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BioAssay: AID 1515

Primary biochemical high throughput screening assay to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9)

Name: Primary biochemical high throughput screening assay to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9) ..more
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 Tested Compounds
 Tested Compounds
All(217964)
 
 
Active(445)
 
 
Inactive(217520)
 
 
 Tested Substances
 Tested Substances
All(218117)
 
 
Active(445)
 
 
Inactive(217672)
 
 
AID: 1515
Data Source: The Scripps Research Institute Molecular Screening Center (RBBP9_INH_FP_1536_%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-02-03
Modify Date: 2010-06-16

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 445
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
1978Fluorescence polarization-based confirmation biochemical high throughput screening assay for inhibitors of the serine hydrolase family member Fam108b.screening
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
1537Confirmation biochemical high throughput screening assay for inhibitors of Retinoblastoma binding protein 9 (RBBP9)screening
1790Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9).summary2
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
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
2175Summary of probe development efforts to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1).summary1
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
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
2200Summary of probe development efforts to identify inhibitors of the serine hydrolase family member Fam108b.summary
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
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
2176Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1).screening
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
2299Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9): Ester Oxime Scaffoldsummary2
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
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
463081Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: Gel-based activity-based protein profiling (ABPP) IC50confirmatory1
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
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
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
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
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: RBBP9_INH_FP_1536_%INH

Name: Primary biochemical high throughput screening assay to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9)

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-β1-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 cell-cycle 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:

RBBP9, retinoblastoma binding protein 9, BOG, cell cycle, cancer, fluorescence polarization, fluorophosphonate rhodamine, FP-Rh, antagonist, inhibitor, primary, 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 identify compounds that act as inhibitors of RBBP9. In this assay, a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family is used to label RBBP9 in the presence of test compounds. 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 RBBP9 inhibitors will prevent RBBP9-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization in the well. Compounds were tested in singlicate at a final nominal concentration of 7.94 micromolar.

Protocol Summary:

Prior to the start of the assay, 4.0 ul of Assay Buffer (0.1% Pluronic acid, 50 mM Tris HCl, 150 mM NaCl, pH 8.0) containing 2.5 uM of RBBP9 protein were dispensed into 1536 microtiter plates. Next, 40 nL of test compound in DMSO or DMSO alone (0.794% 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 ul of 375 nM FP-Rh probe in Assay Buffer to all wells. Plates were centrifuged and after 45 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm). Fluorescence polarization was read for 30 seconds for each polarization plane (parallel and perpendicular). The well fluorescence polarization value (mP) was obtained via the PerkinElmer Viewlux software.

The percent inhibition for each compound was calculated as follows:
Percent inhibition = (Test_Compound_mP -median_Positive_Control_mP) / (median_Negative_Control_mP - median_Positive_Control_mP)*100
Where:
Negative_Control is defined as wells containing RBBP9 and DMSO,
Test_Compound is defined as wells containing RBBP9 in the presence of test compound,
Positive_Control is defined as wells containing no RBBP9 protein.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary 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 was declared active.

The inactive compounds of this assay have activity score range of 0 to 6 and active compounds range of activity score is 6 to 100.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.


List of Reagents:
Recombinant RBBP9 (supplied by Assay Provider)
FP-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)
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.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (7.94μM**)Normalized percent inhibition of the primary screen at a compound concentration of 7.94 micromolar.Float%

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

Data Table (Concise)
Classification
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