrils but do not inhibit fibrillation: a high-resolution NMR study.
      ). This binding mode may, in addition, account for the large REDOR signal detected for the overlapping resonances Val18/Val39 Cγ (Fig. 2a). We cannot exclude docking of sulindac sulfide to the fibril surface because our REDOR and CSP data are also in agreement with the blind docking model proposed by Yesuvadian et al. (
      • Yesuvadian R.
      • Krishnamoorthy J.
      • Ramamoorthy A.
      • Bhunia A.
      Potent γ-secretase inhibitors/modulators interact with amyloid-β fibrils but do not inhibit fibrillation: a high-resolution NMR study.
      ).
      Sulindac sulfide has been reported to form colloidal aggregates above a critical micelle concentration in solution (
      • Barrett P.J.
      • Sanders C.R.
      • Kaufman S.A.
      • Michelsen K.
      • Jordan J.B.
      NSAID-based γ-secretase modulators do not bind to the amyloid-β polypeptide.
      ). This phenomenon is commonly observed for hydrophobic compounds (
      • McGovern S.L.
      • Caselli E.
      • Grigorieff N.
      • Shoichet B.K.
      A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening.
      ,
      • Coan K.E.
      • Shoichet B.K.
      Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors.
      ). The size of small-molecule colloidal particles is typically on the order of 50–600 nm (
      • Barrett P.J.
      • Sanders C.R.
      • Kaufman S.A.
      • Michelsen K.
      • Jordan J.B.
      NSAID-based γ-secretase modulators do not bind to the amyloid-β polypeptide.
      ). These colloids bind unfolded proteins in a promiscuous manner (
      • McGovern S.L.
      • Caselli E.
      • Grigorieff N.
      • Shoichet B.K.
      A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening.
      ,
      • Coan K.E.
      • Shoichet B.K.
      Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors.
      ) and have been shown to lead to precipitation and inhibition of protein function (
      • Hagerman A.E.
      • Butler L.G.
      The specificity of proanthocyanidin-protein interactions.
      ,
      • Feng B.Y.
      • Toyama B.H.
      • Wille H.
      • Colby D.W.
      • Collins S.R.
      • May B.C.
      • Prusiner S.B.
      • Weissman J.
      • Shoichet B.K.
      Small-molecule aggregates inhibit amyloid polymerization.
      • Ono K.
      • Li L.
      • Takamura Y.
      • Yoshiike Y.
      • Zhu L.
      • Han F.
      • Mao X.
      • Ikeda T.
      • Takasaki J.
      • Nishijo H.
      • Takashima A.
      • Teplow D.B.
      • Zagorski M.G.
      • Yamada M.
      Phenolic compounds prevent amyloid β-protein oligomerization and synaptic dysfunction by site-specific binding.
      ). However, the effect of sulindac sulfide on Aβ fibril chemical shifts reported here indicates not a promiscuous but a specific interaction. Aggregates of the compound are observed in TEM images (Fig. 1b), implying the presence of colloids in solution. This is expected because the concentration used (250 μm) lies above the critical micelle concentration of 50–100 μm (
      • Barrett P.J.
      • Sanders C.R.
      • Kaufman S.A.
      • Michelsen K.
      • Jordan J.B.
      NSAID-based γ-secretase modulators do not bind to the amyloid-β polypeptide.
      ). To account for the experimental single set of resonances, we must assume that individual NSAID molecules dissociate from the colloidal complexes and interact specifically with Aβ fibrils.
      Binding of sulindac sulfide in different cavities for different Aβ peptides would result in splitting of the resonances and line-broadening. We observe, however, narrow lines, indicating that the small molecule must either bind simultaneously to different cavities or exchange between these cavities. Relaxation experiments will be carried out in the future to differentiate between these two scenarios.
      In conclusion, we suggest that the NSAID sulindac sulfide is able to interact with Aβ fibrils in a rather specific manner. We find that several cavities can accommodate a sulindac sulfide molecule. This is supported by defined CSPs and 13C-19F REDOR contacts. Sulindac sulfide does not induce drastic architectural changes to the non-toxic fibrillar structure, as indicated by NMR and EM. In addition, the characteristic Asp23-Lys28 salt bridge and the length and positioning of the β-strands are not affected. Molecular modeling suggests that sulindac sulfide intercalates between the two β-strands at, presumably, more than one position. The presented data contribute to the elucidation of the mechanism by which small molecules bind insoluble amyloids. This understanding is crucial for the design of pharmacologically relevant molecules that interfere with Aβ species and that might, in the future, be employed for the treatment of Alzheimer disease.

      Author Contributions

      E. P. expressed and purified isotopically labelled peptide samples, designed and performed NMR experiments, and carried out the data analysis. R. S. implemented the solid-state NMR experiments. J. M. L. d. A. recorded the initial experiments and assisted with data analysis. G. A. O. carried out the fluorine REDOR experiments. G. M. performed biochemical experiments. H. J. B. and P. W. H. performed and analyzed the molecular modeling and docking. All authors discussed the results. B. R. and E. P. conceived the project and wrote the paper with input from all authors.

      Acknowledgments

      This work was performed in the framework of SFB-1035/Project-B07 and SFB740/Project-B6 (German Research Foundation). The computer time necessary for calculation of docking experiments was provided by Norddeutscher Verbund für Hoch- und Höchstleistungsrechner Project bec00085.

      References

        • Bucciantini M.
        • Giannoni E.
        • Chiti F.
        • Baroni F.
        • Formigli L.
        • Zurdo J.
        • Taddei N.
        • Ramponi G.
        • Dobson C.M.
        • Stefani M.
        Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.
        Nature. 2002; 416: 507-511
        • Haass C.
        Take five: BACE and the γ-secretase quartet conduct Alzheimer's amyloid β-peptide generation.
        EMBO J. 2004; 23: 483-488
        • Kang J.
        • Lemaire H.G.
        • Unterbeck A.
        • Salbaum J.M.
        • Masters C.L.
        • Grzeschik K.H.
        • Multhaup G.
        • Beyreuther K.
        • Müller-Hill B.
        The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor.
        Nature. 1987; 325: 733-736
        • Olsson F.
        • Schmidt S.
        • Althoff V.
        • Munter L.M.
        • Jin S.
        • Rosqvist S.
        • Lendahl U.
        • Multhaup G.
        • Lundkvist J.
        Characterization of intermediate steps in amyloid β (Aβ) production under near-native conditions.
        J. Biol. Chem. 2014; 289: 1540-1550
        • Roher A.E.
        • Lowenson J.D.
        • Clarke S.
        • Woods A.S.
        • Cotter R.J.
        • Gowing E.
        • Ball M.J.
        β-Amyloid-(1–42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease.
        Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 10836-10840
        • Prelli F.
        • Castaño E.
        • Glenner G.G.
        • Frangione B.
        Differences between vascular and plaque core amyloid in Alzheimer's disease.
        J. Neurochem. 1988; 51: 648-651
        • Selkoe D.J.
        Soluble oligomers of the amyloid β-protein impair synaptic plasticity and behavior.
        Behav. Brain Res. 2008; 192: 106-113
        • Bieschke J.
        • Russ J.
        • Friedrich R.P.
        • Ehrnhoefer D.E.
        • Wobst H.
        • Neugebauer K.
        • Wanker E.E.
        EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 7710-7715
        • Bieschke J.
        • Herbst M.
        • Wiglenda T.
        • Friedrich R.P.
        • Boeddrich A.
        • Schiele F.
        • Kleckers D.
        • Lopez del Amo J.M.
        • Grüning B.A.
        • Wang Q.
        • Schmidt M.R.
        • Lurz R.
        • Anwyl R.
        • Schnoegl S.
        • Fändrich M.
        • Frank R.F.
        • Reif B.
        • Günther S.
        • Walsh D.M.
        • Wanker E.E.
        Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils.
        Nat. Chem. Biol. 2012; 8: 93-101
        • Akiyama H.
        • Barger S.
        • Barnum S.
        • Bradt B.
        • Bauer J.
        • Cole G.M.
        • Cooper N.R.
        • Eikelenboom P.
        • Emmerling M.
        • Fiebich B.L.
        • Finch C.E.
        • Frautschy S.
        • Griffin W.S.
        • Hampel H.
        • Hull M.
        • Landreth G.
        • Lue L.
        • Mrak R.
        • Mackenzie I.R.
        • McGeer P.L.
        • O'Banion M.K.
        • Pachter J.
        • Pasinetti G.
        • Plata-Salaman C.
        • Rogers J.
        • Rydel R.
        • Shen Y.
        • Streit W.
        • Strohmeyer R.
        • Tooyoma I.
        • Van Muiswinkel F.L.
        • Veerhuis R.
        • Walker D.
        • Webster S.
        • Wegrzyniak B.
        • Wenk G.
        • Wyss-Coray T.
        Inflammation and Alzheimer's disease.
        Neurobiol. Aging. 2000; 21: 383-421
        • McGeer P.L.
        • McGeer E.G.
        The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases.
        Brain Res. Brain Res. Rev. 1995; 21: 195-218
        • in t' Veld B.A.
        • Ruitenberg A.
        • Hofman A.
        • Launer L.J.
        • van Duijn C.M.
        • Stijnen T.
        • Breteler M.M.
        • Stricker B.H.
        Nonsteroidal antiinflammatory drugs and the risk of Alzheimer's disease.
        N. Engl. J. Med. 2001; 345: 1515-1521
        • Weggen S.
        • Eriksen J.L.
        • Sagi S.A.
        • Pietrzik C.U.
        • Ozols V.
        • Fauq A.
        • Golde T.E.
        • Koo E.H.
        Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid β 42 production by direct modulation of γ-secretase activity.
        J. Biol. Chem. 2003; 278: 31831-31837
        • Eriksen J.L.
        • Sagi S.A.
        • Smith T.E.
        • Weggen S.
        • Das P.
        • McLendon D.C.
        • Ozols V.V.
        • Jessing K.W.
        • Zavitz K.H.
        • Koo E.H.
        • Golde T.E.
        NSAIDs and enantiomers of flurbiprofen target γ-secretase and lower Aβ 42 in vivo.
        J. Clin. Invest. 2003; 112: 440-449
        • Takahashi Y.
        • Hayashi I.
        • Tominari Y.
        • Rikimaru K.
        • Morohashi Y.
        • Kan T.
        • Natsugari H.
        • Fukuyama T.
        • Tomita T.
        • Iwatsubo T.
        Sulindac sulfide is a noncompetitive γ-secretase inhibitor that preferentially reduces Aβ 42 generation.
        J. Biol. Chem. 2003; 278: 18664-18670
        • Wanngren J.
        • Ottervald J.
        • Parpal S.
        • Portelius E.
        • Strömberg K.
        • Borgegård T.
        • Klintenberg R.
        • Juréus A.
        • Blomqvist J.
        • Blennow K.
        • Zetterberg H.
        • Lundkvist J.
        • Rosqvist S.
        • Karlström H.
        Second generation γ-secretase modulators exhibit different modulation of Notch β and Aβ production.
        J. Biol. Chem. 2012; 287: 32640-32650
        • Nadezhdin K.D.
        • Bocharova O.V.
        • Bocharov E.V.
        • Arseniev A.S.
        Dimeric structure of transmembrane domain of amyloid precursor protein in micellar environment.
        FEBS Lett. 2012; 586: 1687-1692
        • Chen W.
        • Gamache E.
        • Rosenman D.J.
        • Xie J.
        • Lopez M.M.
        • Li Y.M.
        • Wang C.
        Familial Alzheimer's mutations within APPTM increase Aβ42 production by enhancing accessibility of ϵ-cleavage site.
        Nat. Commun. 2014; 5: 3037
        • Barrett P.J.
        • Song Y.
        • Van Horn W.D.
        • Hustedt E.J.
        • Schafer J.M.
        • Hadziselimovic A.
        • Beel A.J.
        • Sanders C.R.
        The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol.
        Science. 2012; 336: 1168-1171
        • Manrique-Moreno M.
        • Moreno M.M.
        • Garidel P.
        • Suwalsky M.
        • Howe J.
        • Brandenburg K.
        The membrane-activity of Ibuprofen, Diclofenac, and Naproxen: a physico-chemical study with lecithin phospholipids.
        Biochim. Biophys. Acta. 2009; 1788: 1296-1303
        • Hüsch J.
        • Dutagaci B.
        • Glaubitz C.
        • Geppert T.
        • Schneider G.
        • Harms M.
        • Müller-Goymann C.C.
        • Fink L.
        • Schmidt M.U.
        • Setzer C.
        • Zirkel J.
        • Rebmann H.
        • Schubert-Zsilavecz M.
        • Abdel-Tawab M.
        Structural properties of so-called NSAID-phospholipid-complexes.
        Eur. J. Pharm. Sci. 2011; 44: 103-116
        • Lichtenberger L.M.
        • Zhou Y.
        • Jayaraman V.
        • Doyen J.R.
        • O'Neil R.G.
        • Dial E.J.
        • Volk D.E.
        • Gorenstein D.G.
        • Boggara M.B.
        • Krishnamoorti R.
        Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: characterization of interaction of NSAIDs with phosphatidylcholine.
        Biochim. Biophys. Acta. 2012; 1821: 994-1002
        • Kukar T.L.
        • Ladd T.B.
        • Bann M.A.
        • Fraering P.C.
        • Narlawar R.
        • Maharvi G.M.
        • Healy B.
        • Chapman R.
        • Welzel A.T.
        • Price R.W.
        • Moore B.
        • Rangachari V.
        • Cusack B.
        • Eriksen J.
        • Jansen-West K.
        • Verbeeck C.
        • Yager D.
        • Eckman C.
        • Ye W.
        • Sagi S.
        • Cottrell B.A.
        • Torpey J.
        • Rosenberry T.L.
        • Fauq A.
        • Wolfe M.S.
        • Schmidt B.
        • Walsh D.M.
        • Koo E.H.
        • Golde T.E.
        Substrate-targeting γ-secretase modulators.
        Nature. 2008; 453: 925-929
        • Botev A.
        • Munter L.M.
        • Wenzel R.
        • Richter L.
        • Althoff V.
        • Ismer J.
        • Gerling U.
        • Weise C.
        • Koksch B.
        • Hildebrand P.W.
        • Bittl R.
        • Multhaup G.
        The amyloid precursor protein C-terminal fragment C100 occurs in monomeric and dimeric stable conformations and binds γ-secretase modulators.
        Biochemistry. 2011; 50: 828-835
        • Richter L.
        • Munter L.M.
        • Ness J.
        • Hildebrand P.W.
        • Dasari M.
        • Unterreitmeier S.
        • Bulic B.
        • Beyermann M.
        • Gust R.
        • Reif B.
        • Weggen S.
        • Langosch D.
        • Multhaup G.
        Amyloid β 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 14597-14602
        • Khalifa N.B.
        • Van Hees J.
        • Tasiaux B.
        • Huysseune S.
        • Smith S.O.
        • Constantinescu S.N.
        • Octave J.N.
        • Kienlen-Campard P.
        What is the role of amyloid precursor protein dimerization?.
        Cell Adh. Migr. 2010; 4: 268-272
        • Beel A.J.
        • Barrett P.
        • Schnier P.D.
        • Hitchcock S.A.
        • Bagal D.
        • Sanders C.R.
        • Jordan J.B.
        Nonspecificity of binding of γ-secretase modulators to the amyloid precursor protein.
        Biochemistry. 2009; 48: 11837-11839
        • Barrett P.J.
        • Sanders C.R.
        • Kaufman S.A.
        • Michelsen K.
        • Jordan J.B.
        NSAID-based γ-secretase modulators do not bind to the amyloid-β polypeptide.
        Biochemistry. 2011; 50: 10328-10342
        • Hirohata M.
        • Ono K.
        • Naiki H.
        • Yamada M.
        Non-steroidal anti-inflammatory drugs have anti-amyloidogenic effects for Alzheimer's β-amyloid fibrils in vitro.
        Neuropharmacology. 2005; 49: 1088-1099
        • Yesuvadian R.
        • Krishnamoorthy J.
        • Ramamoorthy A.
        • Bhunia A.
        Potent γ-secretase inhibitors/modulators interact with amyloid-β fibrils but do not inhibit fibrillation: a high-resolution NMR study.
        Biochem. Biophys. Res. Commun. 2014; 447: 590-595
        • Prade E.
        • Lopez del Amo J.-M.
        • Reif B.
        Separovic F. Naito A. Advances in Biological Solid-State NMR: Proteins and Membrane-Active Peptides. The Royal Society of Chemistry, Cambridge, UK2014: 533-555
        • Choi J.S.
        • Braymer J.J.
        • Nanga R.P.
        • Ramamoorthy A.
        • Lim M.H.
        Design of small molecules that target metal-Aβ species and regulate metal-induced Aβ aggregation and neurotoxicity.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 21990-21995
        • Rezaei-Ghaleh N.
        • Andreetto E.
        • Yan L.M.
        • Kapurniotu A.
        • Zweckstetter M.
        Interaction between amyloid β peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide.
        PLoS ONE. 2011; 6: e20289
        • Yoo S.I.
        • Yang M.
        • Brender J.R.
        • Subramanian V.
        • Sun K.
        • Joo N.E.
        • Jeong S.H.
        • Ramamoorthy A.
        • Kotov N.A.
        Inhibition of amyloid peptide fibrillation by inorganic nanoparticles: functional similarities with proteins.
        Angew. Chem. Int. Ed. Engl. 2011; 50: 5110-5115
        • Vivekanandan S.
        • Brender J.R.
        • Lee S.Y.
        • Ramamoorthy A.
        A partially folded structure of amyloid-β(1–40) in an aqueous environment.
        Biochem. Biophys. Res. Commun. 2011; 411: 312-316
        • Ramamoorthy A.
        • Lim M.H.
        Structural characterization and inhibition of toxic amyloid-β oligomeric intermediates.
        Biophys. J. 2013; 105: 287-288
        • Lopez del Amo J.M.
        • Fink U.
        • Dasari M.
        • Grelle G.
        • Wanker E.E.
        • Bieschke J.
        • Reif B.
        Structural properties of EGCG-induced, nontoxic Alzheimer's disease Aβ oligomers.
        J. Mol. Biol. 2012; 421: 517-524
        • Masuda Y.
        • Fukuchi M.
        • Yatagawa T.
        • Tada M.
        • Takeda K.
        • Irie K.
        • Akagi K.
        • Monobe Y.
        • Imazawa T.
        • Takegoshi K.
        Solid-state NMR analysis of interaction sites of curcumin and 42-residue amyloid β-protein fibrils.
        Bioorg. Med. Chem. 2011; 19: 5967-5974
        • Mithu V.S.
        • Sarkar B.
        • Bhowmik D.
        • Das A.K.
        • Chandrakesan M.
        • Maiti S.
        • Madhu P.K.
        Curcumin alters the salt bridge-containing turn region in amyloid β(1–42) aggregates.
        J. Biol. Chem. 2014; 289: 11122-11131
        • Sato M.
        • Murakami K.
        • Uno M.
        • Nakagawa Y.
        • Katayama S.
        • Akagi K.
        • Masuda Y.
        • Takegoshi K.
        • Irie K.
        Site-specific inhibitory mechanism for amyloid β42 aggregation by catechol-type flavonoids targeting the Lys residues.
        J. Biol. Chem. 2013; 288: 23212-23224
        • Schütz A.K.
        • Soragni A.
        • Hornemann S.
        • Aguzzi A.
        • Ernst M.
        • Böckmann A.
        • Meier B.H.
        The amyloid-Congo red interface at atomic resolution.
        Angew. Chem. Int. Ed. Engl. 2011; 50: 5956-5960
        • Dasari M.
        • Espargaro A.
        • Sabate R.
        • Lopez del Amo J.M.
        • Fink U.
        • Grelle G.
        • Bieschke J.
        • Ventura S.
        • Reif B.
        Bacterial inclusion bodies of Alzheimer's disease β-amyloid peptides can be employed to study native-like aggregation intermediate states.
        ChemBioChem. 2011; 12: 407-423
        • Walsh D.M.
        • Thulin E.
        • Minogue A.M.
        • Gustavsson N.
        • Pang E.
        • Teplow D.B.
        • Linse S.
        A facile method for expression and purification of the Alzheimer's disease-associated amyloid β-peptide.
        FEBS J. 2009; 276: 1266-1281
        • Lopez del Amo J.M.
        • Schmidt M.
        • Fink U.
        • Dasari M.
        • Fändrich M.
        • Reif B.
        An asymmetric dimer as the basic subunit in Alzheimer's disease amyloid β fibrils.
        Angew. Chem. Int. Ed. Engl. 2012; 51: 6136-6139
        • Bloembergen N.
        On the interaction of nuclear spins in a crystalline lattice.
        Physica. 1949; 15: 386-426
        • Szeverenyi N.M.
        • Sullivan M.J.
        • Maciel G.E.
        Observation of spin exchange by two-dimensional Fourier transform 13C cross polarization-magic-angle spinning.
        J. Magn. Reson. 1982; 47: 462-475
        • Hing A.W.
        • Vega S.
        • Schaefer J.
        Transferred-echo double-resonance NMR.
        J. Magn. Reson. 1992; 96: 205-209
        • Hing A.W.
        • Vega S.
        • Schaefer J.
        Measurement of heteronuclear dipolar coupling by transferred-echo double-resonance NMR.
        J. Magn. Reson. 1993; 103: 151-162
        • Castellani F.
        • van Rossum B.J.
        • Diehl A.
        • Rehbein K.
        • Oschkinat H.
        Determination of solid-state NMR structures of proteins by means of three-dimensional 15N-13C-13C dipolar correlation spectroscopy and chemical shift analysis.
        Biochemistry. 2003; 42: 11476-11483
        • Takegoshi K.
        • Nakamura S.
        • Terao T.
        13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR.
        Chem. Phys. Lett. 2001; 344: 631-637
        • Gullion T.
        • Schaefer J.
        Rotational-echo double-resonance NMR.
        J. Magn. Reson. 1989; 81: 196-200
        • Jaroniec C.P.
        • Filip C.
        • Griffin R.G.
        3D TEDOR NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly 13C,15N-labeled solids.
        J. Am. Chem. Soc. 2002; 124: 10728-10742
        • Petkova A.T.
        • Yau W.M.
        • Tycko R.
        Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils.
        Biochemistry. 2006; 45: 498-512
        • Paravastu A.K.
        • Leapman R.D.
        • Yau W.M.
        • Tycko R.
        Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 18349-18354
        • Goede A.
        • Preissner R.
        • Frömmel C.
        Voronoi cell: new method for allocation of space among atoms: elimination of avoidable errors in calculation of atomic volume and density.
        J. Comput. Chem. 1997; 18: 1113-1123
        • Rother K.
        • Hildebrand P.W.
        • Goede A.
        • Gruening B.
        • Preissner R.
        Voronoia: analyzing packing in protein structures.
        Nucleic Acids Res. 2009; 37: D393-D395
        • Tsai J.
        • Gerstein M.
        Calculations of protein volumes: sensitivity analysis and parameter database.
        Bioinformatics. 2002; 18: 985-995
        • Delaunay B.N.
        Sur la sphère vide.
        B. Acad. Sci. USSR. 1934; 7: 793-800
        • Zhang L.
        • Hermans J.
        Hydrophilicity of cavities in proteins.
        Proteins. 1996; 24: 433-438
        • Sherman W.
        • Day T.
        • Jacobson M.P.
        • Friesner R.A.
        • Farid R.
        Novel procedure for modeling ligand/receptor induced fit effects.
        J. Med. Chem. 2006; 49: 534-553
        • Sherman W.
        • Beard H.S.
        • Farid R.
        Use of an induced fit receptor structure in virtual screening.
        Chem. Biol. Drug Des. 2006; 67: 83-84
      1. Schrödinger Release 2014-1: Maestro, version 9.7.
        Schrödinger, LLC, New York, NY2014
        • Friesner R.A.
        • Banks J.L.
        • Murphy R.B.
        • Halgren T.A.
        • Klicic J.J.
        • Mainz D.T.
        • Repasky M.P.
        • Knoll E.H.
        • Shelley M.
        • Perry J.K.
        • Shaw D.E.
        • Francis P.
        • Shenkin P.S.
        Glide: a new approach for rapid, accurate docking and scoring: 1: method and assessment of docking accuracy.
        J. Med. Chem. 2004; 47: 1739-1749
        • Lu J.X.
        • Qiang W.
        • Yau W.M.
        • Schwieters C.D.
        • Meredith S.C.
        • Tycko R.
        Molecular structure of β-amyloid fibrils in Alzheimer's disease brain tissue.
        Cell. 2013; 154: 1257-1268
        • Petkova A.T.
        • Leapman R.D.
        • Guo Z.
        • Yau W.M.
        • Mattson M.P.
        • Tycko R.
        Self-propagating, molecular-level polymorphism in Alzheimer's β-amyloid fibrils.
        Science. 2005; 307: 262-265
        • Niu Z.
        • Zhao W.
        • Zhang Z.
        • Xiao F.
        • Tang X.
        • Yang J.
        The molecular structure of Alzheimer β-amyloid fibrils formed in the presence of phospholipid vesicles.
        Angew. Chem. Int. Ed. Engl. 2014; 53: 9294-9297
        • Bertini I.
        • Gonnelli L.
        • Luchinat C.
        • Mao J.
        • Nesi A.
        A new structural model of Aβ40 fibrils.
        J. Am. Chem. Soc. 2011; 133: 16013-16022
        • Petkova A.T.
        • Ishii Y.
        • Balbach J.J.
        • Antzutkin O.N.
        • Leapman R.D.
        • Delaglio F.
        • Tycko R.
        A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16742-16747
        • Shen Y.
        • Delaglio F.
        • Cornilescu G.
        • Bax A.
        TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts.
        J. Biomol. NMR. 2009; 44: 213-223
        • Tycko R.
        Molecular structure of amyloid fibrils: insights from solid-state NMR.
        Q. Rev. Biophys. 2006; 39: 1-55
        • Chimon S.
        • Shaibat M.A.
        • Jones C.R.
        • Calero D.C.
        • Aizezi B.
        • Ishii Y.
        Evidence of fibril-like β-sheet structures in a neurotoxic amyloid intermediate of Alzheimer's β-amyloid.
        Nat. Struct. Mol. Biol. 2007; 14: 1157-1164
        • Ahmed M.
        • Davis J.
        • Aucoin D.
        • Sato T.
        • Ahuja S.
        • Aimoto S.
        • Elliott J.I.
        • Van Nostrand W.E.
        • Smith S.O.
        Structural conversion of neurotoxic amyloid-β(1–42) oligomers to fibrils.
        Nat. Struct. Mol. Biol. 2010; 17: 561-567
        • Rauk A.
        • Armstrong D.A.
        • Fairlie D.P.
        Is oxidative damage by β-amyloid and prion peptides mediated by hydrogen atom transfer from glycine α-carbon to methionine sulfur within β-sheets?.
        J. Am. Chem. Soc. 2000; 122: 9761-9767
        • Rauk A.
        • Armstrong D.A.
        Influence of β-sheet structure on the susceptibility of proteins to backbone oxidative damage: preference for αc-centered radical formation at glycine residues of antiparallel β-sheets.
        J. Am. Chem. Soc. 2000; 122: 4185-4192
        • Harmeier A.
        • Wozny C.
        • Rost B.R.
        • Munter L.M.
        • Hua H.
        • Georgiev O.
        • Beyermann M.
        • Hildebrand P.W.
        • Weise C.
        • Schaffner W.
        • Schmitz D.
        • Multhaup G.
        Role of amyloid-β glycine 33 in oligomerization, toxicity, and neuronal plasticity.
        J. Neurosci. 2009; 29: 7582-7590
        • Kanski J.
        • Varadarajan S.
        • Aksenova M.
        • Butterfield D.A.
        Role of glycine-33 and methionine-35 in Alzheimer's amyloid β-peptide 1–42-associated oxidative stress and neurotoxicity.
        Biochim. Biophys. Acta. 2002; 1586: 190-198
        • Brunelle P.
        • Rauk A.
        The radical model of Alzheimer's disease: specific recognition of Gly29 and Gly33 by Met35 in a β-sheet model of Aβ: an ONIOM study.
        J. Alzheimers Dis. 2002; 4: 283-289
        • Hou L.
        • Kang I.
        • Marchant R.E.
        • Zagorski M.G.
        Methionine 35 oxidation reduces fibril assembly of the amyloid aβ-(1–42) peptide of Alzheimer's disease.
        J. Biol. Chem. 2002; 277: 40173-40176
        • McGovern S.L.
        • Caselli E.
        • Grigorieff N.
        • Shoichet B.K.
        A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening.
        J. Med. Chem. 2002; 45: 1712-1722
        • Coan K.E.
        • Shoichet B.K.
        Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors.
        J. Am. Chem. Soc. 2008; 130: 9606-9612
        • Hagerman A.E.
        • Butler L.G.
        The specificity of proanthocyanidin-protein interactions.
        J. Biol. Chem. 1981; 256: 4494-4497
        • Feng B.Y.
        • Toyama B.H.
        • Wille H.
        • Colby D.W.
        • Collins S.R.
        • May B.C.
        • Prusiner S.B.
        • Weissman J.
        • Shoichet B.K.
        Small-molecule aggregates inhibit amyloid polymerization.
        Nat. Chem. Biol. 2008; 4: 197-199
        • Ono K.
        • Li L.
        • Takamura Y.
        • Yoshiike Y.
        • Zhu L.
        • Han F.
        • Mao X.
        • Ikeda T.
        • Takasaki J.
        • Nishijo H.
        • Takashima A.
        • Teplow D.B.
        • Zagorski M.G.
        • Yamada M.
        Phenolic compounds prevent amyloid β-protein oligomerization and synaptic dysfunction by site-specific binding.
        J. Biol. Chem. 2012; 287: 14631-14643