Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the serine hydrolase family member Fam108B.
Name: Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the serine hydrolase family member Fam108B. ..more
BioActive Compounds: 993
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: FAM108B_INH_FP_1536_1X%INH
Name: Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the serine hydrolase family member Fam108B.
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.
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 cellcycle 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.
Fam108, Fam 108B, serine hydrolase, Cgi67 serine protease, alpha/beta serine hydrolase, RBBP9,
retinoblastoma binding protein 9, BOG, cell cycle, cancer, fluorescence polarization, fluorophosphonate rhodamine, FPRh, antagonist, inhibitor, primary, counterscreen, HTS, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that act as inhibitors of the Fam 108B serine hydrolase. This assay also serves as a primary counterscreen for a previous set of experiments entitled, "Primary biochemical high throughput screening assay to identify inhibitors of Retinoblastoma binding protein 9 (RBBP9)" (AID 1515). In this biochemical assay, recombinant Fam108B protein is incubated with test compounds and FP-Rh probe. 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 Fam 108B inhibitors will prevent Fam 108B-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds were tested in singlicate at a final nominal concentration of 9.51 micromolar.
Prior to the start of the assay, 4.0 microliters of Assay Buffer (0.01% Pluronic acid, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT) containing 0.625 micromolar of Fam108B protein were dispensed into 1536 microtiter plates.
Catalytically dead Fam108B (S170A) protein (0.625 micromolar) in Assay Buffer was dispensed to positive control wells. Next, 48 nL of test compound in DMSO or DMSO alone (0.0.951% 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 microliter of 375 nM FP-Rh probe in Assay Buffer to all wells. Plates were centrifuged and after 20 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:
100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Low_Control is defined as the median value of all test compound wells.
Test_Compound is defined as wells containing Fam 108B in the presence of test compound.
High_Control is defined as wells containing catalytically dead Fam 108B (S170A) protein in the presence of DMSO.
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 reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The activity score range for active compounds is 100-11, for inactive 11-0.
List of Reagents:
Recombinant Fam108B (supplied by Assay Provider)
Recombinant Fam108B (S170A) (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)
DTT (Invitrogen, part 15508-013)
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 campaigns 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)