Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of the Steroid Receptor Coactivator 2 (SRC2; NCOA2)
Name: Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of the Steroid Receptor Coactivator 2 (SRC2; NCOA2). ..more
BioActive Compounds: 620
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: SRC2_INH_LUMI_1536_1X%INH PRUN
Name: Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of the Steroid Receptor Coactivator 2 (SRC2; NCOA2).
The steroid receptor coactivator (SRC) family contains three members, SRC-1(1), SRC-2/TIF2/GRIP1(2, 3) and SRC-3/AIB-1/pCIP(4, 5), and each plays broad roles in promoting NR-mediated gene expression. Coactivators are required for NRs to function as transcription factors and play key roles as rheostats that determine the amplitude of biological responses to hormones(6). Consistent with the expansive biological roles that NRs play in reproduction, stress responses, energy metabolism and cell growth, disruption of coactivator biological function results in complex and pleiotropic defects. Small molecule inhibitor (SMI) screens for SRC-1 and SRC-3, which play prominent roles in cancer, have already been performed. While it also has a role in cancer, SRC-2 was found to play a critical role in energy intake by driving the dietary absorption of fat(7). SRC-2 achieves this function in response to post translational modification (PTM)-based activation by the primordial energy-sensing kinase, AMPK (among others) which phosphorylates SRC-2, increasing its intrinsic transcriptional activity. We showed that liver-specific ablation of SRC-2 results in a hypoglycemic phenotype in response to fasting(8). We further demonstrated that that hepatic SRC-2 cooperates with RORalpha to specifically drive expression of glucose-6-phosphatase (G6Pase), an essential rate-limiting enzyme that catalyzes glucose production during fasting. SMIs capable of targeting SRC-2 could serve as novel therapeutic agents to treat obesity and other metabolic diseases.
1 - Onate, S.A., Tsai, S.Y., Tsai, M.J. & O'Malley, B.W. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270, 1354-1357 (1995).
2 - Voegel, J.J., Heine, M.J., Zechel, C., Chambon, P. & Gronemeyer, H. TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J 15, 3667-3675 (1996).
3 - Hong, H., Kohli, K., Trivedi, A., Johnson, D.L. & Stallcup, M.R. GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci U S A 93, 4948-4952 (1996). - PMID: 8643509
4 - Anzick, S.L., et al. AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277, 965-968 (1997).
5 - Torchia, J., et al. The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function. Nature 387, 677-684 (1997).
6 - Lonard, D.M. & O'Malley B, W. Nuclear receptor coregulators: judges, juries, and executioners of cellular regulation. Mol Cell 27, 691-700 (2007).
7 - Chopra, A.R., et al. Cellular energy depletion resets whole-body energy by promoting coactivator-mediated dietary fuel absorption. Cell Metab 13, 35-43 (2011).
8 - Chopra, A.R., et al. Absence of the SRC-2 coactivator results in a glycogenopathy resembling Von Gierke's disease. Science 322, 1395-1399 (2008).
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The purpose of this assay is to identify compounds that act as inhibitors of the steroid receptor coactivator 2 (SRC2), also known as nuclear receptor coactivator 2 (NCOA2). In this assay, HEK293 cells are transfected with a GAL4-responsive reporter plasmid (pG5Luc, Promega) and an expression vector encoding SRC2 fused to the DNA-binding domain of GAL4 (pBIND-SRC-2). 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 SRC2 ability to induce transcription will lead to a decrease in expression of the luciferase gene, resulting in reduced well luminescence. Compounds are tested in singlicate at a final nominal concentration of 8.9 uM.
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 pG5Luc reporter plasmid, 11.5 ug of pBIND-SRC2 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 45 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
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.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally active compounds. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-52, and for inactive compounds 51-0.
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)
pG5luc (Promega, part E2440)
pBIND-SRC2 (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)
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 or stabilize 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)