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Identification of Small Molecule Inhibitors of Tau Aggregation by Targeting Monomeric Tau As a Potential Therapeutic Approach for Tauopathies

View Article: PubMed Central

ABSTRACT

A potential strategy to alleviate the aggregation of intrinsically disordered proteins (IDPs) is to maintain the native functional state of the protein by small molecule binding. However, the targeting of the native state of IDPs by small molecules has been challenging due to their heterogeneous conformational ensembles. To tackle this challenge, we applied a high-throughput chemical microarray surface plasmon resonance imaging screen to detect the binding between small molecules and monomeric full-length Tau, a protein linked with the onset of a range of Tauopathies. The screen identified a novel set of drug-like fragment and lead-like compounds that bound to Tau. We verified that the majority of these hit compounds reduced the aggregation of different Tau constructs in vitro and in N2a cells. These results demonstrate that Tau is a viable receptor of drug-like small molecules. The drug discovery approach that we present can be applied to other IDPs linked to other misfolding diseases such as Alzheimer’s and Parkinson’s diseases.

No MeSH data available.


Related in: MedlinePlus

N2a cells with Tau aggregates stained by Thioflavin S. Fluorescence microscopy shows a representative section of the N2a cell population after 4 days of Tau expression. Cells bearing higher Tau aggregates appear bright green (A) whereas cells with soluble or oligomeric Tau show only background staining (A, green background), similar to cells without any Tau expression (B). (C1 – C5) shows examples of cell distributions by FACS counting after 4 days of incubation, without Tau expression (C1, negative control), with Tau expression (C2, positive control), and with Tau expression in the presence of Tau-aggregation inhibitors ID220149 (C3), ID220218 (C4) and ID220255 (C5). The cell signals were plotted with regard to cell morphology (upper panels: FSC (forward scatter indicating the particle size) vs. SSC (side scatter indicating the particle granularity), and with regard to the amount of cells with Tau aggregates (lower panels: FSC vs. fluorescence intensity measured in the FITC channel). Cell debris would appear as small particles (small FSC values) with high density (high SSC values). Note that the green ThS+ cells are well separated from the cell debris population shown in black. The reduction of aggregate-bearing cells by the inhibitor is apparent from the lower panels.
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Figure 5: N2a cells with Tau aggregates stained by Thioflavin S. Fluorescence microscopy shows a representative section of the N2a cell population after 4 days of Tau expression. Cells bearing higher Tau aggregates appear bright green (A) whereas cells with soluble or oligomeric Tau show only background staining (A, green background), similar to cells without any Tau expression (B). (C1 – C5) shows examples of cell distributions by FACS counting after 4 days of incubation, without Tau expression (C1, negative control), with Tau expression (C2, positive control), and with Tau expression in the presence of Tau-aggregation inhibitors ID220149 (C3), ID220218 (C4) and ID220255 (C5). The cell signals were plotted with regard to cell morphology (upper panels: FSC (forward scatter indicating the particle size) vs. SSC (side scatter indicating the particle granularity), and with regard to the amount of cells with Tau aggregates (lower panels: FSC vs. fluorescence intensity measured in the FITC channel). Cell debris would appear as small particles (small FSC values) with high density (high SSC values). Note that the green ThS+ cells are well separated from the cell debris population shown in black. The reduction of aggregate-bearing cells by the inhibitor is apparent from the lower panels.

Mentions: soluble Tau (Fig. 5A, background) or cells without Tau expression (Fig. 5B, highlighted inset demonstrates the presence of cells). Fig. (5) (C1-C5) shows examples of the quantification of Tau aggregation in uninduced N2a cells (column C1), induced cells (column C2) and 60 µM compound treated cells (column C3 – C5) by FACS analysis. In parallel the cell viability was measured at the same compound concentrations by MTT assay. The extent of Tau4RDΔK280 aggregation and cell viability in the compound-free control is normalized to 100% (results summarized in Table 4). Notably, at least two thirds of the hits had a substantial effect in reducing Tau4RDΔK280 aggregation. The most potent half dozen compounds were between 70-84% compared to control, among which were compounds, ID220149, ID220218 and ID220255 shown in (Fig. 3) and (Table 4), representing three distinct compound series from among the Tau binders identified in the HT-CM-SPR screen. As a reference for aggregation in cells, the Tau aggregation inhibitor BSc3094 (from the phenylthiazolyl-hydrazide class [41]) was tested as well, which reduced aggregation of Tau4RDΔK280 by 93%.


Identification of Small Molecule Inhibitors of Tau Aggregation by Targeting Monomeric Tau As a Potential Therapeutic Approach for Tauopathies
N2a cells with Tau aggregates stained by Thioflavin S. Fluorescence microscopy shows a representative section of the N2a cell population after 4 days of Tau expression. Cells bearing higher Tau aggregates appear bright green (A) whereas cells with soluble or oligomeric Tau show only background staining (A, green background), similar to cells without any Tau expression (B). (C1 – C5) shows examples of cell distributions by FACS counting after 4 days of incubation, without Tau expression (C1, negative control), with Tau expression (C2, positive control), and with Tau expression in the presence of Tau-aggregation inhibitors ID220149 (C3), ID220218 (C4) and ID220255 (C5). The cell signals were plotted with regard to cell morphology (upper panels: FSC (forward scatter indicating the particle size) vs. SSC (side scatter indicating the particle granularity), and with regard to the amount of cells with Tau aggregates (lower panels: FSC vs. fluorescence intensity measured in the FITC channel). Cell debris would appear as small particles (small FSC values) with high density (high SSC values). Note that the green ThS+ cells are well separated from the cell debris population shown in black. The reduction of aggregate-bearing cells by the inhibitor is apparent from the lower panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4997948&req=5

Figure 5: N2a cells with Tau aggregates stained by Thioflavin S. Fluorescence microscopy shows a representative section of the N2a cell population after 4 days of Tau expression. Cells bearing higher Tau aggregates appear bright green (A) whereas cells with soluble or oligomeric Tau show only background staining (A, green background), similar to cells without any Tau expression (B). (C1 – C5) shows examples of cell distributions by FACS counting after 4 days of incubation, without Tau expression (C1, negative control), with Tau expression (C2, positive control), and with Tau expression in the presence of Tau-aggregation inhibitors ID220149 (C3), ID220218 (C4) and ID220255 (C5). The cell signals were plotted with regard to cell morphology (upper panels: FSC (forward scatter indicating the particle size) vs. SSC (side scatter indicating the particle granularity), and with regard to the amount of cells with Tau aggregates (lower panels: FSC vs. fluorescence intensity measured in the FITC channel). Cell debris would appear as small particles (small FSC values) with high density (high SSC values). Note that the green ThS+ cells are well separated from the cell debris population shown in black. The reduction of aggregate-bearing cells by the inhibitor is apparent from the lower panels.
Mentions: soluble Tau (Fig. 5A, background) or cells without Tau expression (Fig. 5B, highlighted inset demonstrates the presence of cells). Fig. (5) (C1-C5) shows examples of the quantification of Tau aggregation in uninduced N2a cells (column C1), induced cells (column C2) and 60 µM compound treated cells (column C3 – C5) by FACS analysis. In parallel the cell viability was measured at the same compound concentrations by MTT assay. The extent of Tau4RDΔK280 aggregation and cell viability in the compound-free control is normalized to 100% (results summarized in Table 4). Notably, at least two thirds of the hits had a substantial effect in reducing Tau4RDΔK280 aggregation. The most potent half dozen compounds were between 70-84% compared to control, among which were compounds, ID220149, ID220218 and ID220255 shown in (Fig. 3) and (Table 4), representing three distinct compound series from among the Tau binders identified in the HT-CM-SPR screen. As a reference for aggregation in cells, the Tau aggregation inhibitor BSc3094 (from the phenylthiazolyl-hydrazide class [41]) was tested as well, which reduced aggregation of Tau4RDΔK280 by 93%.

View Article: PubMed Central

ABSTRACT

A potential strategy to alleviate the aggregation of intrinsically disordered proteins (IDPs) is to maintain the native functional state of the protein by small molecule binding. However, the targeting of the native state of IDPs by small molecules has been challenging due to their heterogeneous conformational ensembles. To tackle this challenge, we applied a high-throughput chemical microarray surface plasmon resonance imaging screen to detect the binding between small molecules and monomeric full-length Tau, a protein linked with the onset of a range of Tauopathies. The screen identified a novel set of drug-like fragment and lead-like compounds that bound to Tau. We verified that the majority of these hit compounds reduced the aggregation of different Tau constructs in vitro and in N2a cells. These results demonstrate that Tau is a viable receptor of drug-like small molecules. The drug discovery approach that we present can be applied to other IDPs linked to other misfolding diseases such as Alzheimer’s and Parkinson’s diseases.

No MeSH data available.


Related in: MedlinePlus