Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: David Frank
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 1 X01 MH079826-01
Grant Proposal PI: David Frank
External Assay ID: STAT1_POT_LUMI_1536_%ACT

Name: Primary cell-based high throughput screening assay to measure STAT1 activation.

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, members of the STAT family (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). Currently available 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 liabilities of the current state of the art for STAT1 modulators necessitate the discovery of higher affinity probes. Due to the diverse roles and potent phenotypes associated with STAT signaling, the identification of selective potentiators of STAT1 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. 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, Loffler 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, signal transducer and activator of transcription 1, transcription factor, Scripps, Scripps Florida, Molecular Library Screening Center Network, MLSCN, HTS, assay, activation, activator, potentiator, potentiation, primary, primary screen, luciferase, luminescence, reporter, 1536.

Assay Overview:

Potentiation of STAT1 activity was measured using a murine NIH 3T3 fibroblast cell line that stably expresses a STAT1-luciferase construct. In this primary assay approximately 195,000 test compounds from the MLSCN library were screened for their ability to increase IFN-gamma-mediated STAT1::luciferase reporter activity. Cells were exposed to test compounds from the MLSCN library, followed by treatment with IFN-gamma. Changes in STAT1::luciferase activity were monitored by measuring luminescence. As designed, a STAT1 potentiator will increase IFN-gamma-mediated STAT1 transcription, thus increasing the activation of the luciferase reporter gene, and increasing luminescence.

Protocol Summary:

This STAT1 potentiator assay was run simultaneously in the same plates as the STAT1 antagonist campaign. 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) 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 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) were dispensed into each well of 1536-well plates. The assay was started by immediately dispensing 28 nL of test compound (5.5 uM final nominal concentration) in DMSO to sample wells, or DMSO alone (0.6% final concentration) to control 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 (final nominal EC80 concentration of 3.0 ng/mL, set as 100% activation) was dispensed to sample and control wells. The plates were then 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.

The percent activity was defined using the following mathematical formula:

% Activity = 100 *( Test_Compound / Median_High_Control )

Where:

Test_Compound is defined as the luminescence value of IFN-gamma-treated wells containing test compound.
Median_High_Control is defined as the median luminescence value of control wells containing IFN-gamma.

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter(127.73%), i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.

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)
SteadyLite HTS Assay Kit (PerkinElmer, part# 6016989)

All data reported were normalized on a per-plate basis. Each plate was normalized using same-plate controls. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter 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 concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

Experiment Date: 12/10/2007

BAO: version:: 1.4b1080

BAO: bioassay specification: assay stage: primary

BAO: bioassay specification: assay biosafety level: bsl1

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: format detail: reagent: inducer: interferon gamma

BAO: meta target: molecular target: protein target: transcription factor

BAO: meta target: biological process target: JAK-STAT cascade

BAO: meta target detail: binding reporter specification: interaction: protein-dna

BAO: design: inducible reporter: luciferase induction

BAO: detection technology: luminescence: bioluminescence

Grant Number: 1 X01 MH079826-01