2254

The Scripps Research Institute Molecular Screening Center

RBBP9_INH_FP_GEL_3XIC50

2010

Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel-based Activity-Based Protein Profiling (ABPP) IC50

Data 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 Center Network (MLPCN)

Grant Proposal Number: 1 2 R01 CA087660-05 Fast Track

Grant Proposal PI: Benjamin Cravatt, TSRI

External Assay ID: RBBP9_INH_FP_GEL_3XIC50

Name: Late stage results from the probe development effort to identify inhibitors of Retinoblastoma Binding Protein 9 (RBBP9): Gel-based Activity-Based Protein Profiling (ABPP) IC50

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. 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 is supported by studies showing that RBBP9 mRNA expression is increased in transformed rat liver cell lines and human liver tumor biopsies (3). Furthermore, 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.

This gel-based activity-based protein profiling (ABPP) inhibition IC50 assay was performed as part of a probe development effort to identify inhibitor probes of RBBP9 belonging to the oxime ester scaffold.

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.

7. Bachovchin, D.A., Brown, S.J., Rosen, H., and Cravatt, B.F., Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat Biotechnol, 2003. 27(4): p. 387-94.

Keywords:

Late stage, Probes, RBBP9, retinoblastoma binding protein 9, BOG, cell cycle, cancer, fluorescence polarization, fluorophosphonate rhodamine, FP-Rh, antagonist, inhibitor, primary, confirmation, gel-based ABPP, HTS, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Please see AIDs 1515 and 1537, Summary AID 1790, and below for all protocols performed in this probe development effort.

Assay Overview:

Please see AIDs 1515 and 1537, Summary AID 1790, and below for all protocols performed in this probe development effort. The purpose of this assay was to confirm activity of compounds identified as active in the uHTS primary (AID 1515) and confirmation screens (AID 1537). 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 products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as RBBP9 inhibitors will prevent RBBP9-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe, leading to low fluorescence polarization in the band in the gel. IC50 values for inhibition of RBBP9 and more than 30 other serine hydrolases (anti-target) were determined

Protocol Summary:

Recombinant RBBP9 (400 nM) in assay buffer was incubated with DMSO or compound (20 mM) for 30 min at 25 degrees Celsius before the addition of FP-rhodamine at a final concentration of 1 uM in 50 ml total reaction volume. The reaction was incubated for 10 min at 25 degrees Celsius, quenched with 2x SDS-PAGE loading buffer, boiled for 5 min at 90 degrees Celsius, separated by SDS-PAGE and visualized in-gel. The percentage activity remaining was determined by measuring the integrated optical density of the bands. IC50 values for inhibition of RBBP9 (Assay Panel 1) and more than 30 other serine hydrolases (anti-target) (Assay Panel 2) were determined from dose-response curves from three trials at each inhibitor concentration (0.1-100 uM).

Activity Score:

The PubChem Activity Score is assigned a value of 100 for probe compounds, 50 for active compounds, and 0 for inactive compounds.

List of Reagents:

Recombinant RBBP9 protein (provided by Assay Provider)

FP-rhodamine (provided by Assay Provider)

Sodium Chloride (Fisher, part 980597)

1M Tris, pH 8.0 (Invitrogen, part T-3038)

This assay was performed in the laboratory of the Assay Provider with compounds ordered as powders. Probes were identified for this project. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed in the Related Bioassays section. Please also see Summary AID 1790. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html. One paper has been published detailing the emetine probe (7).

1515

Screening Assay (RBBP9 inhibitors).

1537

Screening Assay (RBBP9 inhibitors).

1790

Summary AID.

http://mlpcn.florida.scripps.edu

10741

RBBP9 retinoblastoma binding protein 9 [ Homo sapiens ]

602908

9606

1411535

12296629

9697699

19004028

10449909

16272168

19329999

Assay 1: RBBP9 Gel-based ABPP Assay (IC50)

The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.

Assay 1: RBBP9 Gel-based ABPP Assay (Outcome)

The Assay outcome, one of Active, Inactive or Not Tested.

254

Assay 2: Anti-Target Gel-based ABPP Assay (Qualifier)

Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.

Assay 2: Anti-Target Gel-based ABPP Assay (IC50)

The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.

Assay 2: Anti-Target Gel-based ABPP Assay (Outcome)

The Assay outcome, one of Active, Inactive or Not Tested.

254

Probe Molecule Outcome

Indicates if this molecule is a probe or not. One of Probe, Active or inactive.

retinoblastoma binding protein 9 [Homo sapiens]

24119166

Homo sapiens

human

taxon

9606

1 2 R01 CA087660-05

Gel-based Activity-Based Protein Profiling

RBBP9 IC50 Gel-based ABPP Assay

retinoblastoma binding protein 9 [Homo sapiens]

24119166

10741

Anti-Target IC50 Gel-based ABPP Assay

85098567

100

0.635

100

Probe

2010

85098568

1.9

100

Active

2010

85098569

5.7

100

Active

2010

85098570

100

Active

2010

85098571

9.1

100

Active

2010

87225289

1200

100

Inactive

2010

87225290

1500

100

Inactive

2010