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BioAssay: AID 504627

Bcl-2 family members Fluorescence polarization assay with Set1 of powder compounds

Apoptosis is regulated in part by the balance of anti-apoptotic and pro-apoptotic Bcl-2 family members. In humans, six genes have been identified that encode anti-apoptotic proteins characterized by the presence of three conserved motifs designated as Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2000]. These domains form a hydrophobic cleft in the tertiary structure. Pro-apoptotic more ..
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 Tested Compounds
 Tested Compounds
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Unspecified(10)
 
 
 Tested Substances
 Tested Substances
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Unspecified(10)
 
 
AID: 504627
Data Source: NMMLSC (UNMCMD_SBMRI_BCL_SECONDARY_FP_SET1)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-31

Data Table ( Complete ):           View All Data
Tested Compounds:
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: Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interaction
588575SAR analysis of selective Bcl-B inhibitors using fluorescence polarization assayConfirmatory depositor-specified cross reference
588578SAR analysis of selective Bcl-B inhibitors using a Fluorescence Polarization Bcl-XL/Bim-BH3 AssayConfirmatory 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
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:
University of New Mexico Assay Overview:
Assay Support: 1X01 MH079850-01
Project Title: HTS for BCI-2 Family Multiplex
Assay Provider: Larry Sklar/John C. Reed
UNMScreening Center/ PI: UNMCMD/ Larry Sklar
Lead Biologist at Screening Center: Peter Simons PhD
Chemistry Center/ PI: Sanford-Burnham Center for Chemical Genomics / John C. Reed
Chemistry Center Lead: Thomas Chung Ph.D., Robert Ardecky Ph.D., Anton Cheltsov Ph.D.
Assay Implementation: Daiyong Zhai Ph.D.

Assay Background and Significance:

Apoptosis is regulated in part by the balance of anti-apoptotic and pro-apoptotic Bcl-2 family members. In humans, six genes have been identified that encode anti-apoptotic proteins characterized by the presence of three conserved motifs designated as Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2000]. These domains form a hydrophobic cleft in the tertiary structure. Pro-apoptotic family members contain BH3 regions that form an amphipathic helix, and these helices bind in the clefts of the anti-apoptotic proteins. The binding of fluorochrome-conjugated BH3 peptides (including Bim) to Bcl-2 family members thus provides the basis for construction of fluorescence-based assays amenable to flow cytometry high throughput screening for small molecule regulators of these interactions (Zhai et al 2006). The high throughput screen was a multiplexed assay to identify small molecule regulators of protein interactions between the BH3 peptide of Bim and the following six Bcl-2 family members: Bcl-XL, Bcl-W, Bcl-B, Bfl-1, and Mcl-1 and Bcl-2 (the eponymous founding member of the Bcl-2 family). A selective probe candidate scaffold has now emerged as an inhibitor of the interaction of Bcl-B with BIM peptide.

Overall, the screen identified three compounds as specific inhibitors of Bcl-B but not the other Bcl-2 family members; fluorescence polarization and isothermal titration calorimetry data corroborated the flow cytometry findings. 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.
Panel Information
Panel set1 of fluorescence polarization data for Bcl2 family proteins - Panel set1 of fluorescence polarization data for Bcl2 family proteins
    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1BclXL19bcl-2-like protein 1 isoform 1 [Homo sapiens] [gi:20336335]
bcl-xL (bcl-2-like protein 1 isoform 1) [Homo sapiens]Taxonomy id: 9606
2BclB46BCL2-like 10 (apoptosis facilitator) [Homo sapiens] [gi:62739542]
Apoptosis regulator Bcl-B (Bcl-2-like 10 protein) (Bcl2-L-10) (Anti-apoptotic protein NrH) [Homo Sapiens]Taxonomy id: 9606
3Bcl219apoptosis regulator Bcl-2 alpha isoform [Homo sapiens] [gi:72198189]
Apoptosis regulator Bcl-2 alpha isoform [Homo sapiens]Taxonomy id: 9606
4Bfl119bcl-2-related protein A1 isoform 1 [Homo sapiens] [gi:4757840]
BCL2-related protein A1 [Homo Sapiens]Taxonomy id: 9606

§ Panel component ID.
Protocol
Materials:
1. GST-Bcl-2 family proteins are all expressed in E. coli & purified at the Prof. John Reed's laboratory (SBMRI)
a. Bcl-2 family proteins are GST-fusion protein lacking C-terminal transmembrane domains (~20 amino acids)
b. FITC-TR3-R8 (FITC-Ahx-FSRSLHSLL-GX-R8 peptide); Ahx = N-aminohexanoic acid linker; X = N-aminocaproic acid; R8 = 8 residues of D-arginine
2. Assay Buffer: 25 mM HEPES-KOH,20 mM N-glycerophosphate, 0.005% Tween-20, pH 7.5, 1 mM TCEP.
3. 44.4 nM GST-Bcl-2 working solution in assay buffer, freshly prepared and kept on ice prior to use.
4. 44.4 nM : FITC-TR3-R8 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 3-22 of Greiner 384-well black small-volume plates (784076).
3. Columns 1-2 and 23-24 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 23-24 represent negative control wells.
6. Plates were incubated for 1h at 4 degree Celsius.
7. 9 uL of freshly prepared FITC-TR3 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 sigmoidal dose-response equation through non-linear regression using GraphPad Prism (GraphPad Software, San Diego, California). Resulting in estimates of IC50 values.
Active Compounds have a measured fluorescence difference greater than 25. Individual panel member scores were calculated by the following equation:
Individual Score = 100 *(1-IC50/10)
And Total Score was a summation of all the individual scores.
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1BclXL_OUTCOMEBclXL PubChem Outcome1bcl-2-like protein 1 isoform 1 [Homo sapiens]Outcome
2BclXL_SCOREBclXL PubChem Score1Integer
3BclXL_ACTIVITY_QUALIFIERQualifier for the value of IC501String
4BclXL_IC50_MICROMEffective concentration of half maximal inhibition as estimated by curve fit for BclXL1FloatμM
5BclXL_IC50_95CI_LOWLower 95% confidence interval boundary for the IC50 curve fit estimate for BclXL1FloatμM
6BclXL_IC50_95CI_HIGHUpper 95% confidence interval boundary for the IC50 curve fit estimate for BclXL1FloatμM
7BclXL_BOTTOMResponse value at the bottom plateau for BclXL1Float
8BclXL_TOPResponse value at the top plateau for BclXL1Float
9BclXL_HILLSLOPEHillslope estimate for the fitted dose response curve for BclXL1Float
10BclXL_STD_BOTTOMStandard error for the response value at the bottom plateau for BclXL1Float
11BclXL_STD_TOPStandard error for the response value at the top plateau for BclXL1Float
12BclXL_STD_HILLSLOPEStandard error for the Hillslope for BclXL1Float
13BclXL_SPANSpan of dose response calculated from data for BclXL1Float
14BclXL_RSQRCorrelation coefficient for the fitted dose response curve for BclXL1Float
15BclXL_N_POINTSNumber of data points for each dose response curve for BclXL1Integer
16BclXL_RESPONSE_0_MICROM (0μM**)Response with 0 micromolar concentration of test compound for BclXL1Float
17BclXL_RESPONSE_0.1_MICROM (0.1μM**)Response with 0.1 micromolar concentration of test compound for BclXL1Float
18BclXL_RESPONSE_0.2_MICROM (0.2μM**)Response with 0.2 micromolar concentration of test compound for BclXL1Float
19BclXL_RESPONSE_0.39_MICROM (0.39μM**)Response with 0.39 micromolar concentration of test compound for BclXL1Float
20BclXL_RESPONSE_0.78_MICROM (0.78μM**)Response with 0.78 micromolar concentration of test compound for BclXL1Float
21BclXL_RESPONSE_1.56_MICROM (1.56μM**)Response with 1.56 micromolar concentration of test compound for BclXL1Float
22BclXL_RESPONSE_3.12_MICROM (3.12μM**)Response with 3.12 micromolar concentration of test compound for BclXL1Float
23BclXL_RESPONSE_6.25_MICROM (6.25μM**)Response with 6.25 micromolar concentration of test compound for BclXL1Float
24BclXL_RESPONSE_12.5_MICROM (12.5μM**)Response with 12.5 micromolar concentration of test compound for BclXL1Float
25BclXL_RESPONSE_25_MICROM (25μM**)Response with 25 micromolar concentration of test compound for BclXL1Float
26BclXL_RESPONSE_50_MICROM (50μM**)Response with 50 micromolar concentration of test compound for BclXL1Float
27BclB_OUTCOMEBclB PubChem Outcome2BCL2-like 10 (apoptosis facilitator) [Homo sapiens]Outcome
28BclB_SCOREBclB PubChem Score2Integer
29BclB_ACTIVITY_QUALIFIERQualifier for the value of IC502String
30BclB_IC50_MICROMEffective concentration of half maximal inhibition as estimated by curve fit for BclB2FloatμM
31BclB_IC50_95CI_LOWLower 95% confidence interval boundary for the IC50 curve fit estimate for BclB2FloatμM
32BclB_IC50_95CI_HIGHUpper 95% confidence interval boundary for the IC50 curve fit estimate for BclB2FloatμM
33BclB_BOTTOMResponse value at the bottom plateau for BclB2Float
34BclB_TOPResponse value at the top plateau for BclB2Float
35BclB_HILLSLOPEHillslope estimate for the fitted dose response curve for BclB2Float
36BclB_STD_BOTTOMStandard error for the response value at the bottom plateau for BclB2Float
37BclB_STD_TOPStandard error for the response value at the top plateau for BclB2Float
38BclB_STD_HILLSLOPEStandard error for the Hillslope for BclB2Float
39BclB_SPANSpan of dose response calculated from data for BclB2Float
40BclB_RSQRCorrelation coefficient for the fitted dose response curve for BclB2Float
41BclB_N_POINTSNumber of data points for each dose response curve for BclB2Integer
42BclB_RESPONSE_0_MICROM (0μM**)Response with 0 micromolar concentration of test compound for BclB2Float
43BclB_RESPONSE_0.1_MICROM (0.1μM**)Response with 0.1 micromolar concentration of test compound for BclB2Float
44BclB_RESPONSE_0.2_MICROM (0.2μM**)Response with 0.2 micromolar concentration of test compound for BclB2Float
45BclB_RESPONSE_0.39_MICROM (0.39μM**)Response with 0.39 micromolar concentration of test compound for BclB2Float
46BclB_RESPONSE_0.78_MICROM (0.78μM**)Response with 0.78 micromolar concentration of test compound for BclB2Float
47BclB_RESPONSE_1.56_MICROM (1.56μM**)Response with 1.56 micromolar concentration of test compound for BclB2Float
48BclB_RESPONSE_3.12_MICROM (3.12μM**)Response with 3.12 micromolar concentration of test compound for BclB2Float
49BclB_RESPONSE_6.25_MICROM (6.25μM**)Response with 6.25 micromolar concentration of test compound for BclB2Float
50BclB_RESPONSE_12.5_MICROM (12.5μM**)Response with 12.5 micromolar concentration of test compound for BclB2Float
51BclB_RESPONSE_25_MICROM (25μM**)Response with 25 micromolar concentration of test compound for BclB2Float
52BclB_RESPONSE_50_MICROM (50μM**)Response with 50 micromolar concentration of test compound for BclB2Float
53Bcl2_OUTCOMEBcl2 PubChem Outcome3apoptosis regulator Bcl-2 alpha isoform [Homo sapiens]Outcome
54Bcl2_SCOREBcl2 PubChem Score3Integer
55Bcl2_ACTIVITY_QUALIFIERQualifier for the value of IC503String
56Bcl2_IC50_MICROMEffective concentration of half maximal inhibition as estimated by curve fit for Bcl23FloatμM
57Bcl2_IC50_95CI_LOWLower 95% confidence interval boundary for the IC50 curve fit estimate for Bcl23FloatμM
58Bcl2_IC50_95CI_HIGHUpper 95% confidence interval boundary for the IC50 curve fit estimate for Bcl23FloatμM
59Bcl2_BOTTOMResponse value at the bottom plateau for Bcl23Float
60Bcl2_TOPResponse value at the top plateau for Bcl23Float
61Bcl2_HILLSLOPEHillslope estimate for the fitted dose response curve for Bcl23Float
62Bcl2_STD_BOTTOMStandard error for the response value at the bottom plateau for Bcl23Float
63Bcl2_STD_TOPStandard error for the response value at the top plateau for Bcl23Float
64Bcl2_STD_HILLSLOPEStandard error for the Hillslope for Bcl23Float
65Bcl2_SPANSpan of dose response calculated from data for Bcl23Float
66Bcl2_RSQRCorrelation coefficient for the fitted dose response curve for Bcl23Float
67Bcl2_N_POINTSNumber of data points for each dose response curve for Bcl23Integer
68Bcl2_RESPONSE_0_MICROM (0μM**)Response with 0 micromolar concentration of test compound for Bcl23Float
69Bcl2_RESPONSE_0.1_MICROM (0.1μM**)Response with 0.1 micromolar concentration of test compound for Bcl23Float
70Bcl2_RESPONSE_0.2_MICROM (0.2μM**)Response with 0.2 micromolar concentration of test compound for Bcl23Float
71Bcl2_RESPONSE_0.39_MICROM (0.39μM**)Response with 0.39 micromolar concentration of test compound for Bcl23Float
72Bcl2_RESPONSE_0.78_MICROM (0.78μM**)Response with 0.78 micromolar concentration of test compound for Bcl23Float
73Bcl2_RESPONSE_1.56_MICROM (1.56μM**)Response with 1.56 micromolar concentration of test compound for Bcl23Float
74Bcl2_RESPONSE_3.12_MICROM (3.12μM**)Response with 3.12 micromolar concentration of test compound for Bcl23Float
75Bcl2_RESPONSE_6.25_MICROM (6.25μM**)Response with 6.25 micromolar concentration of test compound for Bcl23Float
76Bcl2_RESPONSE_12.5_MICROM (12.5μM**)Response with 12.5 micromolar concentration of test compound for Bcl23Float
77Bcl2_RESPONSE_25_MICROM (25μM**)Response with 25 micromolar concentration of test compound for Bcl23Float
78Bcl2_RESPONSE_50_MICROM (50μM**)Response with 50 micromolar concentration of test compound for Bcl23Float
79Bfl1_OUTCOMEBfl1 PubChem Outcome4bcl-2-related protein A1 isoform 1 [Homo sapiens]Outcome
80Bfl1_SCOREBfl1 PubChem Score4Integer
81Bfl1_ACTIVITY_QUALIFIERQualifier for the value of IC504String
82Bfl1_IC50_MICROMEffective concentration of half maximal inhibition as estimated by curve fit for Bfl14FloatμM
83Bfl1_IC50_95CI_LOWLower 95% confidence interval boundary for the IC50 curve fit estimate for Bfl14FloatμM
84Bfl1_IC50_95CI_HIGHUpper 95% confidence interval boundary for the IC50 curve fit estimate for Bfl14FloatμM
85Bfl1_BOTTOMResponse value at the bottom plateau for Bfl14Float
86Bfl1_TOPResponse value at the top plateau for Bfl14Float
87Bfl1_HILLSLOPEHillslope estimate for the fitted dose response curve for Bfl14Float
88Bfl1_STD_BOTTOMStandard error for the response value at the bottom plateau for Bfl14Float
89Bfl1_STD_TOPStandard error for the response value at the top plateau for Bfl14Float
90Bfl1_STD_HILLSLOPEStandard error for the Hillslope for Bfl14Float
91Bfl1_SPANSpan of dose response calculated from data for Bfl14Float
92Bfl1_RSQRCorrelation coefficient for the fitted dose response curve for Bfl14Float
93Bfl1_N_POINTSNumber of data points for each dose response curve for Bfl14Integer
94Bfl1_RESPONSE_0_MICROM (0μM**)Response with 0 micromolar concentration of test compound for Bfl14Float
95Bfl1_RESPONSE_0.1_MICROM (0.1μM**)Response with 0.1 micromolar concentration of test compound for Bfl14Float
96Bfl1_RESPONSE_0.2_MICROM (0.2μM**)Response with 0.2 micromolar concentration of test compound for Bfl14Float
97Bfl1_RESPONSE_0.39_MICROM (0.39μM**)Response with 0.39 micromolar concentration of test compound for Bfl14Float
98Bfl1_RESPONSE_0.78_MICROM (0.78μM**)Response with 0.78 micromolar concentration of test compound for Bfl14Float
99Bfl1_RESPONSE_1.56_MICROM (1.56μM**)Response with 1.56 micromolar concentration of test compound for Bfl14Float
100Bfl1_RESPONSE_3.12_MICROM (3.12μM**)Response with 3.12 micromolar concentration of test compound for Bfl14Float
101Bfl1_RESPONSE_6.25_MICROM (6.25μM**)Response with 6.25 micromolar concentration of test compound for Bfl14Float
102Bfl1_RESPONSE_12.5_MICROM (12.5μM**)Response with 12.5 micromolar concentration of test compound for Bfl14Float
103Bfl1_RESPONSE_25_MICROM (25μM**)Response with 25 micromolar concentration of test compound for Bfl14Float
104Bfl1_RESPONSE_50_MICROM (50μM**)Response with 50 micromolar concentration of test compound for Bfl14Float

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: 1X01 MH079850-01

Classification
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