Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: David Frank, Dana-Farber Cancer Institute
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 X01 MH079826-01
Grant Proposal PI: David Frank, Dana-Farber Cancer Institute

External Assay ID: STAT1_INH_LUMI_1536_IC50 (CSDRUN)

Name: Dose response counterscreen assay for STAT3 inhibitors: cell-based high throughput assay to measure STAT1 inhibition

Description:

Members of the signal transducer and activator of transcription (STAT) family of transcription factors mediate inflammation, cell survival, differentiation, and proliferation (1, 2). In response to stimuli such as growth factors and cytokines (1-3), cytosolic STATs are activated by phosphorylation by the Janus-activated kinases (Jaks), inducing STAT dimerization, nuclear translocation, and binding to specific enhancer elements in target genes (2). Although structurally similar, STAT proteins possess diverse biological roles (2). For example, STAT1 activity is pro-inflammatory, anti-proliferative and mediates the effects of IFN-gamma, while STAT3 activity is anti-inflammatory, pro-apoptotic, and mediates IL-6 signaling (2, 4). Studies showing that STAT3 is activated in breast and prostate cancers, that genetic inhibition of STAT3 reduces cell proliferation, survival, and wound healing (1, 4, 5), and that disrupting STAT3-EGFR interactions reduces tumor growth (6), suggest that STAT3 activation has broad cellular effects. As a result, the identification of selective STAT3 modulators may provide useful tools for exploring STAT3 biology.


References:

1. Alvarez JV, Febbo PG, Ramaswamy S, Loda M, Richardson A, Frank DA. Identification of a genetic signature of activated signal transducer and activator of transcription 3 in human tumors. Cancer Res. 2005 Jun 15;65(12):5054-62.
2. Schindler C, Levy DE, Decker T. JAK-STAT signaling: from interferons to cytokines. J Biol Chem. 2007 Jul 13;282(28):20059-63.
3. Germain D, Frank DA. Targeting the cytoplasmic and nuclear functions of signal transducers and activators of transcription 3 for cancer therapy. Clin Cancer Res. 2007 Oct 1;13(19):5665-9.
4. Levy DE, Darnell JE Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 2002 Sep;3(9):651-62.
5. Takeda K, Kaisho T, Yoshida N, Takeda J, Kishimoto T, Akira S.1998. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell- specific Stat3-deficient mice. J. Immunol. 161:4652-4660.
6. Buerger C, Nagel-Wolfrum K, Kunz C, Wittig I, Butz K, Hoppe-Seyler F, Groner B. Sequence-specific peptide aptamers, interacting with the intracellular domain of the epidermal growth factor receptor, interfere with Stat3 activation and inhibit the growth of tumor cells. J Biol Chem. 2003 Sep 26;278(39):37610-21.

Keywords:

STAT3, STAT1, acute-phase response factor, APRF, transcription factor, HTS, assay, inhibition, inhibitor, dose response, counterscreen, luciferase, luminescence, reporter, 1536, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assay Overview:

The purpose of this assay is to determine whether a subset of compounds identified as active in a previous set of experiments entitled, "Primary cell-based high throughput screening assay to measure STAT3 inhibition" (PubChem AID 862), and that confirmed activity in a set of experiments entitled, "Confirmation cell-based high throughput screening assay to measure STAT3 inhibition" (AID 1265), were nonselective inhibitors due to inhibition of STAT1. The compounds selected for testing in this AID met the following criteria: 1) they were declared active in AID 862 and AID 1265; 2) they were declared inactive in a set of experiments entitled, "Primary cell-based high throughput screening assay to measure STAT1 inhibition" (AID 920); 3) they were inactive in a set of experiments entitled, "Counterscreen assay for STAT3 inhibitors: cell-based high throughput assay to measure NF-kappaB inhibition" (AID 1308); and 4) they were inactive in a set of experiments entitled, "Counterscreen assay for STAT3 inhibitors: cell-based high throughput assay to measure STAT1 inhibition" (AID 1317).

In this assay STAT1 inhibition was measured using a murine NIH 3T3 fibroblast cell line cell line that stably expresses a STAT1::luciferase construct. Test compounds were screened for their ability to prevent or reduce IFN-gamma-mediated induction of STAT1::luciferase reporter activity. Cells were exposed to test compounds followed by treatment with IFN-gamma. Changes in STAT1::luciferase activity were monitored by measuring well luminescence. As designed, a STAT1 inhibitor will block IFN-gamma-mediated STAT1 transcription, thus preventing or reducing transcription of the luciferase reporter gene, leading to decreased well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series, starting at nominal test concentration of 55.7 uM.

Protocol Summary:The inhibitor and activator dose response counterscreen assays using STAT1::luciferase cells were run simultaneously. NIH 3T3 cells were grown in T-175 flasks in Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% v/v fetal bovine serum and antibiotics (50 micrograms/mL each of penicillin and streptomycin, and 100 micrograms/mL neomycin) at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH).

Prior to the start of the assay, cells were resuspended at a density of 1.88 million cells/mL in phenol red-free growth medium, and filtered through a 0.7 micron filter. Next, 4 ul of well-mixed cell suspension (7,520 cells per well) were dispensed into each well of 1536-well plates. The assay was started by immediately dispensing 28 nL of test compound in DMSO, nifuroxazide (334 uM final nominal concentration) in DMSO, or DMSO alone (0.6% final concentration) to the appropriate wells. The plates were then incubated for 1 hour at 37 degrees C (5% CO2, 95% RH). Next, 1 ul of human recombinant IFN-gamma (3.0 ng/mL final nominal EC80 concentration) was dispensed into all wells. The plates were then incubated for 4 hours at 37 degrees C (5% CO2, 95% RH). The assay was stopped by dispensing 5 microliters of SteadyLite HTS luciferase substrate at room temperature to each well, followed by incubation at room temperature for 15 minutes. Well luminescence was measured on the ViewLux plate reader.

The percent inhibition was defined using the following mathematical formula:

% Inhibition = 100*[1 - ((Test_Compound - Median_High_Control) / (Median_Low_Control - Median_High_Control))]

Where:
Test_Compound is defined as the luminescence value of wells containing IFN-gamma and test compound.

Median_High_Control is defined as the median luminescence of wells containing IFN-gamma and nifuroxazide.

Median_Low_Control is defined as the median luminescence of wells containing IFN-gamma and DMSO.

For each test compound, percent inhibition 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 (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 55.7 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 55.7 uM. Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent inhibition value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent inhibition 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.

List of Reagents:

Dulbecco's Modified Eagle's Media I (Invitrogen, part 11965-092)
Dulbecco's Modified Eagle's Media, no Phenol Red (Invitrogen, part 21063-029)
Fetal Bovine Serum (Hyclone, part SH30088-03)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055).
Recombinant human IFN-gamma (R&D Systems, part 485-MI)
Nifuroxazide (Sigma-Aldrich, part N 2641)
SteadyLite HTS Assay Kit (PerkinElmer, part 6016989)
T175 Flasks (Corning, part 431080)
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 were normalized on a per-plate basis. In this assay the inhibitor nifuroxazide had an IC50 of approximately 80 micromolar. Possible artifacts of this assay can include, but are not limited to dust or lint located in or on wells of the plate, compounds that non-specifically modulate STAT1 or luciferase activity, and compounds that quench or enhance luminescence within the well. All test compound concentrations reported are nominal; the specific test concentration 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 AID.

Grant Number: 1 X01 MH079826-01