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BioAssay: AID 1519

qHTS Assay for Lipid Storage Modulators

NCGC Assay Overview: Storing lipids as a reservoir for energy or the anabolism of elementary metabolites is a common feature of probably all cells and is conserved from bacteria to humans. The universal cellular lipid storage organelle is the so-called lipid storage droplet (LD). Although ubiquitous, LDs share a simple structure composed of a hydrophobic core that harbors the storage lipids, more ..
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 Tested Compounds
 Tested Compounds
All(7979)
 
 
Active(19)
 
 
Inactive(4893)
 
 
Inconclusive(3420)
 
 
 Tested Substances
 Tested Substances
All(8868)
 
 
Active(19)
 
 
Inactive(5264)
 
 
Inconclusive(3585)
 
 
 Related BioAssays
 Related BioAssays
AID: 1519
Data Source: NCGC (LDSA643)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-02-06
Modify Date: 2010-07-06

Data Table ( Complete ):           Active    All
BioActive Compounds: 19
Depositor Specified Assays
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AIDNameTypeProbeComment
488913Confirmation Concentration-Response Assay for lipid storage modulators: for probe SARconfirmatory
504433Secondary Assay for lipid storage modulators: Cytotoxcity for probe SAR round 2confirmatory
488908Secondary Assay for lipid storage modulators: Cytotoxcity for probe SARconfirmatory
1547Secondary Assay for lipid storage modulators. NEFA Incorporation/Release.other
488900Secondary Assay for lipid storage modulators: Hela cell LDSA assay for probe SARconfirmatory
504432Confirmation Concentration-Response Assay for lipid storage modulators: for probe SAR round 2confirmatory
1561Secondary Assay for lipid storage modulators. Cytotoxcityconfirmatory
1569Confirmation Concentration-Response Assay for lipid storage modulatorsconfirmatory
1623qHTS Assay for Lipid Storage Modulators: Summarysummary1
Description:
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: TBA
Assay Submitter (PI): Beller, Mathias; Max-Planck-Institut fur Biophysikalische Chemie

NCGC Assay Overview: Storing lipids as a reservoir for energy or the anabolism of elementary metabolites is a common feature of probably all cells and is conserved from bacteria to humans. The universal cellular lipid storage organelle is the so-called lipid storage droplet (LD). Although ubiquitous, LDs share a simple structure composed of a hydrophobic core that harbors the storage lipids, which is shielded by a droplet-specific phospholipid monolayer to which proteins are attached. The current model of LD biogenesis involves an incorporation of the lipid core into the membrane leaflets of the endoplasmic reticulum (ER) followed by a subsequent budding-like maturation of a LD, which ultimately pinches off. Once released, LD volume can increase by localized lipogenesis or fusion of existing droplets. Storage lipids are re-mobilized enzymatically by lipase activity. Lipase regulation in the adipocyte is heavily studied and involves multiple components including catecholamine signaling, the LD-associated proteins Perilipin and comparative gene identification-58 (CGI-58) and at least two lipases named hormone sensitive lipase (HSL) and adipocyte triglyceride lipase (ATGL). LD regulation outside of the adipocyte is poorly understood and only few components are known. However, there is an urgent need to learn more about ectopic fat depots as mislocalized storage of lipids, for example in the liver or muscle, is an eminent health problem associated with insulin resistance or the metabolic syndrome. We developed a laser-scanning cytometer assay to enable 1,536-well screening and combined the results of the small molecules screen with an RNAi database based on lipid storage [1].
Protocol
NCGC Assay Protocol Summary:
qHTS was performed with embryonic Drosophila S3 cells (Bloomington Drosophila Genomics Resource Center [DGRC]), which showed excellent oleic acid feeding characteristics during RNAi assay development. We dispensed 4 uL of cells at 1.25 x 106 cells/mL into LoBase Aurora COC 1,536-well plates (black walled, clear bottom) with a bottle-valve solenoid-based dispenser (Aurora) to obtain 5,000 cells/well. Triacsin C (Sigma-Aldrich T-4540), an inhibitor of long-chain fatty acyl CoA synthetase that blocks synthesis of triglycerides, was used as a positive control for lipid under-storage. A total of 23 nL of compound solution of different concentrations were transferred to the assay plates using a Kalypsys pin tool equipped with a 1,536-pin array containing 10-nL slotted pins (FP1S10, 0.457-mm diameter, 50.8 mm long; V&P Scientific). One microliter of oleic acid (400 uM) was added, and the plate was lidded with stainless steel rubber gasket lined lids containing pinholes. After 18-24 h incubation at 24 degree celsius and 95% humidity, 4 uL of the dyes BODIPY 493/503 (Molecular Probes) and the Cell Tracker Red CMTPX dye (Molecular Probes) were added to the wells to stain lipid droplets and enumerate cell number, respectively. Fluorescence was detected by excitation of the fluorophores with a 488-nm laser on an Acumen Explorer (TTP Lab Tech). The total intensity in channel 1 (500-530 nm) reflected lipid droplet accumulation. Cells were detected using channel 3 (575-640 nm) with 5-um width and 100-um depth filters. The ratio of the total intensity in PMT channel 1 over total intensity of channel 3 was also calculated. Percent activity was computed relative to an internal control (100% inhibited lipid droplet deposition due to the presence of 20 uM Triacsin C), which was added to 32 wells/plate.
Comment
Compound Ranking:

1. Compounds are first classified as being probable inhibitors (compounds decreasing signal), activators (compounds increasing signal), cytotoxic (artifactual results), inactive, or inconclusive based on the ratio and component channel readouts.

2. Active compounds were ranked by efficacy and potency from 99 to 40. Inconclusive outcome compounds were scored based on the phenotype observed: 20 for less conclusive inhibitors, 10 for inconclusive phenotype classification, and 0 for inactive.
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
5cells-Curve_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 signficant, but below 80% of control.String
6cells-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7cells-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8cells-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9cells-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10cells-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11cells-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12cells-Excluded_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
13cells-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14cells-Activity at 0.0007362069 uM (0.000736207μM**)% Activity at given concentration.Float%
15cells-Activity at 0.00165 uM (0.0016454μM**)% Activity at given concentration.Float%
16cells-Activity at 0.00368 uM (0.00367931μM**)% Activity at given concentration.Float%
17cells-Activity at 0.00823 uM (0.00822759μM**)% Activity at given concentration.Float%
18cells-Activity at 0.018 uM (0.0183966μM**)% Activity at given concentration.Float%
19cells-Activity at 0.041 uM (0.0411345μM**)% Activity at given concentration.Float%
20cells-Activity at 0.092 uM (0.0919764μM**)% Activity at given concentration.Float%
21cells-Activity at 0.206 uM (0.205659μM**)% Activity at given concentration.Float%
22cells-Activity at 0.460 uM (0.459854μM**)% Activity at given concentration.Float%
23cells-Activity at 1.028 uM (1.02823μM**)% Activity at given concentration.Float%
24cells-Activity at 2.299 uM (2.29913μM**)% Activity at given concentration.Float%
25cells-Activity at 5.141 uM (5.14086μM**)% Activity at given concentration.Float%
26cells-Activity at 11.49 uM (11.495μM**)% Activity at given concentration.Float%
27cells-Activity at 25.70 uM (25.7027μM**)% Activity at given concentration.Float%
28cells-Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
29fed-Curve_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 signficant, but below 80% of control.String
30fed-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
31fed-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
32fed-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
33fed-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
34fed-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
35fed-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
36fed-Excluded_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
37fed-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
38fed-Activity at 0.0007362069 uM (0.000736207μM**)% Activity at given concentration.Float%
39fed-Activity at 0.00165 uM (0.0016454μM**)% Activity at given concentration.Float%
40fed-Activity at 0.00368 uM (0.00367931μM**)% Activity at given concentration.Float%
41fed-Activity at 0.00823 uM (0.00822759μM**)% Activity at given concentration.Float%
42fed-Activity at 0.018 uM (0.0183966μM**)% Activity at given concentration.Float%
43fed-Activity at 0.041 uM (0.0411345μM**)% Activity at given concentration.Float%
44fed-Activity at 0.092 uM (0.0919764μM**)% Activity at given concentration.Float%
45fed-Activity at 0.206 uM (0.205659μM**)% Activity at given concentration.Float%
46fed-Activity at 0.460 uM (0.459854μM**)% Activity at given concentration.Float%
47fed-Activity at 1.028 uM (1.02823μM**)% Activity at given concentration.Float%
48fed-Activity at 2.299 uM (2.29913μM**)% Activity at given concentration.Float%
49fed-Activity at 5.141 uM (5.14086μM**)% Activity at given concentration.Float%
50fed-Activity at 11.49 uM (11.495μM**)% Activity at given concentration.Float%
51fed-Activity at 25.70 uM (25.7027μM**)% Activity at given concentration.Float%
52fed-Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
53Ratio-Curve_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 signficant, but below 80% of control.String
54Ratio-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
55Ratio-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
56Ratio-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
57Ratio-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
58Ratio-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
59Ratio-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
60Ratio-Excluded_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
61Ratio-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
62Ratio-Activity at 0.0007362069 uM (0.000736207μM**)% Activity at given concentration.Float%
63Ratio-Activity at 0.00165 uM (0.0016454μM**)% Activity at given concentration.Float%
64Ratio-Activity at 0.00368 uM (0.00367931μM**)% Activity at given concentration.Float%
65Ratio-Activity at 0.00823 uM (0.00822759μM**)% Activity at given concentration.Float%
66Ratio-Activity at 0.018 uM (0.0183966μM**)% Activity at given concentration.Float%
67Ratio-Activity at 0.041 uM (0.0411345μM**)% Activity at given concentration.Float%
68Ratio-Activity at 0.092 uM (0.0919764μM**)% Activity at given concentration.Float%
69Ratio-Activity at 0.206 uM (0.205659μM**)% Activity at given concentration.Float%
70Ratio-Activity at 0.460 uM (0.459854μM**)% Activity at given concentration.Float%
71Ratio-Activity at 1.028 uM (1.02823μM**)% Activity at given concentration.Float%
72Ratio-Activity at 2.299 uM (2.29913μM**)% Activity at given concentration.Float%
73Ratio-Activity at 5.141 uM (5.14086μM**)% Activity at given concentration.Float%
74Ratio-Activity at 11.49 uM (11.495μM**)% Activity at given concentration.Float%
75Ratio-Activity at 25.70 uM (25.7027μM**)% Activity at given concentration.Float%
76Ratio-Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
77Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
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