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BioAssay: AID 652126

Luminescence-based cell-based primary high throughput screening assay to identify activators of the DAF-12 from the parasite S. stercoralis (ssDAF-12)

Name: Luminescence-based cell-based primary high throughput screening assay to identify activators of the DAF-12 from the parasite S. stercoralis (ssDAF-12). ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 652126
Data Source: The Scripps Research Institute Molecular Screening Center (SSDAF12_AG_LUMI_1536_1X%ACT PRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-03-15
Modify Date: 2013-06-25

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 3700
Related Experiments
652133Summary of the probe development effort to identify activators of the DAF-12 from the parasite S. stercoralis (ssDAF-12)Summarydepositor-specified cross reference
720734Luminescence-based cell-based high throughput dose response assay to identify activators of the DAF-12 from the parasite S. stercoralis (ssDAF-12)Confirmatorydepositor-specified cross reference
720737Counterscreen for agonists of the daf-12 abnormal Dauer Formation: Luminescence-based cell-based screening assay to identify agonists of the Liver-X-Receptor (LXR).Confirmatorydepositor-specified cross reference
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: SSDAF12_AG_LUMI_1536_1X%ACT PRUN

Name: Luminescence-based cell-based primary high throughput screening assay to identify activators of the DAF-12 from the parasite S. stercoralis (ssDAF-12).


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].


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.
10. Lok, J.B., Strongyloides stercoralis: a model for translational research on parasitic nematode biology. WormBook, 2007: p. 1-18.


ssDAF12, daf12, daf-12, S. stercoralis, Caenorhabditis elegans, C. elegans, primary screen, primary, PRUN, lumi, luminescence, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Assay Overview:

The purpose of this assay is to identify compounds that act as activators of the nuclear receptor Daf-12 from S.stercoralis. In this assay, HEK293 cells are co-transfected with a DAF12-responsive reporter plasmid (lit1-tk-luc) and expression vectors encoding ssDAF12 and GRIP1. The ability of compounds to increase transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. Compounds were tested in singlicate at a final nominal concentration of 6.8 uM.

Protocol Summary:

HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 1 mL of serum-free OptiMEM containing 8 ug of the ssDAF12-expressing vector, 8 ug of the GRIP1-expressing vector, 20 ug of the lit1-tk-luc reporter plasmid and 80 uL of transfection reagent. Twenty four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 1 million cells per mL in phenol-red free DMEM supplemented as above. Delta7-dafachronic acid (D7-DA), a well-characterized activators for ssDAF12, was used as a positive control.

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 3 uM of D7-DA (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 ) )


High_Control is defined as wells treated with 3 uM Delta7 Dafachronic Acid
Low_Control is defined as wells treated with DMSO only.
Test_Compound is defined as wells treated with test compound.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally active compounds. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 1-0.

List of Reagents:

lit1-tk-luc luciferase reporter plasmid (Assay Provider)
ssDAF12 expressiong plasmid (Assay Provider)
GRIP1 expression 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)
In the absence of a Protein Entry for the DAF-12 protein from S. stercoralis in NCBI's database, links to the target sequence and origin refer to the closest known orthologue from the prototypic parasitic helminth C.elegans.

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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK293
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activation at 6.8 uM (6.8μM**)Percent activation at compound concentration 6.8 uM.Float%

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

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