Source (MLSCN 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 Library Screening Center Network (MLSCN)
Grant Proposal Number: 1 X01 MH079826-01
Grant Proposal PI: David Frank

External Assay ID: STAT3_INH_LUMI_1536_%INH

Name:

Primary cell-based high throughput screening assay to identify inhibitors of STAT3

Description:

The signal transducer and activator of transcription (STAT) family of transcription factors transduce signals from a variety of extracellular stimuli and are important mediators of inflammation, cell survival, differentiation, and proliferation (1, 2). STATs are activated in response to growth factors, cytokines, and G-CSF binding to cell surface receptor tyrosine kinases (1-3). In resting cells STATs are inactive in the cytoplasm. In response to stimuli, STATs are phosphorylated by the Janus-activated kinases (Jaks), which induces STAT dimerization and nuclear translocation, where STATs bind to specific enhancer elements in target genes (2). Although structurally similar, the seven STAT family member (STATs 1, 2, 3, 4, 5a, 5b, and 6) possess diverse biological roles (2). For example, STAT1 activation is pro-inflammatory and anti-proliferative, while STAT3 activation is anti-inflammatory and pro-apoptotic (2). STAT1 is largely responsible for mediating the effects of IFN-gamma, while STAT3 is predominantly involved in IL-6 signaling (4). STAT1 induces expression of genes that inhibit the cell cycle, and thus STAT1 is considered to have tumor suppressor properties (5). Studies show that STAT3 is activated in a majority of breast and prostate cancers, and that STAT3 inhibition using RNA interference or a dominant negative leads to reduced cell proliferation, survival, and wound healing (1, 4, 6). Blocking STAT3 interaction with the epidermal growth factor receptor (EGFR) using peptide aptamers has been shown to reduce tumor growth (7). Due to the diverse roles and potent phenotypes associated with STAT signaling, the identification of selective modulators of STAT1 and STAT3 activity may lead to pharmacological tools for cancer, wound healing, and inflammatory diseases.

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. AK-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. Battle TE, Wierda WG, Rassenti LZ, Zahrieh D, Neuberg D, Kipps TJ, Frank DA. In vivo activation of signal transducer and activator of transcription 1 after CD154 gene therapy for chronic lymphocytic
leukemia is associated with clinical and immunologic response. Clin Cancer Res. 2003 Jun;9(6):2166-72.
6. Takeda, K. 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.
7. 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, signal transducer and activator of transcription 3, acute-phase response factor, APRF, transcription factor, Scripps, Scripps Florida, Molecular Library Screening Center Network, MLSCN, HTS, assay, inhibition, antagonist, primary, primary screen, luciferase, luminescence, reporter, 1536

Assay Overview:

Inhibition of STAT3 transcription was measured using a human U3A fibrosarcoma cell line that stably expresses a human STAT3::luciferase construct. This cell line is deficient in STAT1. In this primary assay 194,700 test compounds from the MLSCN library were screened for their ability to prevent or reduce IL-6-mediated STAT3::luciferase reporter activity. Cells were exposed to test compounds from the MLSCN library, followed by treatment with IL-6 to activate STAT3 transcription. Changes in STAT3::luciferase activity were monitored by measuring luminescence. As designed, a STAT3 antagonist will block IL-6-mediated STAT3 transcription, thus preventing or reducing the activation of the luciferase reporter gene, and decreasing luminescence.

Protocol Summary:

The 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 ug/ml each of penicillin and streptomycin, 100
micrograms/ml neomycin) at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH). Cells were harvested by trypsinization when they passed 70% confluency.

Cells were resuspended in growth medium, prepared in DMEM without phenol red, at a density of 1.88 million cells/ml and filtered through a 0.7 micron filter. Next, 4 ul of well-mixed cell suspension (7,520 cells per well) was dispensed into each well of 1536-well plates. The assay was started by immediately dispensing 28 nL of test compound in DMSO (5.5 uM final nominal concentration; 0.6% DMSO) in sample field wells, while control wells received nifuroxazide in DMSO (100 uM final concentration; 0.6% DMSO final concentration) or DMSO only (0.6% final concentration). The plates were then incubated for 1 hour at 37 degrees C (5% CO2, 95% RH). Next, 1 ul of human recombinant IL-6 (final concentration 33 ng/ml) was dispensed into sample and control wells. This IL-6 concentration
resulted in approximately 80% STAT3::luciferase reporter activity. Next, plates were incubated for 6 hours at 37 degrees C (5% CO2, 95% RH). The assay was stopped by dispensing 5 ul of SteadyLite
HTS luciferase substrate at room temperature to each well, followed by incubation at room temperature for 15 minutes. Luminescence was measured on the ViewLux plate reader.

Prior to % inhibition calculations, luminescence values were measured for each well corrected by subtracting background luminescence measured in wells containing media and substrate only. 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 a well containing test compound
Median_High_Control is defined as the median luminescence of wells containing 100 micromolar nifuroxazide (100% inhibition)
Median_Low_Control is defined as the median luminescence of wells containing 0.6% DMSO (0% inhibition)

A mathematical algorithm was used to determine nominally inhibitory compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active. The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed primary inhibition. Compounds with negative % inhibition values are reported to PubChem as having an activity score of zero.

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).
Neomycin B (Sigma-Aldrich, St. Louis, MO, part 33492)
Recombinant human IL-6 (Peprotech, Rocky Hill, NJ, part# 200-06)
Nifuroxazide (Sigma-Aldrich, part# N 2641)
SteadyLite HTS Assay Kit (PerkinElmer, part# 6016989)

All data were normalized on a per-plate basis. Each plate was normalized using same plate controls. The median value of the high control (maximum inhibition) was set at 100%. In this assay, IL-6 had a 50% effective concentration (EC50) of approximately 10 nM. The inhibitor nifuroxazide had an IC50 in the range of 5-13 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 STAT3 or luciferase activity, and compounds that quench 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.