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BioAssay: AID 651786

Late stage Counterscreen for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): Luminescence-based cell-based dose response assay to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1)

Name: Late stage Counterscreen for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): Luminescence-based cell-based dose response assay to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1). ..more
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Active(3)
 
 
Inactive(5)
 
 
 Tested Substances
 Tested Substances
All(8)
 
 
Active(3)
 
 
Inactive(5)
 
 
AID: 651786
Data Source: The Scripps Research Institute Molecular Screening Center (SRC1_INH_LUMI_1536_4XIC50 MDCSRUN for SRC3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-11-19
Hold-until Date: 2013-11-15
Modify Date: 2013-11-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
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AIDNameTypeComment
588352Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3)Screeningdepositor-specified cross reference: Primary screen (SRC3 inhibitors in singlicate)
588357Summary of the probe development efforts to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3)Summarydepositor-specified cross reference: Summary (SRC3 inhibitors)
588792Luminescence-based cell-based high throughput confirmation assay for inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3)Screeningdepositor-specified cross reference: Confirmation (SRC3 inhibitors in quadruplicate)
588794Counterscreen for inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): 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)
602166Luminescence-based cell-based high throughput dose response assay for inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3)Confirmatorydepositor-specified cross reference: Dose response (SRC3 inhibitors in quadruplicate)
602167Counterscreen for inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)Confirmatorydepositor-specified cross reference: Dose response counterscreen (VP16 inhibitors in quadruplicate)
602168Counterscreen for inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1)Confirmatorydepositor-specified cross reference: Dose response counterscreen (SRC1 inhibitors in quadruplicate)
652066Late stage assay provider assay for the probe development effort to identify SRC-3 cellular protein concentrationConfirmatorydepositor-specified cross reference
652069Late stage assay provider assay for inhibitors of SRC-1 and SRC-3: HeLa Cell growth comparison in SRC-3 wild type and SRC-3 knockout (zinc finger nuclease) conditionsOtherdepositor-specified cross reference
651779Late stage Counterscreen for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): Luminescence-based cell-based dose response assay to identify cytotoxic compoundsConfirmatorysame project related to Summary assay
651781Late stage counterscreen for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): Luminescence-based cell-based dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)Confirmatorysame project related to Summary assay
651782Late stage Counterscreen for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): Luminescence-based cell-based dose response assay to identify inhibitors of the Steroid Receptor Coactivator 2 (SRC2; NCOA2)Confirmatorysame project related to Summary assay
651788Late stage assay for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): Luminescence-based cell-based dose response assay for SRC3 inhibitorsConfirmatorysame 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 MDCSRUN for SRC3

Name: Late stage Counterscreen for the probe development effort to identify inhibitors of the Steroid Receptor Coactivator 3 (SRC3; NCOA3): Luminescence-based cell-based dose response assay to identify inhibitors of the Steroid Receptor Coactivator 1 (SRC1; NCOA1).

Description:

Chemotherapeutic agents that target estrogen receptor alpha (ERa 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:

late stage, powders, purchased, synthesized, MDCSRUN, medchem, SAR, dose response, quadruplicate, counterscreen, steroid receptor coactivator 1, SRC1, nuclear receptor coactivator 1, NCOA1, DBD, LBD, steroid receptor coactivator 3, SRC3, nuclear receptor coactivator 3, NCOA3, amplified in breast cancer 1 protein, AIB1, cancer, breast cancer, inhibit, inhibitor, coactivator, lumi, luminescence, HTS, 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 whether powder samples of compounds identified as SRC3 inhibitor probe candidates are nonselective due to inhibition of SRC1. This assay determines dose response curves.

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 C, 5% CO2 and 95% RH, cells were transfected with 1 mL of preincubated mix of serum-free OptiMEM containing 23 ug of pGL4.31 reporter 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 follow:

%_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 of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-51, and for inactive compounds 1-0.

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.
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
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK293
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
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Chi 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
8RsquareThis 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
9Hill 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
10Response RangeThe range of Y.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 activation 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 % inhibition at 0.002 uM; replicate [1]Float%
14Inhibition at 0.002 uM [2] (0.002μM**)Value of % inhibition at 0.002 uM; replicate [2]Float%
15Inhibition at 0.002 uM [3] (0.002μM**)Value of % inhibition at 0.002 uM; replicate [3]Float%
16Inhibition at 0.002 uM [4] (0.002μM**)Value of % inhibition at 0.002 uM; replicate [4]Float%
17Inhibition at 0.005 uM [1] (0.005μM**)Value of % inhibition at 0.005 uM; replicate [1]Float%
18Inhibition at 0.005 uM [2] (0.005μM**)Value of % inhibition at 0.005 uM; replicate [2]Float%
19Inhibition at 0.005 uM [3] (0.005μM**)Value of % inhibition at 0.005 uM; replicate [3]Float%
20Inhibition at 0.005 uM [4] (0.005μM**)Value of % inhibition at 0.005 uM; replicate [4]Float%
21Inhibition at 0.016 uM [1] (0.016μM**)Value of % inhibition at 0.016 uM; replicate [1]Float%
22Inhibition at 0.016 uM [2] (0.016μM**)Value of % inhibition at 0.016 uM; replicate [2]Float%
23Inhibition at 0.016 uM [3] (0.016μM**)Value of % inhibition at 0.016 uM; replicate [3]Float%
24Inhibition at 0.016 uM [4] (0.016μM**)Value of % inhibition at 0.016 uM; replicate [4]Float%
25Inhibition at 0.049 uM [1] (0.049μM**)Value of % inhibition at 0.049 uM; replicate [1]Float%
26Inhibition at 0.049 uM [2] (0.049μM**)Value of % inhibition at 0.049 uM; replicate [2]Float%
27Inhibition at 0.049 uM [3] (0.049μM**)Value of % inhibition at 0.049 uM; replicate [3]Float%
28Inhibition at 0.049 uM [4] (0.049μM**)Value of % inhibition at 0.049 uM; replicate [4]Float%
29Inhibition at 0.148 uM [1] (0.148μM**)Value of % inhibition at 0.148 uM; replicate [1]Float%
30Inhibition at 0.148 uM [2] (0.148μM**)Value of % inhibition at 0.148 uM; replicate [2]Float%
31Inhibition at 0.148 uM [3] (0.148μM**)Value of % inhibition at 0.148 uM; replicate [3]Float%
32Inhibition at 0.148 uM [4] (0.148μM**)Value of % inhibition at 0.148 uM; replicate [4]Float%
33Inhibition at 0.443 uM [1] (0.443μM**)Value of % inhibition at 0.443 uM; replicate [1]Float%
34Inhibition at 0.443 uM [2] (0.443μM**)Value of % inhibition at 0.443 uM; replicate [2]Float%
35Inhibition at 0.443 uM [3] (0.443μM**)Value of % inhibition at 0.443 uM; replicate [3]Float%
36Inhibition at 0.443 uM [4] (0.443μM**)Value of % inhibition at 0.443 uM; replicate [4]Float%
37Inhibition at 1.3 uM [1] (1.3μM**)Value of % inhibition at 1.3 uM; replicate [1]Float%
38Inhibition at 1.3 uM [2] (1.3μM**)Value of % inhibition at 1.3 uM; replicate [2]Float%
39Inhibition at 1.3 uM [3] (1.3μM**)Value of % inhibition at 1.3 uM; replicate [3]Float%
40Inhibition at 1.3 uM [4] (1.3μM**)Value of % inhibition at 1.3 uM; replicate [4]Float%
41Inhibition at 4.0 uM [1] (4μM**)Value of % inhibition at 4.0 uM; replicate [1]Float%
42Inhibition at 4.0 uM [2] (4μM**)Value of % inhibition at 4.0 uM; replicate [2]Float%
43Inhibition at 4.0 uM [3] (4μM**)Value of % inhibition at 4.0 uM; replicate [3]Float%
44Inhibition at 4.0 uM [4] (4μM**)Value of % inhibition at 4.0 uM; replicate [4]Float%
45Inhibition at 12.0 uM [1] (12μM**)Value of % inhibition at 12.0 uM; replicate [1]Float%
46Inhibition at 12.0 uM [2] (12μM**)Value of % inhibition at 12.0 uM; replicate [2]Float%
47Inhibition at 12.0 uM [3] (12μM**)Value of % inhibition at 12.0 uM; replicate [3]Float%
48Inhibition at 12.0 uM [4] (12μM**)Value of % inhibition at 12.0 uM; replicate [4]Float%
49Inhibition at 35.9 uM [1] (35.9μM**)Value of % inhibition at 35.9 uM; replicate [1]Float%
50Inhibition at 35.9 uM [2] (35.9μM**)Value of % inhibition at 35.9 uM; replicate [2]Float%
51Inhibition at 35.9 uM [3] (35.9μM**)Value of % inhibition at 35.9 uM; replicate [3]Float%
52Inhibition at 35.9 uM [4] (35.9μM**)Value of % inhibition at 35.9 uM; 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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