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BioAssay: AID 621

TR-FRET secondary assay for HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1

Bfl-1, also known as A1 in mice is an anti-apoptotic and NF-kB-inducible member of the Bcl-2 protein family involved in regulation of apoptosis. Due to difficulties with accomplishing targeted gene ablation in mouse models, the endogenous functions of Bfl-1 are largely unknown. Chemical inhibitors of Bfl-1 can be used as research tools for neutralizing Bfl-1 in human and mouse cells. ..more
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 Tested Compounds
 Tested Compounds
All(175)
 
 
Active(82)
 
 
Inactive(95)
 
 
 Tested Substances
 Tested Substances
All(185)
 
 
Active(89)
 
 
Inactive(96)
 
 
AID: 621
Data Source: Burnham Center for Chemical Genomics (SDCCG-A019-Bfl1-TRFRET)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-03-21
Modify Date: 2010-10-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 82
Related Experiments
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AIDNameTypeProbeComment
432HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1Confirmatory depositor-specified cross reference
950Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2.Screening depositor-specified cross reference
951Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-B.Screening depositor-specified cross reference
952Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-W.Screening depositor-specified cross reference
1007Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XL.Screening depositor-specified cross reference
1008Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1Screening depositor-specified cross reference
1009Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1Screening depositor-specified cross reference
1070In Vitro Bfl-1 Dose Response Fluorescence Polarization Assay for SAR StudyConfirmatory depositor-specified cross reference
1231SAR Assays for Anti-Apoptotic Protein Bfl-1 - Summary of the Chloromaleimide SeriesSummary depositor-specified cross reference
1320Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1.Confirmatory depositor-specified cross reference
1322Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XL.Confirmatory depositor-specified cross reference
1327Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-B protein.Confirmatory depositor-specified cross reference
1328Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2Confirmatory depositor-specified cross reference
1329Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1.Confirmatory depositor-specified cross reference
1330Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-W.Confirmatory depositor-specified cross reference
1693Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions via Bim (BCL2-like 11)Summary1 depositor-specified cross reference
2075Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2Confirmatory depositor-specified cross reference
2077Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-BConfirmatory depositor-specified cross reference
2080Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1Confirmatory depositor-specified cross reference
2081Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-WConfirmatory depositor-specified cross reference
2084Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XLConfirmatory depositor-specified cross reference
2086Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1Confirmatory depositor-specified cross reference
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 depositor-specified cross reference
748High Throughput Fluorescence Polarization Screen for Bcl-B Phenotype ConvertersConfirmatory 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
1776Profiling compound fluorescence on GSH Beads with 488 nm excitation and 530 nm emissionOther same project related to Summary assay
504627Bcl-2 family members Fluorescence polarization assay with Set1 of powder compoundsOther same project related to Summary assay
588575SAR analysis of selective Bcl-B inhibitors using fluorescence polarization assayConfirmatory 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:
Sanford-Burnham Center for Chemical Genomics (SBCCG)
Sanford-Burnham Medical Research Institute (San Diego, CA)
NIH Molecular Libraries Screening Centers Network (MLSCN)

Bfl-1, also known as A1 in mice is an anti-apoptotic and NF-kB-inducible member of the Bcl-2 protein family involved in regulation of apoptosis. Due to difficulties with accomplishing targeted gene ablation in mouse models, the endogenous functions of Bfl-1 are largely unknown. Chemical inhibitors of Bfl-1 can be used as research tools for neutralizing Bfl-1 in human and mouse cells.

The current assay was developed at the Sanford-Burnham Center for Chemical Genomics (SBCCG), based on FITC-Bid BH3 peptide binding to GST-Bfl-1 in the presence of Terbium-labeled anti-GST antibody. The assay is aimed to support Bfl-1 chemical probe identification through the TR-FRET assay confirmation of the results obtained in the primary fluorescence polarization (FP) assay (PubChem AID 432) performed at the BCCG.

Bfl-1 screening was performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN). XO1 submission, MH077632-01, Chemical Inhibitors of anti-apoptotic protein Bfl-1, Assay Provider Dr. John C. Read, Sanford-Burnham Medical Research Institute, San Diego, CA
Protocol
Bfl-1 assay materials:
1)Bfl-1 protein and FITC-Bid peptide (FITC-Ahx-EDIIRNIARHLAQVGDSMDR ) were obtained from Prof. John Reed (Sanford-Burnham Medical Research Institute, San Diego, CA)
2)Tb-anti-GST Antibody (Invitrogen, PV4216)
3)Assay Buffer: 25 mM Bis-Tris, pH 7.0, 1 mM TCEP, 0.005% Tween 20.
4)Bfl-1 working solution contained 7.4 nM GST-Bfl-1 in assay buffer
5)FITC-Bid working solution contained 5.6 nM FITC-Bid in assay buffer
6)FITC-Bid/ Tb-Ab working solution contained 5.6 nM FITC-Bid and 2.5 nM Tb-Ab in assay buffer. Solution was prepared fresh and kept on ice prior to use.

Bfl-1 dose-response screening 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. 4 uL of compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well white small-volume plates (784075). Each compound concentration was assayed in duplicate wells. Columns 1-2 and 23-24 contained 4 uL of 10% DMSO.
2)8 uL of assay buffer were added to columns 1-2, which were reserved for positive controls, using a WellMate bulk dispenser (Matrix).
3)8 uL of Bfl-1 working solution was added to columns 3-24 using WellMate bulk dispenser (Matrix). Columns 23-24 represent negative control wells.
4)Plates were incubated for 1h at +4oC.
5)8 uL of freshly prepared FITC-Bid/Tb-Ab working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
6)Plates were incubated for 4h at room temperature protected from direct light.
7)Fluorescence was measured on an M5 plate reader, Molecular Devices (excitation: 340 nm, emission: 490 and 520 nm, cutoff: 475 and 515 nm, respectively) in Time Resolved (TR) mode with signal integrated for 1 ms after initial delay 0.1 ms and averaged from 5 readings. The TR-FRET signal was calculated as the ratio of TR-Fluorescence at 520 nm to TR-Fluorescence at 490 nm.
8)Data analysis was performed using a sigmoidal dose-response equation through non-linear regression.
Comment
Compounds interfering with an assay often appear as positives in screening, thus resulting in false positives. False positives, e.g., artifacts are frequently assay-specific. Therefore, reconfirmation of the positive compounds with an independent assay provides an easy way to separate the true positives hits from assay-specific hits.

The compounds identified as primary screening actives in the Bfl-1 fluorescence polarization assay proceed to the dose-response confirmation stage in both FP and TR-FRET assays performed in parallel from the same compound dilution plate. For the purposes of the TR-FRET assay, compounds that demonstrate IC50 values in the range of analyzed concentrations in the assay are defined as 'active' in the outcome column.

Compounds that failed dose-response confirmation are defined as 'inactive' in the outcome field.

To take the advantage of the results of the two assays available to us, a special activity ranking system was implemented. This system takes into account IC50 values in both assays to provide clear differentiation between the compounds that are active in a single vs. both assays. Details of the scoring system applied to the Bfl-1 project is described below.

Activity Scoring

Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics were devised to take into consideration compound efficacy, the screening stage of the data and the correlation of the results between primary and secondary assays. Details of the Scoring System will be published elsewhere.

Briefly, the outline of the scoring system utilized for the Bfl-1 TR-FRET assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and therefore is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound#s 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 a target- or a pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on the expectation that a compound with a single mode of action that achieved an equilibrium in the assay would demonstrate the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. The score is correlated with the compound potency in both the FP and the TR-FRET assays through the following function that takes into account the information obtained in both assays:
Score=44+3*(pIC50(FP)-3)*QC(FP)*IM(FP)+3*(pIC50(TR-FRET)-3)*QC(TR-FRET),
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units, IM is calculated as described in PubChem AID 432, and indexes (FP) and (TR-FRET) refer to the primary and the secondary assays, respectively. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior in both assays. Compounds that are inactive in either of the assays or whose concentration-dependent behavior is likely to be an artifact of their behavior in the assay will generally have lower score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
2IC50*IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat
5Mean HighMean TR-FRET signals of negative controls in the corresponding plateFloat
6STD Deviation HighStandard deviation (n=16) of TR-FRET signals of negative controls in the corresponding plateFloat
7Mean LowMean TR-FRET signals of negative controls of positive controls in the corresponding plateFloat
8STD Deviation LowStandard deviation (n=16) of TR-FRET signals of positive controls in the corresponding plateFloat

* Activity Concentration.

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