Late 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) conditions
Name: Late 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) conditions. ..more
BioActive Compounds: 6
Depositor Specified Assays
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_MTT-based_Cell-Growth_ WT/KO__MDCSRUN run by assay provider
Name: Late 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) conditions.
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 identify compounds that are able to kill SRC-3 wild type cells while not affecting cells that lack the protein target. HeLa cells that lack a functional copy of the SRC-3 gene were generated using a zinc finger nuclease that targets exon 11 of the SRC-3 gene (SRC-3 KO). SRC-3 KO and wild type parental cells were plated in 96-well plates and treated with Scripps compounds that passed various other secondary screening criteria. Cells were treated with 0.3, 1, 3 or 10 uM of each compound and allowed to grow for five days. Cells were then harvested and proliferation was assessed via MTT assay in a colorimetric-based assay.
Compounds that fail to influence SRC-3 ZFN KO cells at a concentration 3 times higher than that which influence the growth of parental HeLa cells are considered positive. Compound effects on SRC-1 and SRC-2 are expected to reduce the concentration separations to some extent.
5,000 Wild type (parental) HeLa cells and SRC-3 KO HeLa cells were plated at the same time in 96 well plates in DMEM medium with 10% fetal calf serum. Twenty four hours after plating, both groups of cells were treated with indicated compounds disolved in DMSO solvent. Throughout the assay, cells were maintained in an incubator at 37 C, 5% CO2 and 95% RH. Five days after compound addition, cell medium was removed and cell viability was measured using an absorbance-based MTT assay (Invitrogen).
The ratio of cell growth in the presence of test compound in cell growth in the SRC-3 KO cells versus wild type HeLa cells at the indicated concentrations of test compound was calculated.
MTT_Ratio = ( (Mean_Test Compound_SRC-3_KO_Cells / Mean_Negative_Control_SRC-3_KO_Cells ) / ( Mean_Test Compound_WT_Cells / Mean_Negative_Control_WT_Cells )
Test_Compound is defined as wells containing test compound treated cells.
Negative_Control is defined as wells containing DMSO treated cells.
PubChem Activity Outcome and Score:
Compounds which produced a growth ratio of less than 1.2 at a greater concentration than 10 uM were considered inactive. Compounds which produced a growth ratio of greater than or equal to 1.2 at a concentration less than or equal to 10 uM were considered 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, and for inactive compounds 0-0.
List of Reagents:
HeLa cells (ATCC, part CCL-2 )
DMEM media (Invitrogen, part 11965)
Fetal Bovine Serum (Hyclone, part SH30088.03)
This assay was run by the assay provider. Possible artifacts of this assay can include, but are not limited to dust or lint located in or on wells of the plate, compounds that non-specifically modulate kinase signaling or the transcriptional machinery. All test compound concentrations reported are nominal; the specific test concentration for a particular compound may vary based upon the actual sample provided.
** Test Concentration.
Data Table (Concise)