Counterscreen 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).
Name: Counterscreen for agonists of the daf-12 abnormal Dauer Formation: Luminescence-based cell-based screening assay to identify agonists of the Liver-X-Receptor (LXR). ..more
BioActive Compound: 1
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
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_3X%ACT for hcDAF-12 AG
Name: Counterscreen for agonists of the daf-12 abnormal Dauer Formation: Luminescence-based cell-based screening assay to identify agonists of the Liver-X-Receptor (LXR).
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 agonists 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. Ageing 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.
Counterscreen, CSUN, LXR, NR1H2, liver-x-receptor, artifact, daf12, daf-12, Caenorhabditis elegans, C. elegans, lumi, luminescence, HTS, high throughput screen, 1536, 384, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
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 at a nominal concentration of 6.8 uM.
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 ) )
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:
The average percent inhibition and standard deviation of each compound tested were calculated. The sum of these two values was used as a hit cutoff parameter. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated 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 100-100, and for inactive compounds 90-0.
List of Reagents:
lit1-tk-luc luciferase reporter plasmid (Assay Provider)
hcDAF12 expressiong plasmid (Assay Provider)
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)
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.
** Test Concentration.
Data Table (Concise)