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. 2012 Oct 5;287(41):34786-800.
doi: 10.1074/jbc.M112.357665. Epub 2012 Aug 13.

Discovery and structure activity relationship of small molecule inhibitors of toxic β-amyloid-42 fibril formation

Heiko Kroth  1 , Annalisa Ansaloni, Yvan Varisco, Asad Jan, Nampally Sreenivasachary, Nasrollah Rezaei-Ghaleh, Valérie Giriens, Sophie Lohmann, María Pilar López-Deber, Oskar Adolfsson, Maria Pihlgren, Paolo Paganetti, Wolfgang Froestl, Luitgard Nagel-Steger, Dieter Willbold, Thomas Schrader, Markus Zweckstetter, Andrea Pfeifer, Hilal A Lashuel, Andreas Muhs
Affiliations

Discovery and structure activity relationship of small molecule inhibitors of toxic β-amyloid-42 fibril formation

Heiko Kroth et al. J Biol Chem. .

Abstract

Increasing evidence implicates Aβ peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aβ aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aβ42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the β-sheet conformation of Aβ42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aβ42. The efficacy of these compounds on inhibiting Aβ fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aβ42 leading to decreased cell toxicity.

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Figures

FIGURE 1.
FIGURE 1.
A, Shown is the structure of the Ampox (N1,N2-bis(5-methyl-1H-pyrazol-3-yl)oxalamide); Ref. 35) compound, the numbering of the pyrazole moiety, the linker unit, and the possible donor-acceptor-donor interactions of the 3-amino-pyrazole moiety with Aβ42 peptide aggregates having cross-β-sheet conformation. B, structures of the small molecule inhibitors 1–14 of Aβ42 fibrillization containing different aromatic substituents at the 4- or 5-position of the 3-aminopyrazole moiety.
FIGURE 2.
FIGURE 2.
In vitro screening assays using Aβ42 peptide film (A), Aβ42 monomers (B), and Aβ42 protofibrils (C). A, the concentration of Aβ42 peptide film was 33 μm. The test concentration for compounds 1–14 was 330 μm, and the incubation time was 24 h. The data are expressed as the percentage (mean ± S.D.) of control conditions: Aβ42 aggregation with DMSO only. Freshly prepared Aβ42 peptide film (4 μg) was analyzed by SDS-PAGE to confirm the presence of oligomeric Aβ42 present (a, molecular weight marker; b, Aβ42 peptide film). B, the concentration of Aβ42 monomers was 20 μm. C, the concentration of Aβ42 protofibrils was 20 μm. Compounds 1–14 at 10 μm (1:0.5; 1% DMSO), 20 μm (1:1; 2% DMSO), and 40 μm (1:2; 4% DMSO) were co-incubated with Aβ42 monomers or protofibrils for 72 h. The data are expressed as the percentage (mean ± S.D.) of the ThT fluorescence of the 4% DMSO control.
FIGURE 3.
FIGURE 3.
IC50 determination assay using Aβ42 peptide film. The concentration of Aβ42 peptide film was 33 μm. The test concentration for compounds 5, 6, 7, and 10 were 330, 82.5, 20.63, 5.16, 1.29, 0.32, and 0.08 μm with an incubation time of 24 h. The IC50 values were determined from the fluorescence values obtained. The ThT IC50-data are expressed as the mean ± S.D.
FIGURE 4.
FIGURE 4.
Inhibition of Aβ42 aggregation kinetics for compounds 1, 5, 6, 7, and 10 (40 μm) using 10 μm Aβ42 monomers. The total incubation time was 48 h, after which the samples were analyzed with three different assays: ThT fluorescence (A), SDS-PAGE (B), and TEM (C). The ThT-data are expressed as the mean ± S.D.; scale bar for TEM images, 100 nm. a.u., absorbance units.
FIGURE 5.
FIGURE 5.
Inhibition of Aβ42 aggregation kinetics for compounds 1, 5, 6, 7, and 10 (40 μm) using 10 μm Aβ42 protofibrils. The total incubation time was 48 h after which the samples were analyzed with three different assays: ThT fluorescence (A), SDS-PAGE (B), and TEM (C). The ThT data are expressed as the mean ± S.D.; scale bar for TEM images, 100 nm. a.u., absorbance units.
FIGURE 6.
FIGURE 6.
Disaggregation of Aβ42 fibrils by DMSO (40 μm) and compounds 6, 7, and 10 (40 μm) using 100 μm Aβ42 fibrils. The incubation time to form Aβ42 fibrils was 0 h (0hBC) and 48 h before adding DMSO and compounds 6, 7, and 10 (48hBC). Samples were then analyzed after 24 h (24hAC) and 48 h (48hAC) of incubation with DMSO and compounds 6, 7, and 10 using three different assays: SDS-PAGE (A), SDS-PAGE with filtration of the supernatant (B), TEM (C), and ThT fluorescence (D). The ThT data are expressed as the mean ± S.D. Scale bar for TEM images, 100 nm. a.u., absorbance units.
FIGURE 7.
FIGURE 7.
Fluorescence correlation spectroscopy of Aβ42 incubated with compounds 5, 6, 7, and 10. The aggregation of 5 nm Aβ42, N-terminally labeled with Oregon green in PBS and 3% DMSO, was monitored by FCS with or without 200 nm concentrations of compounds 5, 6, 7, and 10. Aggregate formation was quantified by counting the frequency of intensity spikes caused by Aβ42 aggregates passing the detection volume (A) and the height of the intensity spikes (B). Results were normalized to the control aggregation, and the data are expressed as the mean ± S.D.
FIGURE 8.
FIGURE 8.
Ligand-dependent conversion of Aβ42 into oligomers. Intensity changes of proton signals in one-dimensional 1H NMR spectra of Aβ42 upon the addition of compound 6 at various ratios are shown. Changes in the methyl and backbone amide signals are shown separately. Reference values were obtained after the addition of DMSO (without compound 6) at corresponding volumes.
FIGURE 9.
FIGURE 9.
A, shown is the interaction of compounds 6, 7, and 10 with monomeric Aβ42. Shown is the average backbone amide proton and nitrogen chemical shift deviation obtained from two-dimensional 1H,15N heteronuclear single quantum coherence spectra of 15N-labeled Aβ42 in the absence and presence of the compounds 6, 7, and 10. The compounds 6 and 10 were present at a compound/peptide ratio of 30:1, whereas the corresponding value for compound 7 was 24:1. Reference data were obtained after the addition of DMSO (without compounds) at the corresponding volume. B, shown is an illustration of the main interactions of compound 6 with hydrophobic amino acids of monomeric Aβ42 using the NMR structure of Aβ42 fibrils (51) consisting of five peptides (PDB entry 2BEG).
FIGURE 10.
FIGURE 10.
Preferential binding of compound 6 to oligomeric Aβ42. STD spectra of compound 6 in the presence of Aβ42. A, the positive spectra are one-dimensional (1D) 1H NMR spectra of compound 6 in the absence and presence of the peptide. The negative spectra are STD spectra obtained at irradiation frequencies of 0.6, −1, −2, −3, and −5 ppm. B, shown is the STD profile, observed for the ligand peaks at 5.8 and 6.0 ppm as a function of irradiation frequency. Note that the STD intensities obtained with the oligomer-enriched preparation of Aβ42 are larger and extend up to −5 ppm.
FIGURE 11.
FIGURE 11.
Inhibition of crude Aβ42 induced toxicity and inhibition of internalization of crude Aβ42 by compounds 1, 5, 6, 7, and 10 monitored with SH-SY5Y neuroblastoma cells. A, assessment of crude Aβ42 toxicity in the presence of compounds 1, 5, 6, 7, and 10 by employing a MTT reduction assay is shown. Crude Aβ42 was preincubated for 1 h with the compounds a 1:1 molar ratio (10 μm) before the cells were treated with the mixture for 24 h. B, shown is an assessment of the efficacy of compounds 1, 5, 6, 7, and 10 to prevent crude Aβ42 internalization into SH-SY5Y cells. Crude Aβ42 was preincubated for 1 h with the compounds at a 1:1 molar ratio (3 μm) before the cells were treated with the mixture for 2 h. C, shown is correlation between inhibition of internalization of crude Aβ42 and inhibition of crude Aβ42-induced toxicity by compounds 1, 5, 6, 7, and 10. The data are expressed as the mean ± S.D. of three independent experiments.
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