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BioAssay: AID 743037

Counterscreen for agonists of the daf-12 abnormal Dauer Formation: Luminescence-based cell-based dose response assay to identify agonists of the Liver-X-Receptor (LXR).

Name: Counterscreen for agonists of the daf-12 abnormal Dauer Formation: Luminescence-based cell-based dose response assay to identify agonists of the Liver-X-Receptor (LXR). ..more
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 Tested Compounds
 Tested Compounds
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Inactive(247)
 
 
 Tested Substances
 Tested Substances
All(247)
 
 
Inactive(247)
 
 
AID: 743037
Data Source: The Scripps Research Institute Molecular Screening Center (LXR_ACT_LUMI_1536_3XEC50 DCSRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-11-08
Modify Date: 2013-11-14

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
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AIDNameTypeComment
652074Summary of the probe development effort to identify activators of the DAF-12 from the parasite H. contortus (hcDAF-12)Summarydepositor-specified cross reference: Summary AID
687014Luminescence-based cell-based primary high throughput screening assay to identify agonists of the DAF-12 from the parasite H. glycines (hgDAF-12).Screeningdepositor-specified cross reference: Primary Screen (hgDAF12 activators in singlicate)
652067Luminescence-based cell-based primary high throughput screening assay to identify activators of the DAF-12 from the parasite H. contortus (hcDAF-12)Screeningsame project related to Summary assay
743027Counterscreen for agonists of the DAF-12 from the parasite H.contortus (hcDAF-12): Luminescence-based cell-based screening assay to identify agonists of the Liver-X-Receptor (LXR).Screeningsame project related to Summary assay
743032Luminescence-based cell-based high throughput confirmation assay to identify agonists of the DAF-12 from the parasite H. contortus (hcDAF-12)Screeningsame project related to Summary assay
743038Luminescence-based cell-based high throughput dose response assay to identify agonists of the DAF-12 from the parasite H. contortus (hcDAF-12)Confirmatorysame project related to Summary assay
687024Summary of the probe development effort to identify agonists of the DAF-12 from the parasite H. glycines (hgDAF-12).Summarysame project related to Summary assay
743050Luminescence-based cell-based high throughput confirmation assay to identify agonists of the DAF-12 from the parasite H. glycines (hgDAF-12).Screeningsame project related to Summary assay
743051Counterscreen for agonists of the daf-12 abnormal Dauer Formation: Luminescence-based cell-based screening assay to identify agonists of the Liver-X-Receptor (LXR)Screeningsame project related to Summary assay
743052Counterscreen for agonists of the daf-12 abnormal Dauer Formation: Luminescence-based cell-based dose response screening assay to identify agonists of the Liver-X-Receptor (LXR)Confirmatorysame project related to Summary assay
743059Luminescence-based cell-based high throughput dose response assay to identify agonists of the DAF-12 from the parasite H. glycines (hgDAF-12).Confirmatorysame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: UT Southwestern
Assay Provider: David Mangelsdorf, UT Southwestern
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: U19 DK062434
Grant Proposal Pi: David Mangelsdorf, UT Southwestern
External Assay ID: LXR_ACT_LUMI_1536_3XEC50 DCSRUN

Name: Counterscreen for agonists of the daf-12 abnormal Dauer Formation: Luminescence-based cell-based dose response assay to identify agonists of the Liver-X-Receptor (LXR).

Description:

Parasitic helminthes (worms) are a significant health and economic burden: over two billion people are infected by helminthes [1], and parasitic nematodes cause billions of dollars of crop damage each year in the United States [2]. The developmental stages of these organisms are widely studied [3, 4]. One stage, dauer (German for "duration", also known as an alternative L3 larval stage) covers an alternative larval stage in which development stops and the worms enter a hibernation-like state in which they can survive extremely harsh environmental conditions, often for years. In the case of parasitic nematodes, this resting state is quite often the infectious state [5]. As the burden of parasitic nematodes grows in the face of emerging resistance to the few existing antihelminthic agents, it is becoming increasingly important to understand the life cycles of parasitic worms so that new drugs may be developed [1]. The nuclear receptor DAF-12 (for "dauer formation"), first identified in C. elegans, is known to control many nematode species' entry into and exit from the dauer resting state [6]. Daf-12 belongs to a family of over 30 genes which transduce environmental signals to influence the choice between dauer or reproductive development. Favorable environments activate insulin/IGF and TGF-beta pathways converge, leading to production of the steroid hormone dafachronic acid (DA), which binds and activates Daf-12 [7]. Currently available antihelminthic agents, to which resistance is beginning to emerge, act primarily on the feeding stages of the worms and have little effect on the infectious stages [8]. Therefore, pharmacologic activators developed through high-throughput screening would be used both practically as nematicides and academically as tools to characterize the role of DAF-12 in modulating life cycle [8, 9].

References:

1. Jasmer, D.P., A. Goverse, and G. Smant, Parasitic nematode interactions with mammals and plants. Annu Rev Phytopathol, 2003. 41: p. 245-70.
2. Hotez, P.J., J. Bethony, M.E. Bottazzi, S. Brooker, D. Diemert, and A. Loukas, New technologies for the control of human hookworm infection. Trends Parasitol, 2006. 22(7): p. 327-31
3. Mooijaart, S.P., B.W. Brandt, E.A. Baldal, J. Pijpe, M. Kuningas, M. Beekman, B.J. Zwaan, P.E. Slagboom, R.G. Westendorp, and D. van Heemst, C. elegans DAF-12, Nuclear Hormone Receptors and human longevity and disease at old age. Aging Res Rev, 2005. 4(3): p. 351-71
4. Brenner, S., The genetics of Caenorhabditis elegans. Genetics, 1974. 77(1): p. 71-94
5. Motola, D.L., C.L. Cummins, V. Rottiers, K.K. Sharma, T. Li, Y. Li, K. Suino-Powell, H.E. Xu, R.J. Auchus, A. Antebi, and D.J. Mangelsdorf, Identification of ligands for DAF-12 that govern dauer formation and reproduction in C. elegans. Cell, 2006. 124(6): p. 1209-23
6. Antebi, A., W.H. Yeh, D. Tait, E.M. Hedgecock, and D.L. Riddle, daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elegans. Genes Dev, 2000. 14(12): p. 1512-27.
7. Gerisch, B. and A. Antebi, Hormonal signals produced by DAF-9/cytochrome P450 regulate C. elegans dauer diapause in response to environmental cues. Development, 2004. 131(8): p. 1765-76.
8. Wang, Z., X.E. Zhou, D.L. Motola, X. Gao, K. Suino-Powell, A. Conneely, C. Ogata, K.K. Sharma, R.J. Auchus, J.B. Lok, J.M. Hawdon, S.A. Kliewer, H.E. Xu, and D.J. Mangelsdorf, Identification of the nuclear receptor DAF-12 as a therapeutic target in parasitic nematodes. Proc Natl Acad Sci U S A, 2009. 106(23): p. 9138-43
9. Schroeder, F.C., Small molecule signaling in Caenorhabditis elegans. ACS Chem Biol, 2006. 1(4): p. 198-200.

Keywords:

LXR, NR1H2, liver-x-receptor, counterscreen, artifact, daf12, daf-12, Caenorhabditis elegans, C. elegans, screen, counterscreen, CSRUN, lumi, luminescence, 1536, 384, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this assay is to determine non-specific gene reporter activation of compounds found active in a set of experiment entitled "Luminescence-based cell-based primary high throughput screening assay to identify activators of the DAF-12 from the parasite H. contortus (hcDAF-12)" (AID 652067). Compounds are tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 68 uM.
Protocol Summary:
In this assay, HEK293 are transfected with a GAL4-responsive reporter plasmid (tk-luc) and expression vectors encoding Gal4-LXR. The ability of compounds to alter LXR-mediated transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. The LXR agonist T0901317 was used as a positive control for this assay.
The assay was started by dispensing 5 uL of cell suspension into each well of white, solid-bottom 1536-well plates using a flying reagent dispenser (Aurora) and placed in the incubator for 3 hours. Cells were then treated with 34 nL/well of either test compounds, DMSO (Low Control, final concentration 0.68%) or 6.8 uM of T0901317 (High Control). Plates were incubated for 24 hours at 37 C, 5% CO2 and 95%RH and then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of One-Glo reagent to each well. After a 15 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).
The percent activation of each test compound was calculated as follows:
%_Activation = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median__Low_Control ) )
Where:
High_Control is defined as wells treated with 6.8 uM T0901317 compound
Low_Control is defined as wells treated with DMSO only.
Test_Compound is defined as wells treated with test compound.
For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 68 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 68 uM.
PubChem Activity Outcome and Score:
Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for inactive compounds is 0-0, there are no active compounds.
List of Reagents:
MH1000-tk-luc luciferase reporter plasmid (Assay Provider)
Gal4-LXR expressing plasmid (Assay Provider)
List of Consumables:
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 11965)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
TransIT 293 (Mirus Corporation, part MIR-2700)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
One-Glo (Promega, part E6130)
1536-well plates (Greiner part 789173)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate luciferase activity and hence well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK293
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierActivity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
2EC50*The concentration at which 50 percent of the activity in the activator assay is observed; (EC50) shown in micromolar.FloatμM
3LogEC50Log10 of the qualified EC50 (EC50) from the activator assay in M concentrationFloat
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
8Response RangeThe range of Y.Float
9Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
10RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
11Number of DataPointsOverall number of data points of normalized percent activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
12Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
13Activation at 0.0035 uM [1] (0.0035μM**)Value of % activation at 0.004 uM compound concentration; replicate [1]Float%
14Activation at 0.0035 uM [2] (0.0035μM**)Value of % activation at 0.004 uM compound concentration; replicate [2]Float%
15Activation at 0.0035 uM [3] (0.0035μM**)Value of % activation at 0.004 uM compound concentration; replicate [3]Float%
16Activation at 0.01 uM [1] (0.01μM**)Value of % activation at 0.010 uM compound concentration; replicate [1]Float%
17Activation at 0.01 uM [2] (0.01μM**)Value of % activation at 0.010 uM compound concentration; replicate [2]Float%
18Activation at 0.01 uM [3] (0.01μM**)Value of % activation at 0.010 uM compound concentration; replicate [3]Float%
19Activation at 0.031 uM [1] (0.031μM**)Value of % activation at 0.031 uM compound concentration; replicate [1]Float%
20Activation at 0.031 uM [2] (0.031μM**)Value of % activation at 0.031 uM compound concentration; replicate [2]Float%
21Activation at 0.031 uM [3] (0.031μM**)Value of % activation at 0.031 uM compound concentration; replicate [3]Float%
22Activation at 0.093 uM [1] (0.093μM**)Value of % activation at 0.09 uM compound concentration; replicate [1]Float%
23Activation at 0.093 uM [2] (0.093μM**)Value of % activation at 0.09 uM compound concentration; replicate [2]Float%
24Activation at 0.093 uM [3] (0.093μM**)Value of % activation at 0.09 uM compound concentration; replicate [3]Float%
25Activation at 0.28 uM [1] (0.28μM**)Value of % activation at 0.28 uM compound concentration; replicate [1]Float%
26Activation at 0.28 uM [2] (0.28μM**)Value of % activation at 0.28 uM compound concentration; replicate [2]Float%
27Activation at 0.28 uM [3] (0.28μM**)Value of % activation at 0.28 uM compound concentration; replicate [3]Float%
28Activation at 0.84 uM [1] (0.84μM**)Value of % activation at 0.84 uM compound concentration; replicate [1]Float%
29Activation at 0.84 uM [2] (0.84μM**)Value of % activation at 0.84 uM compound concentration; replicate [2]Float%
30Activation at 0.84 uM [3] (0.84μM**)Value of % activation at 0.84 uM compound concentration; replicate [3]Float%
31Activation at 2.5 uM [1] (2.5μM**)Value of % activation at 2.5 uM compound concentration; replicate [1]Float%
32Activation at 2.5 uM [2] (2.5μM**)Value of % activation at 2.5 uM compound concentration; replicate [2]Float%
33Activation at 2.5 uM [3] (2.5μM**)Value of % activation at 2.5 uM compound concentration; replicate [3]Float%
34Activation at 7.6 uM [1] (7.6μM**)Value of % activation at 7.6 uM compound concentration; replicate [1]Float%
35Activation at 7.6 uM [2] (7.6μM**)Value of % activation at 7.6 uM compound concentration; replicate [2]Float%
36Activation at 7.6 uM [3] (7.6μM**)Value of % activation at 7.6 uM compound concentration; replicate [3]Float%
37Activation at 22.7 uM [1] (22.7μM**)Value of % activation at 22.7 uM compound concentration; replicate [1]Float%
38Activation at 22.7 uM [2] (22.7μM**)Value of % activation at 22.7 uM compound concentration; replicate [2]Float%
39Activation at 22.7 uM [3] (22.7μM**)Value of % activation at 22.7 uM compound concentration; replicate [3]Float%
40Activation at 68 uM [1] (68μM**)Value of % activation at 68.0 uM compound concentration; replicate [1]Float%
41Activation at 68 uM [2] (68μM**)Value of % activation at 68.0 uM compound concentration; replicate [2]Float%
42Activation at 68 uM [3] (68μM**)Value of % activation at 68.0 uM compound concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: U19 DK062434

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