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BioAssay: AID 588575

SAR analysis of selective Bcl-B inhibitors using fluorescence polarization assay

Bcl-B is an anti-apoptotic member of the Bcl-2 family that is prominently expressed in plasma and multiple myeloma cells. Prior experiments have suggested that Bcl-B is important for the survival of plasma cell malignancies [Luciano et al., 2007] and several types of solid tumors show pathological elevation of Bcl-B protein, sometimes correlating with poor prognosis [Kajewska et al., 2008]. A specific Bcl-B inhibitor would be useful as a probe for defining the mechanism by which Bcl-B acts and also potentially as a lead compound for therapy. ..more
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 Tested Compounds
 Tested Compounds
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Active(16)
 
 
Inactive(42)
 
 
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 Tested Substances
All(58)
 
 
Active(16)
 
 
Inactive(42)
 
 
AID: 588575
Data Source: Burnham Center for Chemical Genomics (SBCCG-A733-BCL-B-Inhibitor-DryPowder-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-10-20
Hold-until Date: 2012-04-30
Modify Date: 2012-04-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 16
Related Experiments
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AIDNameTypeProbeComment
1693Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions via Bim (BCL2-like 11)Summary1 depositor-specified cross reference
504627Bcl-2 family members Fluorescence polarization assay with Set1 of powder compoundsOther depositor-specified cross reference
432HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1Confirmatory same project related to Summary assay
621TR-FRET secondary assay for HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1Confirmatory same project related to Summary assay
748High Throughput Fluorescence Polarization Screen for Bcl-B Phenotype ConvertersConfirmatory same project related to Summary assay
950Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2.Screening same project related to Summary assay
951Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-B.Screening same project related to Summary assay
952Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-W.Screening same project related to Summary assay
1007Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XL.Screening same project related to Summary assay
1008Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1Screening same project related to Summary assay
1009Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1Screening same project related to Summary assay
1320Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1.Confirmatory same project related to Summary assay
1322Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XL.Confirmatory same project related to Summary assay
1324Profiling Assay to determine GST-GSH interactions in multiplex bead-based assays (HPSMTB buffer)Confirmatory same project related to Summary assay
1327Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-B protein.Confirmatory same project related to Summary assay
1328Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2Confirmatory same project related to Summary assay
1329Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1.Confirmatory same project related to Summary assay
1330Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-W.Confirmatory same project related to Summary assay
1776Profiling compound fluorescence on GSH Beads with 488 nm excitation and 530 nm emissionOther same project related to Summary assay
2075Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2Confirmatory same project related to Summary assay
2077Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-BConfirmatory same project related to Summary assay
2080Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1Confirmatory same project related to Summary assay
2081Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-WConfirmatory same project related to Summary assay
2084Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XLConfirmatory same project related to Summary assay
2086Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1Confirmatory same project related to Summary assay
504598Dose response of powder sourced compounds for small molecule regulators of Bcl-2 family protein interactions, panel upload with wildtype and mutant Bfl1 and wildtype BclB.Other same project related to Summary assay
588578SAR analysis of selective Bcl-B inhibitors using a Fluorescence Polarization Bcl-XL/Bim-BH3 AssayConfirmatory same project related to Summary assay
588716Isothermal titration calorimetry (ITC) with Bcl-B and compounds active in primary screenConfirmatory same project related to Summary assay
720677SAR analysis of selective Bcl-B inhibitors using fluorescence polarization assay, set 2Confirmatory same project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network(MLPCN)
Grant Number: 1X01 MH079850-01
Assay Provider: John C. Reed, Sanford-Burnham Medical Research Institute, San Diego, CA

Bcl-B is an anti-apoptotic member of the Bcl-2 family that is prominently expressed in plasma and multiple myeloma cells. Prior experiments have suggested that Bcl-B is important for the survival of plasma cell malignancies [Luciano et al., 2007] and several types of solid tumors show pathological elevation of Bcl-B protein, sometimes correlating with poor prognosis [Kajewska et al., 2008]. A specific Bcl-B inhibitor would be useful as a probe for defining the mechanism by which Bcl-B acts and also potentially as a lead compound for therapy.

As many other Bcl-2 family members, Bcl-B contains a BH3-binding site. This site is responsible for interaction with other Bcl-2 family members, such as Bim (BCL2L11), a member of the pro-apoptotic Bcl-2 family. This interaction forms the basis for the current fluorescence polarization assay. This assay performed in the laboratory of the assay provider is to study the structure-activity relationship on analogs of the confirmed hits reported in AID 504627. Compounds are either acquired from commercial sources or synthesized internally.

Krajewska, M.; Kitada, S.; Winter, J. N.; Variakojis, D.; Lichtenstein, A.; Zhai, D.; Cuddy, M.; Huang, X.; Luciano, F.; Baker, C. H.; Kim, H.; Shin, E.; Kennedy, S.; Olson, A. H.; Badzio, A.; Jassem, J.; Meinhold- Heerlein, I.; Duffy, M. J.; Schimmer, A. D.; Tsao, M.; Brown, E.; Sawyers, A.; Andreeff, M.; Mercola, D.; Krajewski, S.; Reed, J. C. Bcl-B Expression in Human Epithelial and Nonepithelial Malignancies. Clin. Cancer Res. 2008, 14, 3011-3021.

Luciano, F.; Krajewska, M.; Ortiz-Rubio, P.; Krajewski, S.; Zhai, D.; Faustin, B.; Bruey, J. M.; Bailly-Maitre, B.; Lichtenstein, A.; Kolluri, S. K.; Satterthwait, A. C.; Zhang, X. K.; Reed, J. C. Nur77 Converts Phenotype of Bcl- B, an Antiapoptotic Protein Expressed in Plasma Cells and Myeloma. Blood 2007, 109, 3849-3855
Protocol
Materials:
1. GST-Bcl-B was expressed in E. coli & purified at the Prof. John Reed's laboratory (SBMRI)
a. Bcl-B was a GST-fusion protein lacking C-terminal transmembrane domains (~20 amino acids)
b. FITC-Bim (FITC-Ahx- DMRPEIWIAQELRRIGDEFNAYYAR peptide); Ahx = N-aminohexanoic acid linker; X = N-aminocaproic acid;
2. Assay Buffer: 25 mM HEPES-KOH, 0.005% Tween-20, pH 7.5, 1 mM TCEP.
3. 40 nM GST-Bcl-2 working solution in assay buffer, freshly prepared and kept on ice prior to use.
4. 20 nM : FITC-Bim peptide working solution in assay buffer
Assay Protocol:
1. Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration.
2. 2 uL compounds in 10% DMSO were transferred into columns 5-24 of Greiner 384-well black small-volume plates (784076).
3. Columns 1-2 and 3-4 contained 4 uL of 10% DMSO.
4. Columns 1-2 were reserved for positive controls and 9 uL of assay buffer were added to them using WellMate bulk dispenser (Matrix).
5. 9 uL of Bcl-B working solution was added to columns 2-24 using WellMate bulk dispenser (Matrix). Columns 3-4 represent negative control wells.
6. Plates were incubated for 20 min at 4 degree Celsius.
7. 9 uL of freshly prepared FITC-Bim working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
8. Plates were incubated for 10 min at room temperature protected from direct light.
9. Fluorescence polarization was measured on an Analyst HT plate reader (Molecular Devices, Inc) using fluorescein optics (ex/em - 485/530 nm, dichroic mirror - 505 nm). The signal for each well was acquired for 100 ms.
10. Data analysis was performed using CBIS software (ChemInnovations, Inc) using sigmoidal dose-response equation through non-linear regression.
Comment
Compounds with an IC50 <= 20 uM are defined as actives in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in this assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If the qualifier is ""="", the IC50 result equals the value in that column. If the qualifier is "">"", the IC50 result is greater than that value. If the qualifier is ""<"", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using a sigmoidal dose response equationFloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1IC50 value determined using a sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1Standard Error of the IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% Activity at 20 uM_first_point (20μM**)% Activity at the test concentrationFloat%
9% Activity at 10 uM_first_point (10μM**)% Activity at the test concentrationFloat%
10% Activity at 5 uM_first_point (5μM**)% Activity at the test concentrationFloat%
11% Activity at 2.5 uM_first_point (2.5μM**)% Activity at the test concentrationFloat%
12% Activity at 1.25 uM_first_point (1.25μM**)% Activity at the test concentrationFloat%
13% Activity at 0.625 uM_first_point (0.625μM**)% Activity at the test concentrationFloat%
14% Activity at 0.3125 uM_first_point (0.3125μM**)% Activity at the test concentrationFloat%
15% Activity at 0.15625 uM_first_point (0.15625μM**)% Activity at the test concentrationFloat%
16% Activity at 0.078125 uM_first_point (0.078125μM**)% Activity at the test concentrationFloat%
17% Activity at 0.0390625 uM_first_point (0.0390625μM**)% Activity at the test concentrationFloat%
18Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
19% Activity at 20 uM_second_point (20μM**)% Activity at the test concentrationFloat%
20% Activity at 10 uM_second_point (10μM**)% Activity at the test concentrationFloat%
21% Activity at 5 uM_second_point (5μM**)% Activity at the test concentrationFloat%
22% Activity at 2.5 uM_second_point (2.5μM**)% Activity at the test concentrationFloat%
23% Activity at 1.25 uM_second_point (1.25μM**)% Activity at the test concentrationFloat%
24% Activity at 0.625 uM_second_point (0.625μM**)% Activity at the test concentrationFloat%
25% Activity at 0.3125 uM_second_point (0.3125μM**)% Activity at the test concentrationFloat%
26% Activity at 0.15625 uM_second_point (0.15625μM**)% Activity at the test concentrationFloat%
27% Activity at 0.078125 uM_second_point (0.078125μM**)% Activity at the test concentrationFloat%
28% Activity at 0.0390625 uM_second_point (0.0390625μM**)% Activity at the test concentrationFloat%
29IC50_Qualifier_2This qualifier is to be used with the next TID, IC50_2. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
30IC50_2IC50 value determined using a sigmoidal dose response equationFloatμM
31Std.Err(IC50)_2Standard Error of the IC50 valueFloatμM
32nH_2Hill coefficient determined using sigmoidal dose response equationFloat
33Excluded_Points_third_pointFlags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
34% Activity at 20 uM_third_point (20μM**)% Activity at the test concentrationFloat%
35% Activity at 10 uM_third_point (10μM**)% Activity at the test concentrationFloat%
36% Activity at 5 uM_third_point (5μM**)% Activity at the test concentrationFloat%
37% Activity at 2.5 uM_third_point (2.5μM**)% Activity at the test concentrationFloat%
38% Activity at 1.25 uM_third_point (1.25μM**)% Activity at the test concentrationFloat%
39% Activity at 0.625 uM_third_point (0.625μM**)% Activity at the test concentrationFloat%
40% Activity at 0.3125 uM_third_point (0.3125μM**)% Activity at the test concentrationFloat%
41% Activity at 0.15625 uM_third_point (0.15625μM**)% Activity at the test concentrationFloat%
42% Activity at 0.078125 uM_third_point (0.078125μM**)% Activity at the test concentrationFloat%
43% Activity at 0.0390625 uM_third_point (0.0390625μM**)% Activity at the test concentrationFloat%
44Excluded_Points_fourth_pointFlags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
45% Activity at 20 uM_fourth_point (20μM**)% Activity at the test concentrationFloat%
46% Activity at 10 uM_fourth_point (10μM**)% Activity at the test concentrationFloat%
47% Activity at 5 uM_fourth_point (5μM**)% Activity at the test concentrationFloat%
48% Activity at 2.5 uM_fourth_point (2.5μM**)% Activity at the test concentrationFloat%
49% Activity at 1.25 uM_fourth_point (1.25μM**)% Activity at the test concentrationFloat%
50% Activity at 0.625 uM_fourth_point (0.625μM**)% Activity at the test concentrationFloat%
51% Activity at 0.3125 uM_fourth_point (0.3125μM**)% Activity at the test concentrationFloat%
52% Activity at 0.15625 uM_fourth_point (0.15625μM**)% Activity at the test concentrationFloat%
53% Activity at 0.078125 uM_fourth_point (0.078125μM**)% Activity at the test concentrationFloat%
54% Activity at 0.0390625 uM_fourth_point (0.0390625μM**)% Activity at the test concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1X01 MH079850-01

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