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BioAssay: AID 2176

Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1).

Name: Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1). ..more
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 Tested Compounds
 Tested Compounds
All(2366)
 
 
Active(1284)
 
 
Inactive(1083)
 
 
 Tested Substances
 Tested Substances
All(2374)
 
 
Active(1286)
 
 
Inactive(1088)
 
 
AID: 2176
Data Source: The Scripps Research Institute Molecular Screening Center (GSTO1_INH_FP_1536_3X%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-12-04
Modify Date: 2010-06-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1284
Related Experiments
Show more
AIDNameTypeProbeComment
1515Primary biochemical high throughput screening assay to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9)Screening depositor-specified cross reference: Counterscreen (RBBP9 inhibitors)
1790Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9).Summary2 depositor-specified cross reference: Summary AID.
1974Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1).Screening depositor-specified cross reference: Primary screen (GSTO1 inhibitors)
2175Summary of probe development efforts to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1).Summary1 depositor-specified cross reference
449772Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: LC-MS/MS assay to assess binding of compounds to active siteOther depositor-specified cross reference
449773Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: luminescence-based biochemical dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory depositor-specified cross reference
463081Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: Gel-based activity-based protein profiling (ABPP) IC50Confirmatory depositor-specified cross reference
463093Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): fluorescence-based click chemistry assayOther depositor-specified cross reference
463094Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): fluorescence-based cell-based assayScreening depositor-specified cross reference
463098Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): gel-based activity-based protein profiling (ABPP) percent inhibition assay with endogenous enzymeScreening depositor-specified cross reference
463101Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): fluorescence-based click chemistry assay 2Other depositor-specified cross reference
463102Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): gel-based activity-based protein profiling (ABPP) selectivity assay with endogenous enzymeOther depositor-specified cross reference
463110Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): gel-based activity-based protein profiling (ABPP) percent inhibition assay with recombinant enzymeOther depositor-specified cross reference
463142Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: Gel-based activity-based protein profiling (ABPP) IC50 Set 2Confirmatory depositor-specified cross reference
1537Confirmation biochemical high throughput screening assay for inhibitors of Retinoblastoma binding protein 9 (RBBP9)Screening same project related to Summary assay
1947Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the serine hydrolase family member Fam108B.Screening same project related to Summary assay
1978Fluorescence polarization-based confirmation biochemical high throughput screening assay for inhibitors of the serine hydrolase family member Fam108b.Screening same project related to Summary assay
2243Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Luminescence-based counterscreen assay to identify cytotoxic compoundsConfirmatory same project related to Summary assay
2248Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel-based Activity-Based Protein Profiling (ABPP) Gel Filtration AssaySummary same project related to Summary assay
2254Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel-based Activity-Based Protein Profiling (ABPP) IC50Confirmatory same project related to Summary assay
2269Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel-based Activity-Based Protein Profiling (ABPP) InhibitionScreening same project related to Summary assay
2299Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9): Ester Oxime ScaffoldSummary2 same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Benjamin Cravatt, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R01 CA087660-05 Fast Track
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: GSTO1_INH_FP_1536_3X%INH

Name: Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1).

Description:

The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F (1), which activates genes essential for DNA replication. Hypophosphorylated RB inhibits cell cycle progression by sequestering E2F, thus preventing the activation of genes required for S phase transition. Due to the critical role of RB in regulating the cell cycle, factors that bind and regulate RB activity are considered valuable targets for preventing tumorigenesis. One such protein, RB binding protein 9 (RBBP9), is widely expressed in different tissues and upregulated in certain tumors (2, 3). The RBBP9 protein contains an alpha/beta hydrolase fold which belongs to the DUF1234 domain superfamily of unknown function. Although an enzymatic activity of RBBP9 has not been reported, this protein does react with activity-based probes that target serine hydrolases, suggesting that it is a functional enzyme. Also consistent with this premise, the crystal structure of RBBP9 was recently solved and revealed a well structured active site with a properly arranged catalytic triad (4).

A role for RBBP9 in cellular transformation came from studies showing that RBBP9 mRNA expression is increased in transformed rat liver cell lines and human liver tumor biopsies (3). RBBP9-overexpressing cells form tumors when implanted into immuno-deficient mice (3), and RBBP9 overexpression confers resistance to TGF-#1-induced growth inhibition through its interaction with Rb and displacement of E2F (3, 5). RBBP9 is also suggested to play a role in gender-related differential responses to radiation-induced cell proliferation (6). As a result, the identification of compounds that selectively inhibit RBBP9 activity may provide valuable probes for the study of apoptosis, cell cycle, and tumorigenesis.

References:
1. Nevins, J.R., E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science, 1992. 258(5081): p. 424-9.
2. Chen, J.Z., Yang, Q.S., Wang, S., Meng, X.F., Ying, K., Xie, Y., and Ma, Y.M., Cloning and expression of a novel retinoblastoma binding protein cDNA, RBBP10. Biochem Genet, 2002. 40(7-8): p. 273-82.
3. Woitach, J.T., Zhang, M., Niu, C.H., and Thorgeirsson, S.S., A retinoblastoma-binding protein that affects cellcycle control and confers transforming ability. Nat Genet, 1998. 19(4): p. 371-4.
4. Vorobiev, S.M., Su, M., Seetharaman, J., Huang, Y.J., Chen, C.X., Maglaqui, M., Janjua, H., Proudfoot, M., Yakunin, A., Xiao, R., Acton, T.B., Montelione, G.T., and Tong, L., Crystal structure of human retinoblastoma binding protein 9. Proteins, 2009. 74(2): p. 526-9.
5. Woitach, J.T., Hong, R., Keck, C.L., Zimonjic, D.B., Popescu, N.C., and Thorgeirsson, S.S., Assignment of the Bog gene (RBBP9) to syntenic regions of mouse chromosome 2G1-H1 and human chromosome 20p11.2 by fluorescence in situ hybridization. Cytogenet Cell Genet, 1999. 85(3-4): p. 252-3.
6. Cassie, S., Koturbash, I., Hudson, D., Baker, M., Ilnytskyy, Y., Rodriguez-Juarez, R., Weber, E., and Kovalchuk, O., Novel retinoblastoma binding protein RBBP9 modulates sex-specific radiation responses in vivo. Carcinogenesis, 2006. 27(3): p. 465-74.

Keywords:

GSTO1, oxidoreductase, glutathione S-transferase omega 1, RBBP9, retinoblastoma binding protein 9, Fam 108B, BOG, cell cycle, cancer, fluorescence polarization, sulfonate ester, antagonist, inhibitor, counterscreen, confirmation, HTS, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this assay is to confirm activity of compounds identified as active in a set of previous experiments entitled, "Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1)" (AID 1974). This assay also serves as a counterscreen for a previous set of experiments entitled, "Primary biochemical high throughput screening assay to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9)" (AID 1515). The oxidoreductase GSTO1 has a catalytic cysteine residue and is sensitive to thiol alkylating agents such as Nethylmaleimide. In this assay, recombinant GSTO1 protein is incubated with test compounds and a Rh-conjugated sulfonate ester (SE) activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as GSTO1 inhibitors will prevent GSTO1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in triplicate at a final nominal concentration of 5.96 micromolar.
Protocol Summary:
Prior to the start of the assay, 4.0 microliters of Assay Buffer (0.01% Pluronic acid, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT) containing 1.25 micromolar of GSTO1-1 protein were dispensed into 1536 microtiter plates. Next, 30 nL of test compound in DMSO or DMSO alone (0.59% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 degrees Celsius.
The assay was started by dispensing 1.0 microliter of 375 nM PS-Rh probe in Assay Buffer to all wells. Plates were centrifuged and incubated for 20 hours at 37 degrees Celsius. Prior to reading, plates were equilibrated at room temperature for 10 minutes. Fluorescence polarization was read for 30 seconds for each polarization plane (parallel and perpendicular) on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm). The well fluorescence polarization value (mP) was obtained via the PerkinElmer Viewlux software.
The percent inhibition for each compound was calculated as follows:
100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Where:
Low_Control is defined as wells containing GSTO1-1 and DMSO.
Test_Compound is defined as wells containing GSTO1-1 in the presence of test compound.
High_Control is defined as wells containing no GSTO1-1 enzyme.
A mathematical algorithm was used to determine nominally inhibiting compounds in the screen. 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 calculated for the Primary screen 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 activity score range for active compounds is 100-11, for inactive 11-0.
List of Reagents:
Recombinant GSTO1-1 (supplied by Assay Provider)
PS-Rh probe (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic acid (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)
DTT (Sigma, part D9779)
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, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular 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 AID.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (5.96μM**)Normalized percent inhibition of the screen at a compound concentration of 5.96 uM.Float%
2Standard DeviationStandard deviation of the screen derived from the normalized percent inhibition of the triplicate data for each compound.Float

** Test Concentration.
Additional Information
Grant Number: 1 R01 CA087660-05

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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