Bookmark and Share
BioAssay: AID 432

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
_
   
 Tested Compounds
 Tested Compounds
All(64387)
 
 
Active(17)
 
 
Inactive(64370)
 
 
 Tested Substances
 Tested Substances
All(64394)
 
 
Active(17)
 
 
Inactive(64377)
 
 
AID: 432
Data Source: Burnham Center for Chemical Genomics (SDCCG-A005-Bfl1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-06-16
Modify Date: 2010-08-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 17
Related Experiments
Show more
AIDNameTypeProbeComment
621TR-FRET secondary assay for HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1Confirmatory depositor-specified cross reference
748High Throughput Fluorescence Polarization Screen for Bcl-B Phenotype ConvertersConfirmatory 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
1240Fluorescence Polarization Screen Assay for Bcl-B Phenotype ConvertersScreening depositor-specified cross reference
1243Fluorescence Polarization Dose Response Assay for TR3-Based Bcl-B InhibitorsConfirmatory depositor-specified cross reference
1244Counter Screen for TR3-Based Bcl-B Inhibitors: Fluorescence Polarization Bcl-B/FITC-Bim-BH3 AssayConfirmatory depositor-specified cross reference
1245Counter Screen for TR3-Based Bcl-B Inhibitors: Fluorescence Polarization Bcl-B/FITC-Puma-BH3 AssayConfirmatory 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
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:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Number: XO1 MH077632-01
Assay Provider: Dr. John C. Reed, Sanford-Sanford-Burnham Medical Research Institute


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.

This work's aim is to identify chemical probes of Bfl-1 through a fluorescence polarization assay (FPA) using FITC-Bid BH3 peptide.
Protocol
Bfl-1 assay materials:
1)Bfl-1 protein and FITC-Bid peptide (FITC-Ahx-EDIIRNIARHLAQVGDSMDR ) were obtained from Prof. John Reed (Sanford-Sanford-Burnham Medical Research Institute, San Diego, CA)
2)Assay Buffer: 25 mM Bis-Tris, pH 7.0, 1 mM TCEP, 0.005% Tween 20.
3)Bfl-1 working solution contained 7.4 nM Bfl-1 in assay buffer. Solution was prepared fresh and kept on ice prior to use.
4)FITC-Bid working solution contained 5.6 nM FITC-Bid in assay buffer.

Bfl-1 HTS protocol:
1)4 uL of 100 uM compounds in 10% DMSO were dispensed in columns 3-24 of Greiner 384-well black small-volume plates (784076). Columns 1 and 2 were added with 4 uL of 10% DMSO.
2)Positive control wells, that contained no Bfl-1, were assigned to column 1 and were added 8 uL of assay buffer using WellMate bulk dispenser (Matrix).
3)8 uL of Bfl-1 working solution was added to columns 2-24 using WellMate bulk dispenser (Matrix). Negative control wells that contained no compounds were assigned to column 2.
4)Plates were incubated for 1h at +4C.
5)8 uL of freshly prepared FITC-Bid working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
6)Final concentrations of the components in the assay were as follows:
a.20 mM Bis-Tris-HCl, pH 7.0, 0.8 mM TCEP, 0.004% Tween 20.
b.2.2 nM FITC-Bid (columns 1-24)
c.3.0 nM Bfl-1 (columns 2-24)
d.2 % DMSO (columns 1-24)
e.20 uM compounds (columns 3-24)
7)Plates were incubated for 4h at room temperature protected from direct light.
8)Fluorescence polarization was measured on an Analyst HT plate reader (Molecular Devices, Inc) using fluorescein filters: excitation filter - 485 nM, emission filter 530 nM, dichroic mirror # 505 nM. The signal for each well was acquired for 100 ms.
9)Data analysis was performed using CBIS software (ChemInnovations, Inc).
10)Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.

Bfl-1 secondary 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 compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well black small-volume plates (784076). Columns 1-2 and 23-24 contained 4 uL of 10% DMSO.
2)Columns 1-2 were reserved for positive controls and added with 8 uL of assay buffer using WellMate bulk dispenser (Matrix).
3)8 uL of Bfl-1 working solution was added to columns 2-24 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 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 polarization was measured on an Analyst HT plate reader (Molecular Devices, Inc) using fluorescein filters: excitation filter - 485 nM, emission filter # 530 nM, dichroic mirror # 505 nM. The signal for each well was acquired for 100 ms.
8)Data analysis was performed using sigmoidal dose-response equation through non-linear regression.
Comment
Compounds with greater than 50% displacement of FITC-Bid in the Bfl-1 assay at 20 uM concentration and F_ratio parameter less than 1.5 are defined as actives of the primary screening.

The compounds identified as primary screening actives proceed to the dose-response confirmation stage. Concentrations of compounds that demonstrate a total fluorescence increase above the threshold, F_ratio equal 1.5, are not included in the data analysis. Compounds that demonstrate IC50 values in the range of analyzed concentrations remain "active" in the outcome column. All active compounds demonstrated % displacement at around 80% in the assay. This somewhat lower than expected efficacy, might be speculated to originate from there being no positive control compound. The currently used positive control samples, with no Bfl-1 protein added, may provide an overestimation of the maximal efficacy, since, for example, they would not account for residual non-specific binding of the peptide if it is present.

Compounds that failed dose-response confirmation are downgraded to "inactive" in the outcome field.

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 Bfl-1 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 Bfl-1 assay is as follows:
1)First tier (0-40 range) is reserved for primary screening data-the score is correlated with % displacement in the assay demonstrated by a compound at 20 uM concentration:
a. If primary % displacement is less than 0%, then the assigned score is 0
b. If primary % displacement is greater than 100%, then the assigned score is 40/(1+(F_ratio - 1)^2)
c. If primary % displacement is between 0% and 100%, then the calculated score is (% Displacement)*0.4/(1+(F_ratio-1)^2)
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.Potential interference of fluorescent compounds is factored in via an IM scaling factor that takes into consideration the difference between a compound's potency (IC50) and the lowest concentration of the compound's (FC50) with F_ratio >= 1.5:
IM = 1/([F*IC50/FC50]^F + 1), where F = 2.5 + pIC50/5
This empirical equation results in zero values for fluorescent compounds for which the dose-response behavior is a simple consequence of fluorescence polarization signal interference. For compounds with fluorescence that is less likely to result in signal interference, the value of IM asymptotically approaches unity. IM is equal to 1 for the compounds that show no fluorescence interference (FC50 > maximal concentration tested).
d.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) #C exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target- or pathway-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 the Bfl-1 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 all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC*IM,
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 50 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.
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
Show more
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
5%displacement at 20 uM% displacement of FITC-Bid relative to the controlsFloat
6F_ratioFluorescence intensity normalized to the average fluorescence intensity value of the plate negative contirolsFloat
7Mean HighMean fluorescence polarization signal (ex/em: 485/530) of negative controls in the corresponding plateFloatMP
8STD Deviation HighStandard deviation (n=16) of fluorescence polarization of negative controls in the corresponding plateFloatMP
9Mean LowMean fluorescence polarization signal (ex/em:485/530) of positive controls in the corresponding plateFloatMP
10STD Deviation LowStandard deviation (n=16) of fluorescence polarization of positive controls in the corresponding plateFloatM P

* Activity Concentration.

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