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BioAssay: AID 624116

qHTS Assay for Rab9 Promoter Activators: Hit Validation Using Renilla Luciferase Read-Out for Rab9

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(222)
 
 
Active(10)
 
 
Inactive(190)
 
 
Inconclusive(22)
 
 
 Tested Substances
 Tested Substances
All(222)
 
 
Active(10)
 
 
Inactive(190)
 
 
Inconclusive(22)
 
 
AID: 624116
Data Source: NCGC (Rab9201)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-04-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 10
Related Experiments
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AIDNameTypeComment
485315qHTS Assay for NPC1 Promoter Activators: SummarySummarydepositor-specified cross reference: NPC1 Summary AID
485316qHTS Assay for Rab9 Promoter Activators: SummarySummarydepositor-specified cross reference: Rab9 Summary AID
485313qHTS Assay for NPC1 Promoter ActivatorsConfirmatorysame project related to Summary assay
493203qHTS Assay for NPC1 Promoter Activators: Initial hit validation from the primary screenConfirmatorysame 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
1379Counterscreen for Luciferase (Kinase-Glo TM) InhibitionConfirmatorysame project related to Summary assay
485297qHTS Assay for Rab9 Promoter ActivatorsConfirmatorysame project related to Summary assay
493200qHTS Assay for Rab9 Promoter Activators: Initial hit validation from the primary screenConfirmatorysame 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
720528qHTS Assay for NPC1 Promoter Activators: Hit Validation Using Renila Luciferase Read-Out for Rab9Confirmatorysame project related to Summary assay
Description:
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 renilla luciferase reporter assay in 1536 well format for the identification of up-regulators of the mutant Rab9 promoter. This assay presents data on the validation of hits from the primary screen, which used a firefly luciferase read-out.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: MH089375
Assay Submitter (PI): Yiannis A. Ioannou
Protocol
Huh7 cells stably expressing NPC promoter-luciferase will be seeded at 1,500 cells/well in 5 uL in 1,536-well white opaque plates with 23 nL/well of compound in DMSO solution. The assay plates will be incubated at 37 deg C for 24 hours, after which 3 uL/well BriteLite plus luciferase detection reagent mixture (Perkin Elmer) will be added. Luminescence signal will be measured in a ViewLux plate reader (Perkin Elmer) after a 10 minute incubation at the room temperature.
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 activators are ranked higher than compounds that showed apparent inhibition.
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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0004342330 uM (0.000434233μM**)% Activity at given concentration.Float%
16Activity at 0.00130 uM (0.0013027μM**)% Activity at given concentration.Float%
17Activity at 0.00391 uM (0.0039081μM**)% Activity at given concentration.Float%
18Activity at 0.012 uM (0.0117243μM**)% Activity at given concentration.Float%
19Activity at 0.035 uM (0.0351729μM**)% Activity at given concentration.Float%
20Activity at 0.106 uM (0.105519μM**)% Activity at given concentration.Float%
21Activity at 0.317 uM (0.316556μM**)% Activity at given concentration.Float%
22Activity at 0.950 uM (0.949668μM**)% Activity at given concentration.Float%
23Activity at 2.849 uM (2.849μM**)% Activity at given concentration.Float%
24Activity at 8.547 uM (8.54701μM**)% Activity at given concentration.Float%
25Activity at 25.64 uM (25.641μM**)% Activity at given concentration.Float%
26Activity at 76.92 uM (76.9231μM**)% Activity at given concentration.Float%
27Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH089375

Data Table (Concise)
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