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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

SPR Screening Results. Example of colour coded SPR signals obtained by chemical microarray screening of hTau2N4Rwt under optimized conditions against: (A) a fragment library of 3,000 fragments (out of 25,000 fragments present in the small molecule library screened) spotted in triplicates, and (B) a lead-like library combining (96x96 =9,216) motifs. Reproducibility of screening experiments for chemical microarray with (C) fragments and for (D) lead-like compounds immobilized.
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Figure 2: SPR Screening Results. Example of colour coded SPR signals obtained by chemical microarray screening of hTau2N4Rwt under optimized conditions against: (A) a fragment library of 3,000 fragments (out of 25,000 fragments present in the small molecule library screened) spotted in triplicates, and (B) a lead-like library combining (96x96 =9,216) motifs. Reproducibility of screening experiments for chemical microarray with (C) fragments and for (D) lead-like compounds immobilized.

Mentions: Monomeric hTau2N4Rwt was screened against the Graffinity library using the in-house developed Graffinity SPR Imagers, which allowed recording the SPR minima of four chemical arrays in parallel with 9,216 sensor fields each. For manual analysis of the array data, SPR signals were visualized in coloured two-dimensional fingerprints with the colour representing the intensity of SPR shift upon incubation with protein. To demonstrate this process, (Fig. 2) shows representative examples of hTau2N4Rwt SPR fingerprints in an array view. For fragment libraries compounds were spotted in triplicates, hence hits for the fragment library appear as triplicate spots on a diagonal (Fig. 2A). In the case of lead-like libraries derived from combinatorial synthesis approaches, each row and column consists of a common molecular moiety in combination with 96 other diverse analouges (Fig. 2B), thus hit series identified for hTau2N4Rwt appear as line pattern (Fig. 2B). The plots on the right in (Fig. 2A and B) compare the SPR signals of two independent repetition experiments (different operator, reader, date, same hTau2N4Rwt batch). Multiplicate SPR signals were averaged for each immobilized array compound (e.g. fragments signals: 2 experiments x 3 spots = hexaplicate signals). A filtering step removed potential hits for which the standard deviation of the SPR signal was higher than 0.8 nm (see Fig. 1C). The threshold for selection of Tau-binding compounds was defined as the SPR minimum that was separated from the background noise by at least 1 nm and for which the reproducibility criteria was satisfied. In addition, immobilized compounds that had low purity before spotting to the surface of the array and had low saturation per spot (< 80%) were disregarded.


Identification of Small Molecule Inhibitors of Tau Aggregation by Targeting Monomeric Tau As a Potential Therapeutic Approach for Tauopathies
SPR Screening Results. Example of colour coded SPR signals obtained by chemical microarray screening of hTau2N4Rwt under optimized conditions against: (A) a fragment library of 3,000 fragments (out of 25,000 fragments present in the small molecule library screened) spotted in triplicates, and (B) a lead-like library combining (96x96 =9,216) motifs. Reproducibility of screening experiments for chemical microarray with (C) fragments and for (D) lead-like compounds immobilized.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: SPR Screening Results. Example of colour coded SPR signals obtained by chemical microarray screening of hTau2N4Rwt under optimized conditions against: (A) a fragment library of 3,000 fragments (out of 25,000 fragments present in the small molecule library screened) spotted in triplicates, and (B) a lead-like library combining (96x96 =9,216) motifs. Reproducibility of screening experiments for chemical microarray with (C) fragments and for (D) lead-like compounds immobilized.
Mentions: Monomeric hTau2N4Rwt was screened against the Graffinity library using the in-house developed Graffinity SPR Imagers, which allowed recording the SPR minima of four chemical arrays in parallel with 9,216 sensor fields each. For manual analysis of the array data, SPR signals were visualized in coloured two-dimensional fingerprints with the colour representing the intensity of SPR shift upon incubation with protein. To demonstrate this process, (Fig. 2) shows representative examples of hTau2N4Rwt SPR fingerprints in an array view. For fragment libraries compounds were spotted in triplicates, hence hits for the fragment library appear as triplicate spots on a diagonal (Fig. 2A). In the case of lead-like libraries derived from combinatorial synthesis approaches, each row and column consists of a common molecular moiety in combination with 96 other diverse analouges (Fig. 2B), thus hit series identified for hTau2N4Rwt appear as line pattern (Fig. 2B). The plots on the right in (Fig. 2A and B) compare the SPR signals of two independent repetition experiments (different operator, reader, date, same hTau2N4Rwt batch). Multiplicate SPR signals were averaged for each immobilized array compound (e.g. fragments signals: 2 experiments x 3 spots = hexaplicate signals). A filtering step removed potential hits for which the standard deviation of the SPR signal was higher than 0.8 nm (see Fig. 1C). The threshold for selection of Tau-binding compounds was defined as the SPR minimum that was separated from the background noise by at least 1 nm and for which the reproducibility criteria was satisfied. In addition, immobilized compounds that had low purity before spotting to the surface of the array and had low saturation per spot (< 80%) were disregarded.

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&rsquo;s and Parkinson&rsquo;s diseases.

No MeSH data available.


Related in: MedlinePlus