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: PXR_ACT_LUMI_1536_3XEC50 MDCSRUN

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

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. Lemaire, G., de Sousa, G., and Rahmani, R., A PXR reporter gene assay in a stable cell culture system: CYP3A4 and CYP2B6 induction by pesticides. Biochem Pharmacol, 2004. 68(12): p. 2347-58.

Keywords:

late stage, powders, purchased, synthesized, AHR, bHLHe76, aryl hydrocarbon receptor, receptor, transcription factor, triplicate, dose response, counterscreen, NR1I2, nuclear receptor subfamily 1 group I member 2, BXR, ONR1, PAR, PAR1, PAR2, PARq, PRR, PXR, SAR, SXR, 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 dose response counterscreen is to determine whether powder samples of compounds identified as possible AHR activator probe candidates are nonselective due to activation of PXR. This cell-based assay (14) measures the ability of compounds to activate Pregnane X Receptor (PXR) nuclear signaling. The assay employs human hepatoma (DPX-2) cells co-transfected with a plasmid which expresses full-length human PXR and a reporter plasmid which expresses the firefly luciferase reporter gene under control of a minimal promoter containing a PXR response element (multiple copies of the DR3 motif from mouse CYP3A1 gene). Cells are incubated with test compounds followed by cell lysis and detection of well luminescence using a commercially available luciferase reagent. As designed, compounds that act as PXR agonists will increase PXR activity and nuclear translocation, leading to increased activity of the PXR response element and transcription of the luciferase reporter gene, resulting in increased well luminescence. Compounds are tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 116.1 uM.

Protocol Summary:

The DPX-2 cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). Cells were grown in Puracyp culturing media.

Prior to the start of the assay 5000 cells in a 4 uL volume of Dosing Media were dispensed into each well of 1536-well tissue culture-treated microtiter plates. The assay was started immediately by dispensing 47 nL of test compound in DMSO (1.2 % final DMSO concentration), DMSO alone, or Rifampicin (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 Bright-Glo luciferase substrate to each well, followed by incubation at room temperature for two 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 Rifampicin

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. 116.1 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 116.1 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 inactive compounds100-0. There are no active compounds.

List of Reagents:

DPX-2 cell line (Puracyp)
Rifampicin (Biomol, part GR-306-0001)
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Greiner, part 789072)
DPX-2 Culturing Media (Puracyp, part C-500-100)
Dosing Media (Puracyp, D-500-100)
Bright-Glo Luciferase Assay System (Promega, part E2650)

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, rifampicin had an EC50 of approximately 4 uM. 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