Dose response cell-based assay to measure STAT1 inhibition
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 more ..
BioActive Compounds: 14
Depositor Specified Assays
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
Name: Dose response cell-based assay to measure STAT1 inhibition
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). Due to its activation of genes that inhibit the cell cycle, STAT1 is considered to have tumor suppressor properties (5). As current STAT1 modulators mediate modest effects on STAT-induced transcription, act indirectly by targeting JAK or other kinases activities, or are associated with adverse hematologic or gastrointestinal side effects (6, 7), the discovery of high affinity probes may be useful for elucidating the biological roles of STAT1.
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. Lynch RA, Etchin J, Battle TE, Frank DA. A small-molecule enhancer of signal transducer and activator of transcription 1 transcriptional activity accentuates the antiproliferative effects of IFN-gamma in human cancer cells. Cancer Res. 2007 Feb 1;67(3):1254-61.
7. Johnson S, Smith AG, L#ffler H, Osby E, Juliusson G, Emmerich B, Wyld PJ, Hiddemann W. Multicentre prospective randomised trial of fludarabine versus cyclophosphamide, doxorubicin, and prednisone (CAP) for treatment of advanced-stage chronic lymphocytic leukaemia. The French Cooperative Group on CLL. Lancet. 1996 May 25;347(9013):1432-8.
STAT1, transcription factor, HTS, assay, inhibition, inhibitor, dose response, luciferase, luminescence, reporter, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, "Primary cell-based high throughput screening assay to measure STAT1 inhibition" (PubChem AID 920) and that confirmed activity in a set of experiments entitled, "Confirmation cell-based high throughput screening assay to measure STAT1 inhibition" (AID 1263). The compounds selected for testing in this AID met the following criteria: 1) they were declared active in AID 920 and AID 1263; 2) they were declared inactive in a set of experiments entitled, "Primary cell-based high throughput screening assay to measure STAT3 inhibition" (AID 862); 3) they were inactive in a set of experiments entitled, "Counterscreen assay for STAT1 inhibitors: cell-based high throughput assay to measure NF-kappaB inhibition" (AID 1303); and 4) they were inactive in a set of experiments entitled, "Counterscreen assay for STAT1 inhibitors: cell-based high throughput assay to measure STAT3 inhibition" (AID 1310).
In this assay STAT1 inhibition was measured using a murine NIH 3T3 fibroblast cell line that stably expresses a STAT1::luciferase construct. Test compounds were screened for their ability to prevent or reduce IFN-gamma-mediated 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 a nominal test concentration of 55.7 micromolar.
The STAT1 inhibitor and activator dose response assays 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 50 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 microliters 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 micromolar final nominal concentration, set as 100% inhibition) 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 microliter of human recombinant IFN-gamma (3.0 ng/mL final nominal EC80 concentration, set as 100% activation) was dispensed to 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))]
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 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 55.7 micromolar. Compounds with an IC50 greater than 10 micromolar were considered inactive. Compounds with an IC50 equal to or less than 10 micromolar 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)