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BioAssay: AID 602235

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

Name: Luminescence-based cell-based high throughput dose response assay for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1). ..more
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 Tested Compounds
 Tested Compounds
All(240)
 
 
Active(58)
 
 
Inactive(182)
 
 
 Tested Substances
 Tested Substances
All(240)
 
 
Active(58)
 
 
Inactive(182)
 
 
AID: 602235
Data Source: The Scripps Research Institute Molecular Screening Center (SRC1_INH_LUMI_1536_4XIC50 DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-01-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 58
Related Experiments
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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 screening (SRC1 inhibitors in singlicate)
588362Summary of the probe development efforts to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1)Summarydepositor-specified cross reference: Summary (SRC1 inhibitors)
588820Luminescence-based cell-based high throughput confirmation assay for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1).Screeningdepositor-specified cross reference: Primary screening (SRC1 inhibitors in triplicate)
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).Screeningdepositor-specified cross reference: Counterscreen (VP16 inhibitors in quadruplicate)
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
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)Confirmatorysame project related to Summary assay
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)Confirmatorysame 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_4XIC50 DRUN

Name: Luminescence-based cell-based high throughput dose response 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:

dose response, CRC, titration, 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 determine dose response curves for compounds that confirmed SRC1 inhibitory activity in a set of experiments entitled, "Luminescence-based cell-based high throughput confirmation assay for inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1)" (AID 588820) and were inactive in a set of experiments entitled, "Counterscreen 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)" (AID 588824).

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 using a 10-point 1:3 dilution series starting at a nominal test concentration of 36 uM.

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 C, 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 Cs, 5% CO2 and 95% RH, cells were transfected with 1 mL of preincubated mix of serum-free OptiMEM containing 23 ug of pGL4.31reporter plasmid, 2.3 ug of pBIND-SRC1 vector and 80 uL 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 uL 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 uM final) using a PinTool transfer unit (GNF). Plates were then placed in the incubator at 37 C, 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 uL 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.

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 (Symyx Technologies Inc). 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. 36 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 36 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity 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.

The PubChem Activity Score range for active compounds is 100-79, and for inactive compounds 78-0.

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:
Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit [1]: Mask: Excluded Points
Assay: Dictionary: Version: 0.1
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 footprint: microplate: 1536 well plate
BAO: bioassay specification: assay measurement throughput quality: concentration response multiple replicates
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: bioassay specification: bioassay type: functional: reporter gene
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
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
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 uM 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
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
7Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
8Response RangeThe range of Y.Float
9Chi 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
10RsquareThis 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
11Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
12Number 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
13Inhibition at 0.002 uM [1] (0.002μM**)Value of percent inhibition at 0.002 uM compound concentration; replicate [1]Float%
14Inhibition at 0.002 uM [2] (0.002μM**)Value of percent inhibition at 0.002 uM compound concentration; replicate [2]Float%
15Inhibition at 0.002 uM [3] (0.002μM**)Value of percent inhibition at 0.002 uM compound concentration; replicate [3]Float%
16Inhibition at 0.002 uM [4] (0.002μM**)Value of percent inhibition at 0.002 uM compound concentration; replicate [4]Float%
17Inhibition at 0.005 uM [1] (0.005μM**)Value of percent inhibition at 0.005 uM compound concentration; replicate [1]Float%
18Inhibition at 0.005 uM [2] (0.005μM**)Value of percent inhibition at 0.005 uM compound concentration; replicate [2]Float%
19Inhibition at 0.005 uM [3] (0.005μM**)Value of percent inhibition at 0.005 uM compound concentration; replicate [3]Float%
20Inhibition at 0.005 uM [4] (0.005μM**)Value of percent inhibition at 0.005 uM compound concentration; replicate [4]Float%
21Inhibition at 0.016 uM [1] (0.016μM**)Value of percent inhibition at 0.016 uM compound concentration; replicate [1]Float%
22Inhibition at 0.016 uM [2] (0.016μM**)Value of percent inhibition at 0.016 uM compound concentration; replicate [2]Float%
23Inhibition at 0.016 uM [3] (0.016μM**)Value of percent inhibition at 0.016 uM compound concentration; replicate [3]Float%
24Inhibition at 0.016 uM [4] (0.016μM**)Value of percent inhibition at 0.016 uM compound concentration; replicate [4]Float%
25Inhibition at 0.049 uM [1] (0.049μM**)Value of percent inhibition at 0.049 uM compound concentration; replicate [1]Float%
26Inhibition at 0.049 uM [2] (0.049μM**)Value of percent inhibition at 0.049 uM compound concentration; replicate [2]Float%
27Inhibition at 0.049 uM [3] (0.049μM**)Value of percent inhibition at 0.049 uM compound concentration; replicate [3]Float%
28Inhibition at 0.049 uM [4] (0.049μM**)Value of percent inhibition at 0.049 uM compound concentration; replicate [4]Float%
29Inhibition at 0.148 uM [1] (0.148μM**)Value of percent inhibition at 0.148 uM compound concentration; replicate [1]Float%
30Inhibition at 0.148 uM [2] (0.148μM**)Value of percent inhibition at 0.148 uM compound concentration; replicate [2]Float%
31Inhibition at 0.148 uM [3] (0.148μM**)Value of percent inhibition at 0.148 uM compound concentration; replicate [3]Float%
32Inhibition at 0.148 uM [4] (0.148μM**)Value of percent inhibition at 0.148 uM compound concentration; replicate [4]Float%
33Inhibition at 0.443 uM [1] (0.443μM**)Value of percent inhibition at 0.443 uM compound concentration; replicate [1]Float%
34Inhibition at 0.443 uM [2] (0.443μM**)Value of percent inhibition at 0.443 uM compound concentration; replicate [2]Float%
35Inhibition at 0.443 uM [3] (0.443μM**)Value of percent inhibition at 0.443 uM compound concentration; replicate [3]Float%
36Inhibition at 0.443 uM [4] (0.443μM**)Value of percent inhibition at 0.443 uM compound concentration; replicate [4]Float%
37Inhibition at 1.3 uM [1] (1.3μM**)Value of percent inhibition at 1.3 uM compound concentration; replicate [1]Float%
38Inhibition at 1.3 uM [2] (1.3μM**)Value of percent inhibition at 1.3 uM compound concentration; replicate [2]Float%
39Inhibition at 1.3 uM [3] (1.3μM**)Value of percent inhibition at 1.3 uM compound concentration; replicate [3]Float%
40Inhibition at 1.3 uM [4] (1.3μM**)Value of percent inhibition at 1.3 uM compound concentration; replicate [4]Float%
41Inhibition at 4.0 uM [1] (4μM**)Value of percent inhibition at 4.0 uM compound concentration; replicate [1]Float%
42Inhibition at 4.0 uM [2] (4μM**)Value of percent inhibition at 4.0 uM compound concentration; replicate [2]Float%
43Inhibition at 4.0 uM [3] (4μM**)Value of percent inhibition at 4.0 uM compound concentration; replicate [3]Float%
44Inhibition at 4.0 uM [4] (4μM**)Value of percent inhibition at 4.0 uM compound concentration; replicate [4]Float%
45Inhibition at 12.0 uM [1] (12μM**)Value of percent inhibition at 12.0 uM compound concentration; replicate [1]Float%
46Inhibition at 12.0 uM [2] (12μM**)Value of percent inhibition at 12.0 uM compound concentration; replicate [2]Float%
47Inhibition at 12.0 uM [3] (12μM**)Value of percent inhibition at 12.0 uM compound concentration; replicate [3]Float%
48Inhibition at 12.0 uM [4] (12μM**)Value of percent inhibition at 12.0 uM compound concentration; replicate [4]Float%
49Inhibition at 35.9 uM [1] (35.9μM**)Value of percent inhibition at 35.9 uM compound concentration; replicate [1]Float%
50Inhibition at 35.9 uM [2] (35.9μM**)Value of percent inhibition at 35.9 uM compound concentration; replicate [2]Float%
51Inhibition at 35.9 uM [3] (35.9μM**)Value of percent inhibition at 35.9 uM compound concentration; replicate [3]Float%
52Inhibition at 35.9 uM [4] (35.9μM**)Value of percent inhibition at 35.9 uM compound concentration; replicate [4]Float%

* Activity Concentration. ** 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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