Bookmark and Share
BioAssay: AID 1790

Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9).

Name: Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9). ..more
_
   
 Tested Compounds
 Tested Compounds
All(5)
 
 
Probe(2)
 
 
Active(2)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Probe(2)
 
 
Active(2)
 
 
Inactive(3)
 
 
AID: 1790
Data Source: The Scripps Research Institute Molecular Screening Center (RBBP9_INH_LEADS_SUMMARY)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-05-28
Modify Date: 2011-03-03

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: retinoblastoma binding protein 9 [Homo sapiens]
Description ..   
Protein Family: Esterase_lipase

Gene:RBBP9     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: Chemical Probe: 2    Active: 2
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: Primary screen (RBBP9 inhibitors in singlicate)
1537Confirmation biochemical high throughput screening assay for inhibitors of Retinoblastoma binding protein 9 (RBBP9)Screening depositor-specified cross reference: Confirmation screen (RBBP9 inhibitors in triplicate)
1947Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the serine hydrolase family member Fam108B.Screening depositor-specified cross reference: Primary screen (Fam108B inhibitors in singlicate)
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
1978Fluorescence polarization-based confirmation biochemical high throughput screening assay for inhibitors of the serine hydrolase family member Fam108b.Screening depositor-specified cross reference
2176Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1).Screening depositor-specified cross reference
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 depositor-specified cross reference: Cytotoxicity assay (RBBP9 inhibitors)
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 depositor-specified cross reference: MOA assay (RBBP9 inhibitors, Gel filtration 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 depositor-specified cross reference: Secondary dose response assay (RBBP9 inhibitors, ABPP 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 depositor-specified cross reference: Secondary assay (RBBP9 inhibitors, ABPP assay)
2299Summary of probe development efforts to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9): Ester Oxime ScaffoldSummary2 depositor-specified cross reference: Summary AID (RBBP9 inhibitors, ester oxime scaffold)
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_PROBES_SUMMARY

Name: Summary of probe development efforts 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-beta1-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.

Summary of Probe Development Effort:

Following primary HTS in singlicate to identify RBBP9 inhibitors (AID 1515) and confirmation of hit activity in triplicate (AID 1537), compounds were identified as possible candidates for probe development. The top hits were selected for screening using gel-based ABPP profiling to determine potency, and selectivity in two complex proteomes.

After identification of emetine as an inhibitor of RBBP9, about 80 compounds similar in structure to emetine were purchased, including several other natural product ipecac alkaloids. Only two additional compounds, the natural products cephaeline and tubusoline, inhibited RBBP9, albeit with reduced potency relative to emetine. Notably, dehydroemetine, which only differs from emetine by the presence of one double bond, failed to inhibit RBBP9 at concentrations up to 200 muM. The tight structure-activity relationship of the emetine-RBBP9 interaction suggests that only minor modifications to the emetine structure will preserve/improve activity, and future studies will involve semi-synthetic addition of small moieties to the emetine and cephaeline scaffolds.

The above efforts resulted in the identification of novel, potent, and selective small molecule inhibitors of RBBP9. Two probe reports have been submitted describing compound screening and analog syntheses. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html#RBBP9. One paper has been published detailing these compounds (7). A probe report for SID 855836 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML081. A probe report for SID 85098567 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML114.

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:

Summary, 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.
Protocol
Please see AIDs 1515, 1537, and below for all protocols performed in this probe development effort.
RBBP9 Inhibition Assays (Assays 1 and 2):
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 at a final nominal concentration of 7.94 micromolar in singlicate (AID 1515) and in triplicate (AID 1537).
Gel-based Activity-based Protein Profiling Assay (Assay 3):
This assay was performed in the laboratory of the Assay Provider. The purpose of this assay is to confirm activity of compounds identified as active in a previous set of experiments entitled, "Confirmation biochemical high throughput screening assay for inhibitors of Retinoblastoma binding protein 9 (RBBP9)," (PubChem 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 are determined from dose-response curves from three trials at each inhibitor concentration (0.1-100 mM).
Comment
Probes were identified.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 2
MLP Probe ML# for probe 1: ML081
PubChem Substance ID (SID) for probe 1: 855836
PubChem Compound ID (CID) for probe 1: 6603320
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 50% inhibition at 5 uM in ABBP assay
Target for probe 1: RBBP9 (gi: 24119166)
Anti-target for probe 1: >30 serine proteases
Fold selectivity for probe 1: >200
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK47337/ (ID: 2358180)
Grant number for probe 1: CA087660-05
MLP Probe ML# for probe 2: ML114
PubChem Substance ID (SID) for probe 2: 85098567
PubChem Compound ID (CID) for probe 2: 5934766
Probe type for probe 2: Inhibitor
IC50/EC50 (nM) for probe 2: 630
Target for probe 2: RBBP9 (gi: 24119166)
Disease relevance for probe 2: Cancer
Anti-target for probe 2: >30 serine hydrolases
Fold selectivity for probe 2: >100
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK50690/ (ID: 2376686)
Grant number for probe 2: CA087660
PubMed Publication ID (PMID) for probe 1: 20207142,19329999
PubMed Publication ID (PMID) for probe 2: 19329999
NCBI Book chapter title for probe 1: Probe Report for RBBP9 Inhibitors - Probe 1
NCBI Book chapter title for probe 2: Probe Report for RBBP9 Inhibitors - Probe 2
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML #Unique alphanumeric identifier assigned to a chemical probe molecule within the Molecular Libraries Probe Production Centers Network (MLPCN).String
2Assay 1: RBBP9 Inhibition PRUN (AID 1515) (7.94μM**)Normalized percent inhibition of the primary screen at a compound concentration of 7.94 micromolar.Float%
3Assay 1: OutcomeThe Assay outcome, one of Active, Inactive or Not Tested.String
4Assay 2: RBBP9 Inhibitor CRUN (AID 1537) (7.94μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 7.94 micromolar.Float%
5Assay 2: OutcomeThe Assay outcome, one of Active, Inactive or Not Tested .String
6Assay 3: QualifierActivity 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.String
7Assay 3: IC50 Gel-based ABPP AssayThe concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
8Assay 3: OutcomeThe Assay outcome, one of Active, Inactive or Not Tested .String

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

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