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A novel Aβ-fibrinogen interaction inhibitor rescues altered thrombosis and cognitive decline in Alzheimer's disease mice.

Ahn HJ, Glickman JF, Poon KL, Zamolodchikov D, Jno-Charles OC, Norris EH, Strickland S - J. Exp. Med. (2014)

Bottom Line: To determine if the Aβ-fibrinogen interaction could be targeted as a potential new treatment for AD, we designed a high-throughput screen and identified RU-505 as an effective inhibitor of the Aβ-fibrinogen interaction.Furthermore, long-term treatment of RU-505 significantly reduced vascular amyloid deposition and microgliosis in the cortex and improved cognitive impairment in mouse models of AD.Our studies suggest that inhibitors targeting the Aβ-fibrinogen interaction show promise as therapy for treating AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Neurobiology and Genetics and High Throughput Screening Resource Center, The Rockefeller University, New York, NY 10065.

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The chemical structure and dose–response curve of Aβ–fibrinogen interaction inhibitors. (A) TAMRA–labeled Aβ peptide was bound to fibrinogen and the test compound, and the anisotropy of TAMRA–Aβ–fibrinogen binding was determined by FP. (B) Biotin-labeled Aβ42, which binds a streptavidin donor, was incubated with fibrinogen, which binds a protein A acceptor bead coated with antifibrinogen antibody. Aβ42 and fibrinogen interactions bring the beads in close proximity, resulting in the excitation of the donor beads and release of singlet oxygen molecules that triggers light emission in acceptor beads (AlphaLISA [AL]). (C) The half-maximal inhibitory concentration (IC50) values of the indicated compounds were determined by dose–response FP and AL experiments and are indicated inside the panel (red, FP; blue, AL). A quenching test was also performed to calculate how much each hit compound interfered with the AL signal at 10 µM concentration. Quenching values are indicated below the dose–response curve. n = 3–4 repeats per assay and all error bars indicate SEM. Data are representative of at least three independent experiments.
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fig1: The chemical structure and dose–response curve of Aβ–fibrinogen interaction inhibitors. (A) TAMRA–labeled Aβ peptide was bound to fibrinogen and the test compound, and the anisotropy of TAMRA–Aβ–fibrinogen binding was determined by FP. (B) Biotin-labeled Aβ42, which binds a streptavidin donor, was incubated with fibrinogen, which binds a protein A acceptor bead coated with antifibrinogen antibody. Aβ42 and fibrinogen interactions bring the beads in close proximity, resulting in the excitation of the donor beads and release of singlet oxygen molecules that triggers light emission in acceptor beads (AlphaLISA [AL]). (C) The half-maximal inhibitory concentration (IC50) values of the indicated compounds were determined by dose–response FP and AL experiments and are indicated inside the panel (red, FP; blue, AL). A quenching test was also performed to calculate how much each hit compound interfered with the AL signal at 10 µM concentration. Quenching values are indicated below the dose–response curve. n = 3–4 repeats per assay and all error bars indicate SEM. Data are representative of at least three independent experiments.

Mentions: To investigate this idea, we designed a high-throughput screen (HTS) to identify small molecules that inhibit the interaction between Aβ and fibrinogen. Low molecular weight compounds were screened using fluorescence polarization (FP) and AlphaLISA assays in a complementary fashion to cross check the activity of the hit compounds and to ensure the removal of false-positive artifacts. Primarily, ∼93,000 compounds were screened using FP, which measured the changes in the anisotrophy induced by binding of a 5-carboxy-tetramethylrhodamine (TAMRA)–labeled Aβ peptide to fibrinogen (Fig. 1 A). Then, hits from FP were screened using AlphaLISA to independently confirm the activity of the inhibitors identified in the FP assay (Fig. 1 B). After both steps, we selected only drug-like compounds using Lipinski’s Rule of Five, which allowed us to determine which chemical compounds have pharmacological properties that would make them likely orally active drugs in humans (Lipinski et al., 2001). We also filtered out artifactual compounds using a quenching assay, which identifies insoluble compounds, singlet oxygen quenchers, and biotin mimetics interfering with the AlphaLISA signal. We identified several candidate compounds with half-maximal inhibitions (IC50) between 10 and 50 µM from the dose-response assays using both FP and AlphaLISA assays (Table 1).


A novel Aβ-fibrinogen interaction inhibitor rescues altered thrombosis and cognitive decline in Alzheimer's disease mice.

Ahn HJ, Glickman JF, Poon KL, Zamolodchikov D, Jno-Charles OC, Norris EH, Strickland S - J. Exp. Med. (2014)

The chemical structure and dose–response curve of Aβ–fibrinogen interaction inhibitors. (A) TAMRA–labeled Aβ peptide was bound to fibrinogen and the test compound, and the anisotropy of TAMRA–Aβ–fibrinogen binding was determined by FP. (B) Biotin-labeled Aβ42, which binds a streptavidin donor, was incubated with fibrinogen, which binds a protein A acceptor bead coated with antifibrinogen antibody. Aβ42 and fibrinogen interactions bring the beads in close proximity, resulting in the excitation of the donor beads and release of singlet oxygen molecules that triggers light emission in acceptor beads (AlphaLISA [AL]). (C) The half-maximal inhibitory concentration (IC50) values of the indicated compounds were determined by dose–response FP and AL experiments and are indicated inside the panel (red, FP; blue, AL). A quenching test was also performed to calculate how much each hit compound interfered with the AL signal at 10 µM concentration. Quenching values are indicated below the dose–response curve. n = 3–4 repeats per assay and all error bars indicate SEM. Data are representative of at least three independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4042638&req=5

fig1: The chemical structure and dose–response curve of Aβ–fibrinogen interaction inhibitors. (A) TAMRA–labeled Aβ peptide was bound to fibrinogen and the test compound, and the anisotropy of TAMRA–Aβ–fibrinogen binding was determined by FP. (B) Biotin-labeled Aβ42, which binds a streptavidin donor, was incubated with fibrinogen, which binds a protein A acceptor bead coated with antifibrinogen antibody. Aβ42 and fibrinogen interactions bring the beads in close proximity, resulting in the excitation of the donor beads and release of singlet oxygen molecules that triggers light emission in acceptor beads (AlphaLISA [AL]). (C) The half-maximal inhibitory concentration (IC50) values of the indicated compounds were determined by dose–response FP and AL experiments and are indicated inside the panel (red, FP; blue, AL). A quenching test was also performed to calculate how much each hit compound interfered with the AL signal at 10 µM concentration. Quenching values are indicated below the dose–response curve. n = 3–4 repeats per assay and all error bars indicate SEM. Data are representative of at least three independent experiments.
Mentions: To investigate this idea, we designed a high-throughput screen (HTS) to identify small molecules that inhibit the interaction between Aβ and fibrinogen. Low molecular weight compounds were screened using fluorescence polarization (FP) and AlphaLISA assays in a complementary fashion to cross check the activity of the hit compounds and to ensure the removal of false-positive artifacts. Primarily, ∼93,000 compounds were screened using FP, which measured the changes in the anisotrophy induced by binding of a 5-carboxy-tetramethylrhodamine (TAMRA)–labeled Aβ peptide to fibrinogen (Fig. 1 A). Then, hits from FP were screened using AlphaLISA to independently confirm the activity of the inhibitors identified in the FP assay (Fig. 1 B). After both steps, we selected only drug-like compounds using Lipinski’s Rule of Five, which allowed us to determine which chemical compounds have pharmacological properties that would make them likely orally active drugs in humans (Lipinski et al., 2001). We also filtered out artifactual compounds using a quenching assay, which identifies insoluble compounds, singlet oxygen quenchers, and biotin mimetics interfering with the AlphaLISA signal. We identified several candidate compounds with half-maximal inhibitions (IC50) between 10 and 50 µM from the dose-response assays using both FP and AlphaLISA assays (Table 1).

Bottom Line: To determine if the Aβ-fibrinogen interaction could be targeted as a potential new treatment for AD, we designed a high-throughput screen and identified RU-505 as an effective inhibitor of the Aβ-fibrinogen interaction.Furthermore, long-term treatment of RU-505 significantly reduced vascular amyloid deposition and microgliosis in the cortex and improved cognitive impairment in mouse models of AD.Our studies suggest that inhibitors targeting the Aβ-fibrinogen interaction show promise as therapy for treating AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Neurobiology and Genetics and High Throughput Screening Resource Center, The Rockefeller University, New York, NY 10065.

Show MeSH
Related in: MedlinePlus