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BioAssay: AID 485313

qHTS Assay for NPC1 Promoter Activators

Niemann Pick Type C (NPC) is a rare neurodegenerative lipidosis that is characterized by lipid storage in the endosomal/lysosomal system. Treatment modalities for this devastating disease are currently non-existent due to the severe obstacles associated with accessing the central nervous system with proteins or genes. The majority of mutations causing NPC disease are missense mutations that are more ..
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 Tested Compounds
 Tested Compounds
All(320689)
 
 
Active(7576)
 
 
Inactive(304199)
 
 
Inconclusive(8936)
 
 
 Tested Substances
 Tested Substances
All(321376)
 
 
Active(7586)
 
 
Inactive(304846)
 
 
Inconclusive(8944)
 
 
AID: 485313
Data Source: NCGC (NPC1542)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-09-29
Modify Date: 2010-10-04

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 7576
Related Experiments
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AIDNameTypeComment
485315qHTS Assay for NPC1 Promoter Activators: SummarySummarydepositor-specified cross reference
493203qHTS Assay for NPC1 Promoter Activators: Initial hit validation from the primary screenConfirmatorydepositor-specified cross reference
485316qHTS Assay for Rab9 Promoter Activators: SummarySummarysame project related to Summary assay
624116qHTS Assay for Rab9 Promoter Activators: Hit Validation Using Renilla Luciferase Read-Out for Rab9Confirmatorysame project related to Summary assay
624117qHTS Assay for Rab9 Promoter Activators: Hit Validation Using Renilla Luciferase Read-OutConfirmatorysame project related to Summary assay
624493qHTS Assay for Rab9 Promoter Activators: Hit Validation Using Firefly Luciferase Read-Out for NPC1Confirmatorysame project related to Summary assay
624501qHTS Assay for Rab9 Promoter Activators: Hit Validation Using Firefly Luciferase Read-Out for Rab9Confirmatorysame project related to Summary assay
720525qHTS Assay for NPC1 Promoter Activators: Hit Validation Using Firefly Luciferase Read-Out for Rab9Confirmatorysame project related to Summary assay
720526qHTS Assay for NPC1 Promoter Activators: Hit Validation Using Renila Luciferase Read-Out for NPC1Confirmatorysame project related to Summary assay
720527qHTS Assay for NPC1 Promoter Activators: Hit Validation Using Firefly Luciferase Read-Out for NPC1Confirmatorysame project related to Summary assay
720528qHTS Assay for NPC1 Promoter Activators: Hit Validation Using Renila Luciferase Read-Out for Rab9Confirmatorysame project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: MH089375-01A1
Assay Submitter (PI): Yiannis A. Ioannou

NCGC Assay Overview:
Niemann Pick Type C (NPC) is a rare neurodegenerative lipidosis that is characterized by lipid storage in the endosomal/lysosomal system. Treatment modalities for this devastating disease are currently non-existent due to the severe obstacles associated with accessing the central nervous system with proteins or genes. The majority of mutations causing NPC disease are missense mutations that are distributed throughout the length of the NPC1 protein, with the most prevalent being the I1061T allele. Although NPC1 mutant proteins may be functional, they are typically trapped in the ER due to misfolding. Furthermore, over-expression of some mutant NPC1 proteins can rescue the disease phenotype, suggesting that up-regulation of the endogenous NPC1 mutant protein is a new drug treatment modality of the disorder.
We have developed and optimized a cell based luciferase reporter assay in 1536 well format for the identification of up-regulators of the mutant NPC1 promoter.
Protocol
The NPC1 promoter is cloned in front of the luciferase reporter gene, therefore NPC promoter activation results in luciferase expression. Frozen stock of the NPC1 promoter transfected Huh7 cells were obtained from the laboratory of Dr. Yiannis Ioannou (The Mount Sinai School of Medicine, New York, NY). Cells were propagated and maintained at NCGC in medium containing RPMI-1640 supplemented with 10% FBS, 2mM L-glutamine, and 50ug/ml penicillin/streptomycin. Cells were harvested in and plated for assays in OP-MEM I reduced-serum medium supplemented with 10% FBS. The Amplite luciferase reporter gene assay kit was purchased from ABD Bioquest and prepared/stored according to manufacturers recommendations.
qHTS Assay Setup:
1.#Dispensed 5ul of Huh7 cells stably expressing NPC1-luciferase at a density of 1500 cells per well 1536-well plates.
2.#Incubate cells at 37C, 5% CO2, and 95% humidity for 2 hours.
3.#Transferred 23nl per well of compound in DMSO solution.
4.#Incubated at 37C, 5% CO2, and 95% humidity for 24 hours.
5.#Dispensed 3ul per well of Amplite reagent(ABD Bioquest) per well.
6.#Incubate at ambient for 10 minutes.
7.#Read using the luminescence protocol on the ViewLux plate reader (Perkin Elmer).
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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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 0.00295 uM (0.00295μM**)% Activity at given concentration.Float%
15Activity at 0.015 uM (0.0147μM**)% Activity at given concentration.Float%
16Activity at 0.074 uM (0.0737422μM**)% Activity at given concentration.Float%
17Activity at 0.369 uM (0.369μM**)% Activity at given concentration.Float%
18Activity at 1.840 uM (1.84μM**)% Activity at given concentration.Float%
19Activity at 9.220 uM (9.22μM**)% Activity at given concentration.Float%
20Activity at 46.10 uM (46.1μM**)% Activity at given concentration.Float%
21Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH089375-01A1

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