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BioAssay: AID 1688

qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Aggregate Formation (GFP)

Huntington's disease is a neurodegenerative disorder caused by a trinucleotide repeat expansion in Exon 1 of the Huntingtin gene. The CAG trinucleotide encodes glutamine and polyglutamine expansions cause cell death in selective areas of the brain. Huntington polyglutamine repeats have the tendency to form aggregates which are observable in later stages of the disease. The exact nature of the aggregates, whether toxic, protective, or insignificant, is unclear. ..more
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 Tested Compounds
 Tested Compounds
All(220577)
 
 
Active(2380)
 
 
Inactive(201933)
 
 
Inconclusive(16308)
 
 
 Tested Substances
 Tested Substances
All(221029)
 
 
Active(2385)
 
 
Inactive(202329)
 
 
Inconclusive(16315)
 
 
AID: 1688
Data Source: NCGC (HTT010)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-04-21

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 2380
Depositor Specified Assays
AIDNameTypeProbeComment
1471qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection (ATP)confirmatory
1482Quantitative High-Throughput Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Summarysummary2
2669qHTS Assay Multiplex Screening to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection (Protease relase)confirmatory
2672qHTS Assay Multiplex Screening to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection in HTT Q25 expressing cells (ATP)confirmatory
2673qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection confirmation (ATP)confirmatory
2713qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytotoxicity in HTT Striatal Cellsother
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: R03MH084839-01A1
Assay Submitter (PI): Wei Zheng

NCGC Assay Overview:

Huntington's disease is a neurodegenerative disorder caused by a trinucleotide repeat expansion in Exon 1 of the Huntingtin gene. The CAG trinucleotide encodes glutamine and polyglutamine expansions cause cell death in selective areas of the brain. Huntington polyglutamine repeats have the tendency to form aggregates which are observable in later stages of the disease. The exact nature of the aggregates, whether toxic, protective, or insignificant, is unclear.

A stable PC12 cell line containing a gene fusion of Exon 1 of the Huntingtin gene linked to GFP under the control of the inducible ecdysone promoter were used as the cell-based model of Huntington Disease for high throughput screening. Exon 1 of the Huntingtin gene contained an expansion of 103 polyglutamines (Q103) which, when expressed, caused cell death and distinct, bright GFP aggregates. The amount of cell death and the size and intensity of GFP aggregates increased over time and induction level. The cell death were quantified with a measurement of ATP content in the cells. A maximal 40-50% of cell death was observed when the Huntington gene was induced in this cell line.

We optimized this assay in a homogeneous 1536 well format. A quantitative high throughput screen (qHTS) was conducted on the PC12 cells which were induced to express the toxic fusion construct. Small molecules which reduced aggregate formation were identified bu using a laser scanning cytometer calibrated to detect the aggregates in cell lysates.
Protocol
PC12 cells stably containing a construct with an ecdysone inducible promoter expressing GFP fused to the exon 1 of HTTQ103 (Huntington protein which containing a 103 ployglutamine expansion) were used.

NCGC Assay Protocol Summary:
Sequence, Parameter, Description
1, Reagent, 5 uL 1500 cells/well 1536 TC treated White clear bottom plate
2, Compound, 23 nL Tebufenozide inducer (100 nM final) to all columns
3, Compound, 23 nL 40 uM to 0.5 nM Libraries
4, Time, 40 hours at 37C
5, Reagent, 5 ul ATPLite cell death assay reagent
6, Detector, Aggregate quantitation by Acumen Explorer

Media consisted of phenol red free DMEM + 5% equine serum (Hyclone # SH30072.03 ), 5% supplemented Bovine Calf Serum (Hyclone # SH30074.03 ), 1x pen/strep, no G418
Acumen Explorer was used to quantitate GFP aggregates using the following criteria: 488 nM laser excitation, 500-530 nM emission, the PMT was set at a voltage of 450 volts, and objects ranging in size of 1 to 25 m in size having a signal of 6 STDEV above background were collected.
Control columns: 1 = DMSO, 2= 2 uM teb dilns (1:1), 4 = 100 nM Teb inducer. Sample columns received 100 nM Teb.
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, apparent inhibitors are ranked higher than compounds that showed apparent activation.
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 0.0000590000 uM (5.9e-05μM**)% Activity at given concentration.Float%
15Activity at 0.0001320000 uM (0.000132μM**)% Activity at given concentration.Float%
16Activity at 0.0002739534 uM (0.000273953μM**)% Activity at given concentration.Float%
17Activity at 0.0006009766 uM (0.000600977μM**)% Activity at given concentration.Float%
18Activity at 0.00134 uM (0.00134389μM**)% Activity at given concentration.Float%
19Activity at 0.00301 uM (0.00300564μM**)% Activity at given concentration.Float%
20Activity at 0.00671 uM (0.00671402μM**)% Activity at given concentration.Float%
21Activity at 0.015 uM (0.0149857μM**)% Activity at given concentration.Float%
22Activity at 0.034 uM (0.0336201μM**)% Activity at given concentration.Float%
23Activity at 0.074 uM (0.073815μM**)% Activity at given concentration.Float%
24Activity at 0.168 uM (0.168025μM**)% Activity at given concentration.Float%
25Activity at 0.369 uM (0.368986μM**)% Activity at given concentration.Float%
26Activity at 0.840 uM (0.839578μM**)% Activity at given concentration.Float%
27Activity at 1.842 uM (1.84229μM**)% Activity at given concentration.Float%
28Activity at 4.198 uM (4.19789μM**)% Activity at given concentration.Float%
29Activity at 9.218 uM (9.21832μM**)% Activity at given concentration.Float%
30Activity at 20.60 uM (20.6μM**)% Activity at given concentration.Float%
31Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
32Activity at 46.10 uM (46.1μM**)% Activity at given concentration.Float%
33Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084839-01A1

Data Table (Concise)
Classification
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