Late stage assay provider assay for the probe development effort to identify SRC-3 cellular protein concentration
Name: Late stage assay provider assay for the probe development effort to identify SRC-3 cellular protein concentration. ..more
BioActive Compounds: 6
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: SRC3_INH_MCF-7_cells_WESTERN BLOT_PROTEIN REDUCTION_MDCSRUN Run by assay provider
Name: Late stage assay provider assay for the probe development effort to identify SRC-3 cellular protein concentration.
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.
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.
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The purpose of this assay is to determine whether powder samples of compounds identified as SRC3 inhibitor probe candidates can reduce SRC-3 protein level in the cell .
MCF-7 breast cancer cells which overexpress SRC-3 at high levels were chosen for this assay. MCF-7 cells were plated in 6 well tissue culture plates in DMEM with 10% fetal bovine serum. Twenty four hours after plating, test compounds or DMSO vehicle control was delivered to tissue culture wells and cells were harvested and lysates prepared twenty four hour therafter. Levels of SRC-3 in whole cell extracts were determined by Western blotting.
%_SRC-3_Protein_Reduction = ( 1 - ( SRC-3_Protein_Level_Test_Compound / SRC-3_Protein_Level_Negative_Control ) * 100
Test_Compound is defined as SRC-3 protein level in presence of test compound normalized against actin
Negative_Control is defined as SRC-3 protein level in the presence of DMSO control vehicle normalized against actin
This assay measures a compound's effect on SRC-3 cellular protein concentration. MCF-7 cells grown in standard growth medium were treated with compounds identified from previous SRC-3 KO/SRC-3 WT growth assays for twenty four hours. Cells were harvested and lysed in a SRC-3 sample buffer (25 mM Tris pH 8.0, 150 mM NaCl, 0.05% Triton X-100). Western blotting for SRC-3 and Actin were performed to identify compounds that are able to promote SRC-3 protein down regulation.
PubChem Activity Outcome and Score:
Compounds which were able to reduce SRC-3 protein concentration 50% at 10 uM or lower concentration were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of inactive. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score of active.
Active compounds were given a score of 100 and inactive compounds a score of 0.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
MCF-7 cells (ATCC, part HTB-22)
DMEM media (Invitrogen, part 11965)
Fetal Bovine Serum (Hyclone, part SH30088.03)
6 well tissue culture plates (Falcon, part 353046)
SRC-3 antibody, rabbit monoclonal 5E11 (Cell Signaling, part 2126)
ECL detection reagents (Pierce, part 32132)
This assay was run by the assay provider. Compounds that non-specifically modulate kinase signaling or protein translation could generate false positive results. All test compound concentrations reported are nominal; the specific test concentration for a particular compound may vary based upon the actual sample provided.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)