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BioAssay: AID 588820

Luminescence-based cell-based high throughput confirmation assay for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1).

Name: Luminescence-based cell-based high throughput confirmation assay for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1). ..more
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 Tested Compounds
 Tested Compounds
All(2321)
 
 
Active(485)
 
 
Inactive(1836)
 
 
 Tested Substances
 Tested Substances
All(2323)
 
 
Active(486)
 
 
Inactive(1837)
 
 
AID: 588820
Data Source: The Scripps Research Institute Molecular Screening Center (SRC1_INH_LUMI_1536_3X%INH CRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-11-23

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 485
Related Experiments
Show more
AIDNameTypeComment
588354Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1)Screeningdepositor-specified cross reference: Primary screen (SRC1 inhibition)
588362Summary of the probe development efforts to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1)Summarydepositor-specified cross reference: Summary (SRC1 inhibition)
602234Counterscreen for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3)Confirmatorydepositor-specified cross reference
602235Luminescence-based cell-based high throughput dose response assay for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1)Confirmatorydepositor-specified cross reference
602236Counterscreen for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)Confirmatorydepositor-specified cross reference
651794Late stage Counterscreen for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1): Luminescence-based cell-based dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)Confirmatorydepositor-specified cross reference
651796Late stage assay for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1): Luminescence-based cell-based dose response assay for SRC1 inhibitorsConfirmatorydepositor-specified cross reference
651797Late stage Counterscreen for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1): Luminescence-based cell-based dose response assay to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3)Confirmatorydepositor-specified cross reference
651799Late stage Counterscreen for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1): Luminescence-based cell-based dose response assay to identify inhibitors of the Steroid Receptor Coactivator 2 (SRC2; NCOA2)Confirmatorydepositor-specified cross reference
588824Counterscreen for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1): Luminescence-based cell-based high throughput assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16).Screeningsame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Bert O'Malley, Baylor College of Medicine
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number 5U19DK062434-09
Grant Proposal PI: Bert O'Malley, Baylor College of Medicine
External Assay ID: SRC1_INH_LUMI_1536_3X%INH CRUN

Name: Luminescence-based cell-based high throughput confirmation assay for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1).

Description:

Chemotherapeutic agents that target estrogen receptor alpha (ERalpha and growth factor signaling systems have been extensively pursued and developed for a long time [1-4]. However, one of the most pressing limitations of currently established chemotherapeutic agents for breast cancer is the fact that breast cancers frequently acquire resistance to antiestrogens [5, 6]. Nuclear receptors (NR) and other hormone receptors mediate their cellular effects in part through the interaction with coactivators which increase their transcriptional activity. The best characterized coactivator family is the steroid receptor coactivator (SRC) family [7]. Given the central role that SRC-3 plays in breast and other cancers, the search for small molecule agents that target SRC-1 and SRC-3 represent an innovative and potentially effective strategy to identify agents to treat hormone-refractory breast cancers and other cancers where these coactivators are overexpressed. Compounds that target the function of steroid receptor coactivator 3 (SRC-3) protein promise to be different because cancer cells are less likely to bypass the comprehensive disruption of multiple growth factor signaling systems that result from the loss of SRC-3 function. In contrast to the goal of screens that seek to interfere with NR-coactivator interactions, the work proposed here aims to identify compounds that specifically target the coactivators themselves. This approach offers to be more broadly applicable. For instance, SRC-1 or SRC-3 typically remains overexpressed in ER negative cancers or acts as a coactivator for other oncogenic transcription factors [8]. SMIs that target ERa, on the other hand are largely predicted to duplicate the biological action of antiestrogens such as tamoxifen.

References:

1. Arteaga, C.L., A.K. Tandon, D.D. Von Hoff, and C.K. Osborne, Transforming growth factor beta: potential autocrine growth inhibitor of estrogen receptor-negative human breast cancer cells. Cancer Res, 1988. 48(14): p. 3898-904.
2. Ciardiello, F., T. Troiani, F. Caputo, M. De Laurentiis, G. Tortora, G. Palmieri, F. De Vita, M.R. Diadema, M. Orditura, G. Colantuoni, C. Gridelli, G. Catalano, S. De Placido, and A.R. Bianco, Phase II study of gefitinib in combination with docetaxel as first-line therapy in metastatic breast cancer. Br J Cancer, 2006. 94(11): p. 1604-9.
3. Goldstein, D., S.M. Bushmeyer, P.L. Witt, V.C. Jordan, and E.C. Borden, Effects of type I and II interferons on cultured human breast cells: interaction with estrogen receptors and with tamoxifen. Cancer Res, 1989. 49(10): p. 2698-702.
4. Riggins, R.B., A. Zwart, R. Nehra, and R. Clarke, The nuclear factor kappa B inhibitor parthenolide restores ICI 182,780 (Faslodex; fulvestrant)-induced apoptosis in antiestrogen-resistant breast cancer cells. Mol Cancer Ther, 2005. 4(1): p. 33-41.
5. Chen, F.L., W. Xia, and N.L. Spector, Acquired resistance to small molecule ErbB2 tyrosine kinase inhibitors. Clin Cancer Res, 2008. 14(21): p. 6730-4.
6. Riggins, R.B., M.M. Mazzotta, O.Z. Maniya, and R. Clarke, Orphan nuclear receptors in breast cancer pathogenesis and therapeutic response. Endocr Relat Cancer, 2010. 17(3): p. R213-31.
7. Lonard, D.M., R. Kumar, and B.W. O'Malley, Minireview: the SRC family of coactivators: an entree to understanding a subset of polygenic diseases? Mol Endocrinol, 2010. 24(2): p. 279-85.
8. Xu, J., R.C. Wu, and B.W. O'Malley, Normal and cancer-related functions of the p160 steroid receptor co-activator (SRC) family. Nat Rev Cancer, 2009. 9(9): p. 615-30.

Keywords:

confirmation, confirmatory, quadruplicate, steroid receptor coactivator 1, SRC1, nuclear receptor coactivator 1, NCOA1, amplified in breast cancer 1 protein, AIB1, cancer, breast cancer, inhibit, inhibitor, coactivator, lumi, luminescence, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this assay is to confirm activity of compounds identified as active in a set of experiments entitled, "Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1)" (PubChem AID 588354).
In this assay, HEK293 cells are transfected with a GAL4-responsive reporter plasmid (pGL4.31, Promega) and an expression vector encoding SRC1 fused to the DNA-binding domain of GAL4 (pBIND-SRC-1). The ability of compounds to reduce coactivator transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. As designed, compounds that inhibit SRC1 ability to induce transcription will lead to a decrease in expression of the luciferase gene, resulting in reduced well luminescence. Compounds are tested in quadruplicate at a final nominal concentration of 3.6 micromolar.
Protocol Summary:
Seven million HEK293 cells were seeded in T-175 flasks 23 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v Anti-Anti. Flasks were then incubated for 48 hours at 37 degrees Celsius, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, cells were harvested using TrypLE, resuspended in fresh media at a density of 1 million cells per mL and seeded into new T-175 flasks (23 mL per flask). After being allowed to attach for one hour at 37 degrees Celsius, 5% CO2 and 95% RH, cells were transfected with 1 mL of preincubated mix of serum-free OptiMEM containing 23 micrograms of pGL4.31reporter plasmid, 2.3 micrograms of pBIND-SRC1 vector and 80 microliters of transfection reagents. Twenty four hours post transfection, cells were harvested using 5 mL of TrypLE and resuspended at a concentration of 750,000 cells per mL in phenol-red free DMEM media supplemented as described above.
The assay was started by dispensing 5 microliters of cell suspension into each well of a white, solid-bottom 1536-well plate using a flying reagent dispenser (i.e. 3,750 cells per well). The first two columns received cells transfected with the reporter plasmid and an empty pBIND vector as a control for background luminescence. Cells were then treated with 18 nL/well of test compounds, DMSO as a negative control (final concentration 0.36%) or Gossypol as a positive control (36 micromolar final) using a PinTool transfer unit (GNF). Plates were then placed in the incubator at 37 degrees Celsius, 5% CO2 and 95%RH. Twenty four hours later, plates were removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase was detected by adding 5 microliters per well of ONE-Glo luciferase detection reagent. After a 15 minutes incubation time, light emission was measured with the ViewLux reader (PerkinElmer). The percent inhibition of each test compound was calculated as follows:
%Inhibition = ( 1 - ( median_positive_control - test_compound ) / ( median_positive_control - median_negative_control ) * 100
Where:
Test_Compound is defined as wells containing test compound treated cells..
Positive_Control is defined as wells containing Gossypol treated cells.
Negative_Control is defined as wells containing DMSO treated cells.
A mathematical algorithm was used to determine nominally active compounds. The average percent activation and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the average plus three standard deviation of the Negative_Control wells (i.e. DMSO treated, calculated at 46.68%) was declared active.
PubChem Activity Outcome and Score:
The inactive compounds of this assay have an activity score range of 0 to 46 and the active compounds have an activity score range of 46 to 100.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
List of Reagents:
HEK-293 cells (ATCC, part CRL-1573)
DMEM media (Invitrogen, part 11965)
Fetal Bovine Serum (Hyclone, part SH30088.03)
Anti-Anti (Gibco, part 15240)
TrypLE (Invitrogen, part 12604)
T-175 flasks (Falcon, part 353112)
pGL4.31 (Promega, part C935A)
pBIND-SRC1 (Assay Provider)
TransIT 293 transfection reagent (Mirus Corporation, part MIR-2700)
ONE-Glo luciferase reagent (Promega, part E6130)
White, solid-bottom 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. 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 reported 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 microtiter plate, cytotoxic compounds, compounds that perturb the UAS/GAL4 reporter system, and compounds that quench, inhibit, stabilize, or emit 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. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: enzyme reporter: enzyme activity: enzyme inhibition
BAO: assay format: cell-based format
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: bioassay specification: assay stage: confirmatory
BAO: detection technology: luminescence: chemiluminescence
BAO: meta target: biological process target: regulation of transcription
BAO: meta target: molecular target: protein target: transcription factor
BAO: version: 1.4b1090
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK293
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Inhibition at 3.6 uM (3.6μM**)Average of normalized percent inhibition of the confirmation screen at a compound concentration of 3.6 micromolar.Float%
2Standard DeviationStandard deviation derived from the normalized percent inhibition of the quadruplicate data for each compound.Float
3Inhibition at 3.6 uM [1] (3.6μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 3.6 micromolar; replicate 1.Float%
4Inhibition at 3.6 uM [2] (3.6μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 3.6 micromolar; replicate 2.Float%
5Inhibition at 3.6 uM [3] (3.6μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 3.6 micromolar; replicate 3.Float%
6Inhibition at 3.6 uM [4] (3.6μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 3.6 micromolar; replicate 4.Float%

** Test Concentration.
Additional Information
Grant Number: 5U19DK062434-09

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