Counterscreen assay for STAT3 inhibitors: Cell-based high throughput assay to measure NF-kappaB inhibition.
Name: Counterscreen assay for STAT3 inhibitors: Cell-based high throughput assay to measure NF-kappaB inhibition. ..more
BioActive Compounds: 438
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Prem Subramaniam, The Scripps Research Institute Molecular Screening Center (SRIMSC)
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 1 X01 MH079826-01
Grant Proposal PI: David Frank
External Assay ID: NFkB_INH_LUMI_1536_3X%INH (STAT3CS)
Name: Counterscreen assay for STAT3 inhibitors: Cell-based high throughput assay to measure NF-kappaB inhibition.
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.
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. 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.
STAT3, signal transducer and activator of transcription 3, acute-phase response factor, APRF, transcription factor, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, MLSCN, HTS, assay, inhibition, inhibitor, NF-kappaB, NFkB, counterscreen, luciferase, luminescence, reporter, 1536
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) were nonselective due to inhibition of NF-kappaB. The compounds selected for testing in this AID met at least the two following criteria: 1) they were declared active in AID 862; and 2) they were declared inactive in a previous set of experiments entitled, "Primary cell-based high throughput screening assay to measure STAT1 inhibition" (PubChem AID 920).
In this assay inhibition of NF-kB transcription was measured using a human HEK 293T cell line that stably expresses a human NF-kB::luciferase construct. Test compounds were screened for their ability to prevent or reduce TNF-alpha-mediated induction of NF-kB::luciferase reporter activity. Cells were exposed to test compounds, followed by treatment with TNF-alpha to activate NF-kB transcription. Changes in NF-kB::luciferase activity were monitored by measuring luminescence. An inhibitor will block TNF-alpha-mediated NF-kB transcription, thus preventing or reducing the activation of the luciferase reporter gene, and decreasing well luminescence. As designed, test compounds that inhibit NF-kB activity are considered non-selective inhibitors.
The STAT3 inhibitor and activator counterscreen assays using NF-kB::luciferase cells were run simultaneously. HEK 293T 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, 100 micrograms/mL neomycin and 1 microgram/mL of puromycin) at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH).
Prior to the start of the assay the cells were resuspended at a density of 0.94 million cells/mL in phenol red-free growth medium, and filtered through a 0.7 micron filter. Next, 4 microliters of cell suspension (3,760 cells per well) were dispensed into each well of 1536-well plates. Cells were allowed to adhere to plates by incubation at 37 degrees C, 5%CO2, and 95% RH for 18 hours. The assay was started by dispensing 28 nL of test compound (5.7 micromolar final nominal concentration) in DMSO to sample wells, while High Control wells received MG-132 (100 micromolar final nominal concentration; set as 100% inhibition) in DMSO, and Low Control wells received DMSO (0.6% final concentration; set as 0% inhibition). The plates were then incubated for 1 hour at 37 degrees C, 5% CO2, and 95% RH. Next, 1 microliter of human recombinant TNF-alpha (5.6 ng/ml final concentration) was dispensed into sample and control wells. This TNF-alpha concentration resulted in approximately 80% NF-kappaB::luciferase reporter activity. Next, the plates were incubated for 6 hours at 37 degrees C, 5% CO2, and 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. 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))]
Test_Compound is defined as the luminescence value of a well containing TNF-alpha and test compound.
Median_High_Control is defined as the median luminescence of wells containing TNF-alpha and MG-132.
Median_Low_Control is defined as the median luminescence of wells containing TNF-alpha and DMSO.
Test compounds were assayed in triplicate at a single concentration of 5.7 micromolar. A mathematical algorithm was used to determine nominally inhibiting compounds. Two values were calculated: (1) the average percent inhibition of all 1280 wells defined as #Median_Low_Control# (i.e. a #DMSO plate#), and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter: 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 inhibition. Negative % inhibition values were assigned 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, part 33492)
Puromycin (Sigma-Aldrich, part P8833)
Recombinant human TNF-alpha (Invitrogen, part PHC3016)
MG-132 (Tocris Cookson Ltd, part 1748-5)
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 MLSCN compound library, 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. All data 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 plate, compounds that non-specifically modulate NF-kappaB 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.
Active compounds of this assay fall into the activity score range of 13 to 100 and inactive compounds have range of activity score from 0 to 13.
** Test Concentration.
Data Table (Concise)