Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Michael Denison, University of California, Davis
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1-X01-DA026558-01
Grant Proposal PI: Michael Denison
External Assay ID: AHR_ACT_LUMI_1536_3XEC50 MDRUN

Name: Late stage results for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based cell-based dose response assay for AHR activators.

Description:

Transcription factors are critical regulators of gene expression (1). Under conditions such as environmental stress and exposure to endogenous toxins, transcription factors can rapidly modulate the transcription of genes whose products regulate cell proliferation and metabolism. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor of the basic helix-loop-helix protein superfamily involved in the biological response to aromatic hydrocarbons, and regulates the expression of xenobiotic-metabolizing enzymes such as cytochrome P450, aldehyde dehydrogenase, quinone reductase, and other phase I and phase II detoxification genes (2, 3). In response to various compounds, including the environmental pollutants dioxins, benzo(a)pyrene, dietary contaminants, grapefruit juice, endogenous toxins, and plant products such as carotinoids, nicotine and caffeine (2, 4-6), cytosolic AHR complexes with chaperones hsp90, p23, and XAP2, translocates to the nucleus where it dimerizes with the AHR nuclear translocator (ARNT) to influence target gene transcription (7, 8). Gain-of-function studies in mice reveal the oncogenic potential of AHR (9), while other reports show roles for AHR in diverse biologic events such as organ development (10, 11), immune function and allergy (12), and estrogen responsiveness (13). The identification of agonists of AHR will provide useful tools to elucidate the roles of this receptor in cell metabolism, transcriptional control, and tumor formation.

References:

1. Ptashne, M., Regulation of transcription: from lambda to eukaryotes. Trends Biochem Sci, 2005. 30(6): p. 275-9.
2. McMillan, B.J. and Bradfield, C.A., The aryl hydrocarbon receptor sans xenobiotics: endogenous function in genetic model systems. Mol Pharmacol, 2007. 72(3): p. 487-98.
3. Puga, A., Tomlinson, C.R., and Xia, Y., Ah receptor signals cross-talk with multiple developmental pathways. Biochem Pharmacol, 2005. 69(2): p. 199-207.
4. Bock, K.W. and Kohle, C., Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions. Biochem Pharmacol, 2006. 72(4): p. 393-404.
5. Denison, M.S. and Nagy, S.R., Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu Rev Pharmacol Toxicol, 2003. 43: p. 309-34.
6. de Waard, P.W., Peijnenburg, A.A., Baykus, H., Aarts, J.M., Hoogenboom, R.L., van Schooten, F.J., and de Kok, T.M., A human intervention study with foods containing natural Ah-receptor agonists does not significantly show AhR-mediated effects as measured in blood cells and urine. Chem Biol Interact, 2008.
7. Hankinson, O., The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol, 1995. 35: p. 307-40.
8. Petrulis, J.R. and Perdew, G.H., The role of chaperone proteins in the aryl hydrocarbon receptor core complex. Chem Biol Interact, 2002. 141(1-2): p. 25-40.
9. Andersson, P., McGuire, J., Rubio, C., Gradin, K., Whitelaw, M.L., Pettersson, S., Hanberg, A., and Poellinger, L., A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors. Proc Natl Acad Sci U S A, 2002. 99(15): p. 9990-5.
10. Ramos, K.S., Transcriptional profiling and functional genomics reveal a role for AHR transcription factor in nephrogenesis. Ann N Y Acad Sci, 2006. 1076: p. 728-35.
11. Walisser, J.A., Glover, E., Pande, K., Liss, A.L., and Bradfield, C.A., Aryl hydrocarbon receptor-dependent liver development and hepatotoxicity are mediated by different cell types. Proc Natl Acad Sci U S A, 2005. 102(49): p. 17858-63.
12. Lawrence, B.P., Denison, M.S., Novak, H., Vorderstrasse, B.A., Harrer, N., Neruda, W., Reichel, C., and Woisetschlager, M., Activation of the aryl hydrocarbon receptor is essential for mediating the anti-inflammatory effects of a novel low-molecular-weight compound. Blood, 2008. 112(4): p. 1158-65.
13. Ohtake, F., Takeyama, K., Matsumoto, T., Kitagawa, H., Yamamoto, Y., Nohara, K., Tohyama, C., Krust, A., Mimura, J., Chambon, P., Yanagisawa, J., Fujii-Kuriyama, Y., and Kato, S., Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature, 2003. 423(6939): p. 545-50.
14. Zhao, B., Baston, D.S., Hammock, B., and Denison, M.S., Interaction of diuron and related substituted phenylureas with the Ah receptor pathway. J Biochem Mol Toxicol, 2006. 20(3): p. 103-13.
15. Garrison, P.M., Tullis, K., Aarts, J.M., Brouwer, A., Giesy, J.P., and Denison, M.S., Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam Appl Toxicol, 1996. 30(2): p. 194-203.
16. Han, D., Nagy, S.R., and Denison, M.S., Comparison of recombinant cell bioassays for the detection of Ah receptor agonists. Biofactors, 2004. 20(1): p. 11-22.

Keywords:

late stage, powders, purchased, synthesized, AHR, bHLHe76, aryl hydrocarbon receptor, receptor, transcription factor, triplicate, dose response, HTS, high throughput screen, 1536, activator, agonist, activation, luciferase, luminescence, reporter, Scripps, 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 determine dose response curves for powder samples of compounds identified as possible AHR activator probe candidates. This cell-based assay monitors the ability of compounds to activate AHR signaling. The assay employs human hepatoma (HepG2) cells stably transfected with the AHR-dependent pGudLuc6.1-DRE plasmid (HG2L6.1c3 cell line), which expresses the firefly luciferase reporter gene under control of a minimal promoter containing a synthetic dioxin response element (DRE) (14-16). Cells are incubated with test compounds for 24 hours, followed by cell lysis and detection of well luminescence using a commercially available luciferase reagent. As designed, compounds that act as AHR agonists will increase AHR activity and nuclear translocation, leading to increased activity of the DRE, increased transcription of the luciferase reporter gene, and increased well luminescence. Compounds are tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 91.7 uM.

Protocol Summary:

The HG2L6.1c3 cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Minimum Essential Medium (MEM) alpha supplemented with 10% v/v premium fetal bovine serum, 400 ug/mL G418 (Geneticin), and 1X antibiotic mix (penicillin, streptomycin, and neomycin).

Prior to the start of the assay 5000 cells in a 4 uL volume of assay media (growth media as above except without geneticin) were dispensed into each well of 1536-well tissue culture-treated microtiter plates. The assay was started immediately by dispensing 37 nL of test compound in DMSO (0.9% final DMSO concentration), DMSO alone, or Indirubin (60 uM final concentration) to the appropriate wells. Next, the plates were incubated for 24 hours at 37 C (5% CO2, 95% RH). After equilibrating the plates to room temperature for 30 minutes, the assay was stopped by dispensing 4 uL of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 5 minutes. Well luminescence was measured on the ViewLux plate reader.

The percent activation for each compound was calculated using the following mathematical formula:

% Activation = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control- Median_Low_Control ) )

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing Indirubin

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. 91.7 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 91.7 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, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-43, and for inactive compounds 1-1.

List of Reagents:

HG2L6.1c3 AHR cell line (provided by Assay provider)
Indirubin (Amplachem, part Aa-31440)
Minimum Essential Medium alpha (Invitrogen, part 12561072)
G418 sulfate powder (Gemini Bio-Products, part 400-11P)
Trypsin-EDTA solution (Invitrogen, part 15400054)
Fetal Bovine Serum, Premium (Atlanta Biologicals, part S11150)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
SteadyLite HTS Assay Kit (PerkinElmer, part 6016989)
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Greiner, part 789072)

This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. In this assay, Indirubin had an EC50 of approximately 420 nM. 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, compounds that modulate 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.

Grant Number: 1-X01-DA026558-01