AID 2299 - PubChem BioAssay Summary

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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 Ester Oxime Scaffold

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.

Identification of initial RBBP9 probe (emetine):

These efforts resulted in the identification of emetine (MLS SID 855836; SCRIPPS SID 87235993) as a probe, with an IC50 of 7.8 uM. These results can be found in our probe report on page 5. Following the emetine discovery, collaborative efforts by the assay provider and the SRIMSC were pursued to improve upon emetine's potency and selectivity (>200-fold), and to identify related scaffolds for probe development. 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 uM. The tight structure-activity relationship of the emetine-RBBP9 interaction suggests that only minor modifications to the emetine structure will preserve/improve activity. Please refer to Summary AID 1790 for further details regarding this first probe.

Identification of second RBBP9 probe (ester oxime scaffold):

Two related compounds with ester oxime cores were identified as the most potent and selective of the primary screening hits. An initial round of SAR by purchase on these chemically tractable scaffolds revealed that modification of the oxime leaving group resulted in a dramatic loss in potency. A second round of SAR by purchase and synthesis in the Cravatt lab with compounds that retained the oxime leaving group identified the probe compound (SID 85098567), the most potent (0.63 uM) and selective (>100-fold) of the compounds tested. The probe compound SID 85098567 is a 10-fold more potent inhibitor of RBBP9 than is emetine, and it has a mechanism of action distinct from that of emetine; unlike emetine, probe compound SID 85098567 covalently inhibits RBBP9. In addition, whereas emetine is highly cytotoxic, the probe compound SID 85098567 exhibits no cytotoxicity.

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. Two papers have been published detailing these compounds (7,8). 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. 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.

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.
8. Bachovchin DA, Wolfe MR, Masuda K, Brown SJ, Spicer TP, Fernandez-Vega V, Chase P, Hodder PS, Rosen H, Cravatt BF., Oxime esters as selective, covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9). Bioorg Med Chem Lett, 2010. 20(7): p. 2254-8.

Keywords:

Late stage, Summary, probes, ester oxime, ester, oxime, oxime ester, RBBP9, retinoblastoma binding protein 9, BOG, cell cycle, cancer, fluorescence polarization, fluorophosphonate rhodamine, FP-Rh, antagonist, inhibitor, primary, confirmation, gel-based ABPP, reversible binding, gel filtration, HEK 293T cells, cytotoxicity, luminescence, viability, HTS, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.