Late 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 Assay
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) Gel Filtration Assay ..more
BioActive Compound: 1
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_FILTRATION
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) Gel Filtration Assay
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) gel filtration assay was performed as part of a probe development effort to identify inhibitor probes of RBBP9 belonging to the oxime ester scaffold.
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.
Late stage, reversible binding, gel filtration, 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.
The purpose of this assay is to assess reversibility of binding of inhibitor compounds belonging to the oxime ester scaffold. In this assay, a fraction of the enzyme-inhibitor mixture is passaged over a Sephadex G-25M column (GE Healthcare) before reaction with 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 irreversible RBBP9 inhibitors will prevent RBBP9-probe interactions after gel filtration, thereby increasing the proportion of free (unbound) fluorescent probe, leading to low fluorescence polarization in the band in the gel. The compound's reversibility of inhibition of RBBP9 was assessed.
Recombinant RBBP9 (2mM) in assay buffer was incubated with DMSO or inhibitor (100 mM) for 30 min at 25 degrees Celsius and each reaction was split into two fractions. One fraction was reacted directly with FP-Rh, and the other was passaged over a Sephadex G-25M column (GE Healthcare) and then reacted with FP-Rh at a final concentration of 75 nM in 50 ml total reaction volume. The reaction was incubated for 45 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 PubChem Activity Scores in this assay were determined as follows; 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)
Sephadex G-25 (GE Healthcare, part 17-0851-01)
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).
Data Table (Concise)