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BioAssay: AID 1694

Confirmation Concentration-Response Assay for Inhibitors of Tau Fibril Formation, Total Fluorescence Counterscreen for Fluorescence Polarization

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(134)
 
 
Active(2)
 
 
Inactive(103)
 
 
Inconclusive(30)
 
 
 Tested Substances
 Tested Substances
All(138)
 
 
Active(2)
 
 
Inactive(106)
 
 
Inconclusive(30)
 
 
AID: 1694
Data Source: NCGC (TAU2930)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-04-24

Data Table ( Complete ):           Active    All
Target
Sequence: Microtubule-associated protein tau [Homo sapiens]
Description ..   
Protein Family: Tau and MAP protein, tubulin-binding repeat

Gene:MAPT     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 2
Depositor Specified Assays
AIDNameTypeComment
1463Counterscreen qHTS for Inhibitors of Tau Fibril Formation, Fluorescence Polarizationconfirmatory
1475Quantitative High-Throughput Screen for Inhibitors of Tau Fibril Formation: Summarysummary
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 by fluorescence polarization (FP) of Alexa 594-labeled K18 P301L, which does not fibrillize readily but incorporates into growing filaments of unlabeled tau. Total fluorescence (555 nm excitation and 632 nm emission) was measured to identify fluorescent or absorbent compounds at this wavelength. Such compounds may be artifacts that interfere with the Alexa 594 Tau fluorescence polarization assay.


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.

Li, W. and V. M. Lee (2006). "Characterization of two VQIXXK motifs for tau fibrillization in vitro." Biochemistry 45(51): 15692-701.

von Bergen, M., S. Barghorn, et al. (2001). "Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure." J Biol Chem 276(51): 48165-74.
Protocol
NCGC Assay Protocol Summary:
Two K18 mutants were produced: P301L, which fibrillizes faster than the wild-type form (von Bergen et al. 2001) and K311D, which does not fibrillize (Li and Lee 2006) and was thus used as non-fibrillizing control in the assay. For screening, 2 uL/well human K18 P301L (15 uM unlabeled and 0.24 uM Alexa 594-labeled tau final concentrations) in reagent buffer (100 mM sodium acetate pH 7) was dispensed into black solid 1536-well plates (Grenier) using a solenoid-based dispenser. Following transfer of 23 nL compound or DMSO vehicle by a pin tool, 2 uL/well heparin (20 uM final concentration) in reagent buffer was added and the plate centrifuged 15 s at 1000 RPM. Plates were incubated 6 hr at 37 C and then 1 uL/well ThT (30 uM final concentration) was added. After a 1 hr ambient incubation, the plates were read by an Envision (Perkin Elmer) to monitor Alexa 594 FP (555 nm excitation and 632 nm emission).


Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, NCGC, qHTS, tau, tauopathies, Alzheimer's disease, fibril, neurodegeneration, aggregation
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, compounds that showed apparent activation are likely fluorescent and apparent inhibitors are likely absorbent compounds.

2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.049 uM (0.0488281μM**)% Activity at given concentration.Float%
15Activity at 0.111 uM (0.111275μM**)% Activity at given concentration.Float%
16Activity at 0.223 uM (0.223114μM**)% Activity at given concentration.Float%
17Activity at 0.391 uM (0.390625μM**)% Activity at given concentration.Float%
18Activity at 0.446 uM (0.446286μM**)% Activity at given concentration.Float%
19Activity at 0.890 uM (0.890198μM**)% Activity at given concentration.Float%
20Activity at 1.786 uM (1.78604μM**)% Activity at given concentration.Float%
21Activity at 3.125 uM (3.125μM**)% Activity at given concentration.Float%
22Activity at 3.571 uM (3.57143μM**)% Activity at given concentration.Float%
23Activity at 6.250 uM (6.25μM**)% Activity at given concentration.Float%
24Activity at 7.143 uM (7.14286μM**)% Activity at given concentration.Float%
25Activity at 14.27 uM (14.2667μM**)% Activity at given concentration.Float%
26Activity at 28.53 uM (28.5333μM**)% Activity at given concentration.Float%
27Activity at 50.00 uM (50μM**)% Activity at given concentration.Float%
28Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
29Activity at 100.00 uM (100μM**)% Activity at given concentration.Float%
30Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: X01 MH083262-01

Data Table (Concise)
Classification
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