Luminescence-based cell-based high throughput dose response assay to identify agonists of the DAF-12 from the parasite H. contortus (hcDAF-12)
Name: Luminescence-based cell-based high throughput dose response assay to identify agonists of the DAF-12 from the parasite H. contortus (hcDAF-12). ..more
Depositor Specified Assays
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: HCDAF12_ACT_LUMI_1536_3XEC50 DRUN
Name: Luminescence-based cell-based high throughput dose response assay to identify agonists of the DAF-12 from the parasite H. contortus (hcDAF-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 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.
DRUN, triplicate, dose response, titration, dilution, daf12, daf-12, Caenorhabditis elegans, C. elegans, endpoint, end-point, lumi, luminescence, HTS, high throughput screen, 1536, 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 the nuclear receptor Daf-12 from H.contortus agonists dose response curves for compounds that confirmed activity in a set of previous experiments 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). In this assay, HEK293 cells are co-transfected with a DAF12-responsive reporter plasmid (lit1-tk-luc) and expression vector encoding hcDAF12. The ability of compounds to increase transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. Compounds are tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 68 uM.
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 hcDAF12-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 agonist for hcDAF12, 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.
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 (Accelrys 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.
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:
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)