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BioAssay: AID 493200

qHTS Assay for Rab9 Promoter Activators: Initial hit validation from the primary screen

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 (Ioannou, 2000). We have developed a new paradigm, termed "Orphan Receptor more ..
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 Tested Compounds
 Tested Compounds
All(222)
 
 
Active(47)
 
 
Inactive(137)
 
 
Inconclusive(38)
 
 
 Tested Substances
 Tested Substances
All(222)
 
 
Active(47)
 
 
Inactive(137)
 
 
Inconclusive(38)
 
 
AID: 493200
Data Source: NCGC (RAB9544)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-02-11

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 47
Related Experiments
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AIDNameTypeComment
485297qHTS Assay for Rab9 Promoter ActivatorsConfirmatorydepositor-specified cross reference: Primary screen for Rab9 activators.
485316qHTS Assay for Rab9 Promoter Activators: SummarySummarydepositor-specified cross reference: Summary assay for project.
1379Counterscreen for Luciferase (Kinase-Glo TM) InhibitionConfirmatorysame project related to Summary assay
485315qHTS Assay for NPC1 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
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: MH089537-01
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 (Ioannou, 2000). We have developed a new paradigm, termed "Orphan Receptor Bypass Therapy" (ORByT), to address these disorders. This paradigm posits the existence of endogenous "suppressor" proteins whose expression can dramatically improve LSD pathogenesis. The goal is the identification/discovery of small chemical compounds that can modulate the expression of these proteins and provide a new treatment modality for these devastating disorders. One such protein whose overexpression reverses aspects of the NPC phenotype is Rab9.
We have developed and optimized a cell based luciferase reporter assay in 1536 well format for the identification of up-regulators of the Rab9 promoter. This assay presents data on the validation of hits from the primary screen.
Protocol
The Rab9 promoter is cloned in front of the luciferase reporter gene, therefore NPC promoter activation results in luciferase expression. Frozen stock of the Rab9 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 Rab9-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 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.
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 0.0002601396 uM (0.00026014μM**)% Activity at given concentration.Float%
15Activity at 0.0007804188 uM (0.000780419μM**)% Activity at given concentration.Float%
16Activity at 0.00234 uM (0.00234μM**)% Activity at given concentration.Float%
17Activity at 0.00702 uM (0.00702μM**)% Activity at given concentration.Float%
18Activity at 0.021 uM (0.021μM**)% Activity at given concentration.Float%
19Activity at 0.063 uM (0.063μM**)% Activity at given concentration.Float%
20Activity at 0.190 uM (0.19μM**)% Activity at given concentration.Float%
21Activity at 0.569 uM (0.569μM**)% Activity at given concentration.Float%
22Activity at 1.707 uM (1.707μM**)% Activity at given concentration.Float%
23Activity at 5.120 uM (5.12μM**)% Activity at given concentration.Float%
24Activity at 15.36 uM (15.36μM**)% Activity at given concentration.Float%
25Activity at 46.08 uM (46.08μM**)% Activity at given concentration.Float%
26Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH089537-01

Data Table (Concise)
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