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BioAssay: AID 1475

Quantitative High-Throughput Screen for Inhibitors of Tau Fibril Formation: Summary

The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly, to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other more ..
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 Tested Compounds
 Tested Compounds
All(18)
 
 
Probe(1)
 
 
Active(1)
 
 
Inactive(2)
 
 
Inconclusive(15)
 
 
 Tested Substances
 Tested Substances
All(21)
 
 
Probe(1)
 
 
Active(1)
 
 
Inactive(2)
 
 
Inconclusive(18)
 
 
AID: 1475
Data Source: NCGC (TAU2466)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-01-02
Modify Date: 2010-06-29

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: Chemical Probe: 1    Active: 1
Related Experiments
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AIDNameTypeComment
1460qHTS for Inhibitors of Tau Fibril Formation, Thioflavin T BindingConfirmatorydepositor-specified cross reference: Primary screen tested at 6 concentrations from 56 uM to 18 nM, Thioflavin T fluorescence (ThT) reado
1463Counterscreen qHTS for Inhibitors of Tau Fibril Formation, Fluorescence PolarizationConfirmatorydepositor-specified cross reference: Primary counterscreen tested at 6 concentrations from 56 uM to 18 nM, total fluorescence readout mea
1468qHTS for Inhibitors of Tau Fibril Formation, Fluorescence PolarizationConfirmatorydepositor-specified cross reference: Primary screen tested at 6 concentrations from 56 uM to 18 nM, Fluorescence Polarization (FP) readou
1558Confirmation Concentration-Response Assay for for Inhibitors of Tau Fibril Formation, Thioflavin T BindingConfirmatorydepositor-specified cross reference: Confirmation assay: retest of selected compounds, ThT readout
1559Confirmation Concentration-Response Assay for Inhibitors of Tau Fibril Formation, Fluorescence PolarizationConfirmatorydepositor-specified cross reference: Confirmation assay: retest of selected compounds, FP readout
1694Confirmation Concentration-Response Assay for Inhibitors of Tau Fibril Formation, Total Fluorescence Counterscreen for Fluorescence PolarizationConfirmatorydepositor-specified cross reference: Confirmation assay: retest of selected compounds, total fluorescence counterscreen measured by FP
1709Assay for Inhibitors of Tau-Mediated Microtubule AssemblyOtherdepositor-specified cross reference: Secondary assay 2: Inhibition of tau-mediated microtubule assembly
1711Concentration-Response Counterscreen for Tau: Redox Active Inhibitors of Caspase-1Confirmatorydepositor-specified cross reference: Secondary assay 5: Caspase-1 counterscreen for redox artifacts
1712Assay for Inhibitors of the fibrillization of the Beta-Amyloid Protein Fragment, A-beta 1-42Confirmatorydepositor-specified cross reference: Secondary assay: Inhibition of the fibrillization of the beta-amyloid protein fragment, A-beta 1-42
1719Transmission Electron Microscopy Assay for Inhibitors of Tau FibrillizationOtherdepositor-specified cross reference: Secondary assay 4: Transmission Electron Microscopy
1720Sedimentation Assay for Inhibitors of Tau FibrillizationOtherdepositor-specified cross reference: Secondary assay 3: Sedimentation
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLPCN Grant: X01 MH083262-01
Assay Provider: Carlo Ballatore, University of Pennsylvania


NCGC Assay Overview:

The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly, to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other essential cellular constituents to travel along the axons. Under pathological conditions, tau becomes sequestered into insoluble aggregates called neurofibrillary tangles. This phenomenon is believed to have pathological consequences by promoting axonal transport deficits that ultimately lead to synaptic dysfunction and neuronal loss.

To identify inhibitors of tau aggregation, a heparin-induced tau fibril formation assay was used that employed a recombinantly expressed fragment of tau, K18 (Q242-E372), bearing a P301L mutation (Gustke et al., 1994; Hong et al., 1998)). This assay monitors tau fibrillization in two ways, Thioflavin T (ThT) fluorescence and fluorescence polarization (FP) of Alexa 594-labeled K18 P301L, which does not fibrillize readily but incorporates into growing filaments of unlabeled tau. The assay was screened against the MLSMR in a quantitative high-throughput screen (qHTS, (Inglese et al., 2006)) of six library concentrations where ThT (AID 1460), FP mP (AID 1468) and FP total fluorescence (AID 1463) readouts were collected. The total fluorescence measurement for the FP assay served as a counterscreen to identify fluorescent compounds. The titration-response data were curve-fit and classified to identify actives and nascent SAR analysis was performed.

For follow-up, 134 compounds were selected based on the following criteria: active with quality curve-fit in one or both of the ThT and FP measurements, little or no activity in the total fluorescence readout, absence of unwanted series bearing reactive or unstable functional groups, and lacking promiscuous activity in other screens. The compounds were tested in the original screening assay where ThT (AID 1558), FP mP (AID 1559) and FP total fluorescence (AID 1694) readouts were collected. Assay samples were also tested in a sedimentation assay (AID 1720) where tau filaments pellet upon centrifugation while tau monomers remain in the supernatant. Assay samples where the compound inhibited tau sedimentation by 40% or more were examined by transmission electron microscopy (AID 1719) to visualize the size and morphology of the remaining tau filaments.

An aminothienopyridazine series that showed good performance in the follow-up assays and suitable chemical tractability was chosen for probe optimization. Analogs from this series were tested in several secondary assays to determine the selectivity of tau filament inhibition. The analogs did not inhibit tau-mediated microtubule assembly (AID 1709) or filament formation of the beta-amyloid protein fragment, Abeta 1-42 (AID 1712). Series members showed little or no inhibition in a caspase-1 enzymatic assay (AID 1711), which is sensitive to nonspecific redox active compounds. These assays indicated the aminothienopyridazine series were selective inhibitors of tau filament formation in vitro. The most active compound (ML103) from this series was nominated as the probe for Tau fibril inhibition.


Gustke, N., B. Trinczek, et al. (1994). "Domains of tau protein and interactions with microtubules." Biochemistry 33(32): 9511-22.

Hong, M., V. Zhukareva, et al. (1998). "Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17." Science 282(5395): 1914-7.

Inglese, J, Auld, DS, Jadhav, A, Johnson, RL, Simeonov, A, Yasgar, A, Zheng, W, Austin, CP (2006). Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A 103: 11473-8.
Protocol
Please refer to other AIDs (1460, 1468, 1463, 1559, 1558, 1694, 1712, 1709, 1720, 1719, 1711) for detailed assay protocols.
Comment
This summary is written for the purposes of summarizing the probe activities from the project. MLPCN probes are given a score of 100. Molecules in the prior art are given a score of 80. Other, less active molecules in the same chemical series as the probe molecules are given a score of 50. Molecules pending validation are given a score of 10. Inactive analogues from these series are given a score of 0. The present results represent compounds from a probe series.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity SummaryNature of interaction of the sample with the Tau protein.String
2Thioflavin T Measured Potency_uMThe concentration of sample yielding half-maximal inhibition of the Tau protein fibril formation as measured by Thioflavin T (ThT) fluorescence.FloatμM
3Fluorescence Polarization Measured Potency_uMThe concentration of sample yielding half-maximal inhibition of the Tau protein fibril formation as measured by fluorescence polarization (FP) of Alexa 594-labeled K18 P301L.FloatμM
4Total Fluorescence Measured PhenotypeIndicates type of activity observed as measured by sample total fluorescence: fluorescent, quencher, inactive, or inconclusive.String
5A-beta PhenotypeIndicates type of activity observed as measured in the beta-Amyloid protein fibrillization inhibition assay: inhibitor, activator, inactive, or inconclusive.String
6A-beta Potency_uMThe concentration of sample yielding half-maximal inhibition of the beta-Amyloid protein fibrillization, A-beta. FloatμM
7MT Assembly PhenotypeIndicates type of activity observed as measured in the Tau-mediated microtubule assembly inhibition assay: inhibitor, activator, inactive, or inconclusive.String
8Sedimentation Inhibition (%)Percent inhibition at 50 or 80 uM of the Tau protein fibrillization as measured by the sedimentation assay. Float%
9EM PhenotypeIndicates type of activity observed as measured in the transmission electron microscopy assay for Tau fibrillization inhibition: inhibitor, activator, inactive, or inconclusive.String
10Caspase-1 Potency_uMThe concentration of sample yielding half-maximal inhibition of the caspase-1 enzyme. Activity observed for sample in this assay may indicate redox activity.FloatμM
11ML numberMLPCN probe ML numberString
Additional Information
Grant Number: X01 MH083262-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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