Counterscreen for inhibitors of the Steroid Receptor Coactivator 2 (SRC2; NCOA2): Luminescence-based cell-based high throughput assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)
Name: Counterscreen for inhibitors of the Steroid Receptor Coactivator 2 (SRC2; NCOA2): Luminescence-based cell-based high throughput assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16). ..more
BioActive Compounds: 424
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: VP16_INH_LUMI_1536_3X%INH CSRUN for SRC2
Name: Counterscreen for inhibitors of the Steroid Receptor Coactivator 2 (SRC2; NCOA2): Luminescence-based cell-based high throughput assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16).
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.
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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).
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 determine whether the compounds that were available among the top 2500 most active compounds previously identified in a set of experiments entitled, "Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of the Steroid Receptor Coactivator 2 (SRC2; NCOA2)" (AID 651957) are nonselective or promiscuous transcriptional modulators.
In this counterscreen, the strong transactivation domain of the herpes simplex virus Virion Protein 16 (VP16) is fused to the yeast GAL4 DNA Binding Domain (DBD) in place of the original SRC2 moiety used in the SRC2 assay. Cells are co-transfected with the pGL5 luciferase reporter plasmid containing UAS repeats. As designed, compounds that repress VP16 transcriptional activity and/or prevent GAL4 binding to the UAS sequence will lead to a reduced expression of luciferase driven by the pGL5 reporter plasmid, resulting in decreased well luminescence. These compounds are likely to be nonselective inhibitors or cytotoxic. Compounds are tested in quadruplicate 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, 230 ng of pBIND-VP16 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 (3,750 cells per well). The first two columns received cells transfected with 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 activity was detected by adding 5 uL per well of ONE-Glo luciferase detection reagent. After a 15 minute incubation time, light emission was measured using the ViewLux plate 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
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:
The average percent activation and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the average plus three standard deviation of the Negative_Control wells (i.e. DMSO treated, calculated at 35.30%) was declared active.
The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-35, and for inactive compounds 35-0.
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-VP16 (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, 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.
** Test Concentration.
Data Table (Concise)