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BioAssay: AID 686995

Counterscreen for activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM): Luminescence-based cell-based high throughput dose response assay to identify non-selective compounds using the VP16 reporter assay

Name: Counterscreen for activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM): Luminescence-based cell-based high throughput dose response assay to identify non-selective compounds using the VP16 reporter assay. ..more
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AID: 686995
Data Source: The Scripps Research Institute Molecular Screening Center (VP16_ACT_LUMI_1536_3XEC50 DCSRUN for BRM ACT)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-05-21

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
652017Luminescence-based cell-based primary high throughput screening assay to identify activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM)screeningPrimary Screen (BRM ACT in singlicate)
652022Summary of the probe development effort to identify activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM)summarySummary (BRM ACT)
686939Counterscreen for activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM): Luminescence-based cell-based high throughput screening assay to identify non-selective compounds using the VP16 reporter assayscreeningCounterscreen(SW13 ACT in triplicate)
720660On Hold
720661On Hold
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: David Reisman, University of Florida
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number 1R03DA028854-01
Grant Proposal PI: David Reisman
External Assay ID: VP16_ACT_LUMI_1536_3XEC50 DCSRUN for BRM ACT

Name: Counterscreen for activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM): Luminescence-based cell-based high throughput dose response assay to identify non-selective compounds using the VP16 reporter assay.

Description:

The protein encoded by the BRM gene (SMARCA2, SNF2, SWI2) is a member of the SWI/SNF family of proteins and is highly similar to the brahma protein of Drosophila (1). Members of this family have helicase and ATPase activities and are thought to regulate transcription of certain genes by altering the chromatin structure around those genes (2). The encoded protein is part of the large ATP-dependent chromatin remodeling complex SNF/SWI, which is required for transcriptional activation of genes normally repressed by chromatin. BRM is an epigenetically suppressed anti-cancer gene, which is silenced in wide variety of solid tumors (2). Because BRM function is key for growth control, restoring its expression routinely inhibits cancer cell growth. For this reason, restoring BRM has potential as an anticancer therapeutic modality (3-4). Its expression prevents cancer development as seen in murine models system where BRM loss potentiates cancer development 10-fold (5). It is known from preliminary studies that histone deacetylase (HDAC) inhibitors are found to restore BRM expression in cancer cell lines, but not its function (2). However, BRM is involved in many pathways and required by numerous transcription factors which control development, differentiation, DNA repair, adhesion, and growth control (6). As such, the impact of inactivating BRM and/or restoring its expression goes well beyond growth control (7).

Although pan-histone deacetylase (HDAC) inhibitors are found to restore BRM expression, but not its function in cancer cell lines, specific inhibitors of either HDAC3 or HDAC9, as well as the transcription factor GATA3 and/or MEF2D, induce a functional BRM. These other constituents involved in BRM regulation may also be the molecular targets. However, since HDAC9 and GATA3 are highly overexpressed and given the limited scope of expression of HDAC9, these proteins would be preferred targets. The fact that each are highly over-expressed is akin to EGFR in lung cancer or HER2 in breast cancer. Candidate compounds that are positive on the primary screen will be re-screened using our counter-screen, where BRM has been suppressed using anti-BRM shRNA (MG2-KDM). We have found that even the most potent inducers of BRM, are blocked by at least 50% or more by this assay. Hence, false positives will yield readouts of luciferase activity that show >50% inductions, closely approximating the levels found in the primary BRM (MG213) screen and will not have inductions <50% as observed with essentially all BRM-specific inducers.

Compounds will be then be verified as positive hits by a series of assays beginning with a third confirmatory (dose response) screening. Following the dose response screening, hits will be screened by directly measuring BRM induction by qPCR since the BRM gene is controlled by transcription. Additionally, we will determine the relative specificity the hits identified by screening each for its potential to induce a number (~10) of BRM-dependent genes. Since each of these BRM-dependent genes are controlled by different signal transduction pathways, interference in one or more cellular pathways (due to lack of specificity for BRM) will be demonstrated by a lack or reduced induction of one or more of the BRM-dependent genes. Moreover, the level of induction observed for each BRM-dependent gene is an indirect marker for the potency of BRM induction.

A secondary goal of this project is to group these verified compounds based upon their relative site of action in the pathway of BRM induction. We will use a secondary assay to differentiate compounds affecting upstream and downstream sections in the regulatory pathway. A possible additional screen will then be optionally performed using cells harboring anti-HDAC2 shRNA (MGH2KD cells). Since the deacetylation of BRM is a requirement for function thus generating a luciferase signal in these reporter cells, only the compound(s) that affect higher-level regulatory genes (such as MAP kinase inhibition) will be identified with a positive result using this assay. For those compounds having an impact lower in the pathway, we will use them to determine if they inhibit BRN2, GATA3, HDAC3, MEF2D, or perhaps HDAC9, thereby assisting us in determining which motifs of the compound underlie the re-expression of BRM. After this series of screens, all hits will then undergo a series of secondary assays to determine how well they restore BRM expression and its function in BRM-deficient cell lines. The assay provider will explore these secondary MOA studies in additional collaboration beyond the scope of this CPDP.

References:

1. Bourachot, B., M. Yaniv, and C. Muchardt, The activity of mammalian brm/SNF2alpha is dependent on a high-mobility-group protein I/Y-like DNA binding domain. Mol Cell Biol, 1999. 19(6): p. 3931-9.
2. Glaros, S., et al., The reversible epigenetic silencing of BRM: implications for clinical targeted therapy. Oncogene, 2007. 26(49): p. 7058-66.
3. Reisman, D., S. Glaros, and E.A. Thompson, The SWI/SNF complex and cancer. Oncogene, 2009. 28(14): p. 1653-68.
4. Reyes, J.C., et al., Altered control of cellular proliferation in the absence of mammalian brahma (SNF2alpha). EMBO J, 1998. 17(23): p. 6979-91.
5. Liu, G., et al., Two novel BRM insertion promoter sequence variants are associated with loss of BRM expression and lung cancer risk. Oncogene, 2011. 30(29): p. 3295-304.
6. Coisy-Quivy, M., et al., Role for Brm in cell growth control. Cancer Res, 2006. 66(10): p. 5069-76.
7. Gramling, S., et al., Pharmacologic reversal of epigenetic silencing of the anticancer protein BRM: a novel targeted treatment strategy. Oncogene, 2011. 30(29): p. 3289-94.

Keywords:

DCSRUN, dose response, counterscreen, triplicate, VP16, herpes, transactivator, Gal4, SW13 cell-based, alternate, BRM, SMARCA, SMARCA2, SWI/SNF, ATPase, gene, transcription, regulator, reporter, activate, activator, inducer, increase, lumi, luc, luminescence, luciferase, cell, cell-based, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, 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 compounds identified as active in the BRM Primary screen are luminescence artifacts or are promiscuous transcriptional modulators. Any compound active in this will not be pursued. In this counterscreen, cells are transiently transfected with an expression plasmid containing a strong transactivation domain of the herpes simplex virus Virion Protein 16 (VP16) fused to the yeast GAL4 DNA Binding Domain (DBD). Cells are co-transfected with the pG5luc luciferase reporter plasmid containing UAS repeats. As designed, compounds that activate VP16 transcriptional activity and/or allow GAL4 binding to the UAS sequence will lead to an increased expression of the pG5-luciferase reporter gene, resulting in increased well luminescence. These compounds are likely to be nonselective activators or fluorescent artifacts. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 91.2 uM.

Protocol Summary:


293 cells are routinely cultured in DMEM with 10% FBS and 1X penicillin-streptomycin. The 293 cells are harvested from T175 flasks with trypsin. The 293 cells are counted and resuspended in Maxycyte electroporation buffer at 1e8 cells/ml. 200ug/ml pGL4.31 and 1ug/ml Gal4-VP16 is added to the cells and the cells are electroporated using the maxcyte electroporator using a CL-2 Bag. Remove the cells from the bag and let incubate in a 6 well for 20 minutes at 37C, 5%CO2. The cells are then plated in T175 flasks at 15 million cells per flask in 30ml of growth media and incubated overnight at 37C, 5%CO2. The cells can then be frozen down to use in multiple experiments. The cells are frozen in 90%FBS, 10% DMSO at 15 million per ml. The Cells are then thawed from a cryovial into a T175 per 15 million cells in 30ml growth media. The cells are incubated for 24 hours at 37C, 5%CO2. The next day, The cells are removed from the flasks with trypsin, and counted. The cells are resuspended in growth media at 2,500 cells per well in 5uL per well of growth media in 1536 well plates. The cells are briefly spun down and the compounds and controls are pinned at 46nL per well with Kalypsis pintool. The cells are incubated for 24 hours at 37C, 5% CO2. The next day 5ul/well of One GLO luciferase substrate is added to each well and incubated at room temperature for 15 minutes. The Luminescence is detected on the Viewlux.

The percent activation for each compound was calculated as follows:

%Activation = ( 1 - ( ( Test_Compound - Median_High_Control ) / ( Median_Low_Control - Median_High_Control ) ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing SAHA

PubChem Activity Outcome and Score:

For each test compound, percent activation 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 (Accelrys Inc). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 91.2uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 91.2 uM.

Compounds with an EC50 greater than 10 uM were considered inactive.
Compounds with an EC50 equal to or less than 10 uM were considered active.

The PubChem Activity Score range for inactive compounds is 0-0, there are no active compounds.

List of Reagents:

293 cell line (ATCC, part 1573)
DMEM medium (Invitrogen, part 11995)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16000-044)
One Glo Assay Kit (Promega, part E6130/QTE30675 )
SAHA (cayman Chemicals, part 10009929)
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Corning, part 789173)
Maxcyte reagents and Maxycyte electroporator (Maxcyte)
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, compounds that non-specifically modulate luciferase activity, and compounds that quench 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.
Categorized Comment
Assay: Dictionary: Version: 0.1

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

Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierActivity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
2EC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (EC50) shown in micromolar.FloatμM
3LogEC50Log10 of the qualified EC50 (EC50) 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
7Hill 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
8Response RangeThe range of Y.Float
9Chi 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
10RsquareThis 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
11Number 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
12Excluded 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
13Activation at 0.0046 uM [1] (0.0046μM**)Value of %activation at 0.0046 micromolar activator concentration; replicate [1]Float%
14Activation at 0.0046 uM [2] (0.0046μM**)Value of %activation at 0.0046 micromolar activator concentration; replicate [2]Float%
15Activation at 0.0046 uM [3] (0.0046μM**)Value of %activation at 0.0046 micromolar activator concentration; replicate [3]Float%
16Activation at 0.014 uM [1] (0.014μM**)Value of %activation at 0.014 micromolar activator concentration; replicate [1]Float%
17Activation at 0.014 uM [2] (0.014μM**)Value of %activation at 0.014 micromolar activator concentration; replicate [2]Float%
18Activation at 0.014 uM [3] (0.014μM**)Value of %activation at 0.014 micromolar activator concentration; replicate [3]Float%
19Activation at 0.042 uM [1] (0.042μM**)Value of %activation at 0.042 micromolar activator concentration; replicate [1]Float%
20Activation at 0.042 uM [2] (0.042μM**)Value of %activation at 0.042 micromolar activator concentration; replicate [2]Float%
21Activation at 0.042 uM [3] (0.042μM**)Value of %activation at 0.042 micromolar activator concentration; replicate [3]Float%
22Activation at 0.12 uM [1] (0.12μM**)Value of %activation at 0.12 micromolar activator concentration; replicate [1]Float%
23Activation at 0.12 uM [2] (0.12μM**)Value of %activation at 0.12 micromolar activator concentration; replicate [2]Float%
24Activation at 0.12 uM [3] (0.12μM**)Value of %activation at 0.12 micromolar activator concentration; replicate [3]Float%
25Activation at 0.13 uM [1] (0.13μM**)Value of %activation at 0.13 micromolar activator concentration; replicate [1]Float%
26Activation at 0.13 uM [2] (0.13μM**)Value of %activation at 0.13 micromolar activator concentration; replicate [2]Float%
27Activation at 0.13 uM [3] (0.13μM**)Value of %activation at 0.13 micromolar activator concentration; replicate [3]Float%
28Activation at 0.37 uM [1] (0.37μM**)Value of %activation at 0.37 micromolar activator concentration; replicate [1]Float%
29Activation at 0.37 uM [2] (0.37μM**)Value of %activation at 0.37 micromolar activator concentration; replicate [2]Float%
30Activation at 0.37 uM [3] (0.37μM**)Value of %activation at 0.37 micromolar activator concentration; replicate [3]Float%
31Activation at 0.38 uM [1] (0.38μM**)Value of %activation at 0.38 micromolar activator concentration; replicate [1]Float%
32Activation at 0.38 uM [2] (0.38μM**)Value of %activation at 0.38 micromolar activator concentration; replicate [2]Float%
33Activation at 0.38 uM [3] (0.38μM**)Value of %activation at 0.38 micromolar activator concentration; replicate [3]Float%
34Activation at 1.1 uM [1] (1.1μM**)Value of %activation at 1.1 micromolar activator concentration; replicate [1]Float%
35Activation at 1.1 uM [2] (1.1μM**)Value of %activation at 1.1 micromolar activator concentration; replicate [2]Float%
36Activation at 1.1 uM [3] (1.1μM**)Value of %activation at 1.1 micromolar activator concentration; replicate [3]Float%
37Activation at 3.4 uM [1] (3.4μM**)Value of %activation at 3.4 micromolar activator concentration; replicate [1]Float%
38Activation at 3.4 uM [2] (3.4μM**)Value of %activation at 3.4 micromolar activator concentration; replicate [2]Float%
39Activation at 3.4 uM [3] (3.4μM**)Value of %activation at 3.4 micromolar activator concentration; replicate [3]Float%
40Activation at 10.1 uM [1] (10.1μM**)Value of %activation at 10.1 micromolar activator concentration; replicate [1]Float%
41Activation at 10.1 uM [2] (10.1μM**)Value of %activation at 10.1 micromolar activator concentration; replicate [2]Float%
42Activation at 10.1 uM [3] (10.1μM**)Value of %activation at 10.1 micromolar activator concentration; replicate [3]Float%
43Activation at 30.4 uM [1] (30.4μM**)Value of %activation at 30.4 micromolar activator concentration; replicate [1]Float%
44Activation at 30.4 uM [2] (30.4μM**)Value of %activation at 30.4 micromolar activator concentration; replicate [2]Float%
45Activation at 30.4 uM [3] (30.4μM**)Value of %activation at 30.4 micromolar activator concentration; replicate [3]Float%
46Activation at 91.2 uM [1] (91.2μM**)Value of %activation at 91.2 micromolar activator concentration; replicate [1]Float%
47Activation at 91.2 uM [2] (91.2μM**)Value of %activation at 91.2 micromolar activator concentration; replicate [2]Float%
48Activation at 91.2 uM [3] (91.2μM**)Value of %activation at 91.2 micromolar activator concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03DA028854-01

Data Table (Concise)
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