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BioAssay: AID 488913

Confirmation Concentration-Response Assay for lipid storage modulators: for probe SAR

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(152)
 
 
Active(66)
 
 
Inactive(24)
 
 
Inconclusive(62)
 
 
 Tested Substances
 Tested Substances
All(153)
 
 
Active(67)
 
 
Inactive(24)
 
 
Inconclusive(62)
 
 
 Related BioAssays
 Related BioAssays
AID: 488913
Data Source: NCGC (LDSA002)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-26
Hold-until Date: 2011-10-25
Modify Date: 2011-10-25

Data Table ( Complete ):           Active    All
BioActive Compounds: 66
Depositor Specified Assays
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AIDNameTypeProbeComment
1519qHTS Assay for Lipid Storage Modulatorsconfirmatory old qHTS screen
1547Secondary Assay for lipid storage modulators. NEFA Incorporation/Release.other NEFA assay
1561Secondary Assay for lipid storage modulators. Cytotoxcityconfirmatory S3 cell-titer glo cytotoxicity
1569Confirmation Concentration-Response Assay for lipid storage modulatorsconfirmatory S3 LDSA confirmation assay
1623qHTS Assay for Lipid Storage Modulators: Summarysummary1 Summary AID
2685qHTS Assay for Lipid Storage Modulators in Drosophila S3 Cellsconfirmatory qHTS rescreen
504432Confirmation Concentration-Response Assay for lipid storage modulators: for probe SAR round 2confirmatory
504433Secondary Assay for lipid storage modulators: Cytotoxcity for probe SAR round 2confirmatory
492960Secondary Assay for lipid storage modulators: HepG2 cell LDSA assay for probe SARconfirmatory
504434Secondary Assay for lipid storage modulators: AML12 cell LDSA assay for probe SARconfirmatory
Description:
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1 R03 MH085686-01
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:
Confirmation concentration-response assay 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 celcius 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.Data is the Fed objects (BODIPY 493/503 fluorophore).
2. 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".
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 0.0002530838 uM (0.000253084μM**)% Activity at given concentration.Float%
15Activity at 0.0005205393 uM (0.000520539μM**)% Activity at given concentration.Float%
16Activity at 0.0007804188 uM (0.000780419μM**)% Activity at given concentration.Float%
17Activity at 0.00156 uM (0.00156162μM**)% Activity at given concentration.Float%
18Activity at 0.00234 uM (0.00234126μM**)% Activity at given concentration.Float%
19Activity at 0.00468 uM (0.00468485μM**)% Activity at given concentration.Float%
20Activity at 0.00702 uM (0.00702377μM**)% Activity at given concentration.Float%
21Activity at 0.014 uM (0.0140546μM**)% Activity at given concentration.Float%
22Activity at 0.021 uM (0.0210713μM**)% Activity at given concentration.Float%
23Activity at 0.042 uM (0.0421637μM**)% Activity at given concentration.Float%
24Activity at 0.063 uM (0.0632139μM**)% Activity at given concentration.Float%
25Activity at 0.126 uM (0.126491μM**)% Activity at given concentration.Float%
26Activity at 0.190 uM (0.189642μM**)% Activity at given concentration.Float%
27Activity at 0.379 uM (0.379473μM**)% Activity at given concentration.Float%
28Activity at 0.569 uM (0.568925μM**)% Activity at given concentration.Float%
29Activity at 1.138 uM (1.13842μM**)% Activity at given concentration.Float%
30Activity at 1.707 uM (1.70678μM**)% Activity at given concentration.Float%
31Activity at 3.415 uM (3.41526μM**)% Activity at given concentration.Float%
32Activity at 5.120 uM (5.12033μM**)% Activity at given concentration.Float%
33Activity at 10.25 uM (10.2458μM**)% Activity at given concentration.Float%
34Activity at 15.36 uM (15.361μM**)% Activity at given concentration.Float%
35Activity at 30.74 uM (30.7373μM**)% Activity at given concentration.Float%
36Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
37Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH085686-01

Data Table (Concise)
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