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BioAssay: AID 1396

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 ..
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 Tested Compounds
 Tested Compounds
All(53)
 
 
Active(14)
 
 
Inactive(39)
 
 
 Tested Substances
 Tested Substances
All(53)
 
 
Active(14)
 
 
Inactive(39)
 
 
AID: 1396
Data Source: The Scripps Research Institute Molecular Screening Center (STAT1_INH_LUMI_1536_IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2008-09-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 14
Related Experiments
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AIDNameTypeComment
862Primary cell-based high throughput screening assay to measure STAT3 inhibitionScreeningdepositor-specified cross reference
920Primary cell-based high throughput screening assay to measure STAT1 inhibitionScreeningdepositor-specified cross reference
1263Confirmation cell-based high throughput screening assay to measure STAT1 inhibitionScreeningdepositor-specified cross reference
1303Counterscreen assay for STAT1 inhibitors: Cell-based high throughput assay to measure NF-kappaB inhibitionScreeningdepositor-specified cross reference
1310Counterscreen assay for STAT1 inhibitors: Cell-based high throughput assay to measure STAT3 inhibitionScreeningdepositor-specified cross reference
1803Summary of probe development efforts to identify inhibitors of signal transducer and activator of transcription 1 (STAT1)Summarydepositor-specified cross reference
2771Late stage counterscreen results from the probe development effort to identify STAT1 inhibitors: luminescence-based cell-based dose response assay for STAT3 inhibitorsConfirmatorydepositor-specified cross reference
2772Late stage results from the probe development effort to identify STAT1 inhibitors: luminescence-based cell-based dose response assay for STAT1 inhibitorsConfirmatorydepositor-specified cross reference
449758Late stage assay provider counterscreen results from the probe development effort to identify STAT1 inhibitors: fluorescence-based cell-based quantitative PCR assay to identify inhibitors of IRF-1 gene expressionOtherdepositor-specified cross reference
1409Dose response counterscreen for STAT1 inhibitors: cell-based high throughput assay to measure STAT3 inhibitionConfirmatorysame project related to Summary assay
Description:
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

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). 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.


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. 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.

Keywords:

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.
Protocol
Assay Overview:

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.

Protocol Summary:

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))]

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 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)
Comment
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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curveFloat
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 3.0 nM (0.003μM**)Value of %inhibition at 3.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
12Inhibition at 8.0 nM (0.008μM**)Value of %inhibition at 8.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
13Inhibition at 30.0 nM (0.03μM**)Value of %inhibition at 30.0 nanomolar inhibitor concentration; average of triplicate measurement. Float%
14Inhibition at 80.0 nM (0.08μM**)Value of %inhibition at 80.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
15Inhibition at 200.0 nM (0.2μM**)Value of %inhibition at 200.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 0.7 uM (0.7μM**)Value of %inhibition at 0.7 micromolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 2.0 uM (2μM**)Value of %inhibition at 2.0 micromolar inhibitor concentration; average of triplicate measurement.Float%
18Inhibition at 6.0 uM (6μM**)Value of %inhibition at 6.0 micromolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 20.0 uM (20μM**)Value of %inhibition at 20.0 micromolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 60.0 uM (60μM**)Value of %inhibition at 60.0 micromolar inhibitor concentration; average of triplicate measurement.Float%

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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