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BioAssay: AID 504598

Dose 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.

One arm of apoptosis is regulated 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 conserved motifs designated as three Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2004]. These domains form a hydrophobic cleft in tertiary structure. Pro-apoptotic more ..
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 Tested Compounds
 Tested Compounds
All(22)
 
 
Active(3)
 
 
Inactive(19)
 
 
 Tested Substances
 Tested Substances
All(22)
 
 
Active(3)
 
 
Inactive(19)
 
 
AID: 504598
Data Source: NMMLSC (UNMCMD_BCL_DOSERRESPONSE_PANEL_BCLB_BFL1_BFL1CA_POWDERCOMPOU..)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-28

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 3
Related Experiments
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AIDNameTypeProbeComment
432HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1Confirmatory depositor-specified cross reference: HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1
621TR-FRET secondary assay for HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1Confirmatory depositor-specified cross reference: TR-FRET secondary assay for HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1
748High Throughput Fluorescence Polarization Screen for Bcl-B Phenotype ConvertersConfirmatory depositor-specified cross reference: High Throughput Fluorescence Polarization Screen for Bcl-B Phenotype Converters
1008Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1Screening depositor-specified cross reference: Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interaction
1693Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions via Bim (BCL2-like 11)Summary1 depositor-specified cross reference: Summary report of Bcl-2 family protein interactions via Bim
2008Leishmania major promastigote EC50 determinationsConfirmatory depositor-specified cross reference: Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regula
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
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
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:
University of New Mexico Assay Overview:
Assay Support: NIH 1X01 MH079850-01
HTS to identify small molecule regulators of Bcl-2 family protein interactions
PI: Larry Sklar, Ph.D.
Assay Implementation: Peter Simons Ph.D, Susan Young MS, Anna Waller Ph.D, Mark Carter MS
Assay Collaborator: John Reed,Ph.D.

Dose Response Assay Background and Significance:

One arm of apoptosis is regulated 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 conserved motifs designated as three Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2004]. These domains form a hydrophobic cleft in 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. Overexpression of pro-apoptotic BH3 peptides has been shown to increase apoptosis of leukemia cells using in vitro and animal model studies [Holinger, et al. 1999; Wang et al. 2000; Walensky et al. 2004]. 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. The screen is 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).

23 compounds were found active against Bfl-1 in AID 2080, a confirmatory dose response. Subsequent fluorescence polarization assay results declared all of these 23 compounds to be inactive. Two possibilities for this discrepancy were tested. One possibility was that a cysteine residue in the binding pocket of Bfl-1 was reacting covalently with the compounds, so a mutant was made in which the cysteine was replaced by an alanine at amino acid 55, denoted Bfl-1CA. The second possibility was that the original preparation of Bfl-1 had degraded during the year in a -80 degreeC freezer between the original screen (AID 1008) and the first confirmatory dose response assay (AID 2080). The present confirmatory dose response assay revealed that none of the compounds in AID 2080 were active using the fresh Bfl-1 or the fresh Bfl-1CA. We therefore declare that the results of AID 2080 are invalid, probably due to deterioration of the original preparation of Bfl-1.
Panel Information
Panel of dose response for Bcl-2 family proteins with powder compounds - Panel of dose response for Bcl-2 family proteins with powder compounds
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1Wildtype Bfl1121bcl-2-related protein A1 isoform 1 [Homo sapiens] [gi:4757840]
BCL2-related protein A1 isoform 1 [Homo sapiens]
2Mutant Bfl122bcl-2-related protein A1 isoform 1 [Homo sapiens] [gi:4757840]
Mutated cysteine to alanine BCL2-related protein A1 isoform 1 [Homo sapiens]
3Wildtype BclB220RecName: Full=Bcl-2-like protein 10; Short=Bcl2-L-10; AltName: Full=Anti-apoptotic protein NrH; AltName: Full=Apoptosis regulator Bcl-B [gi:23396469]
Apoptosis regulator Bcl-B (Bcl-2-like 10 protein) (Bcl2-L-10) (Anti-apoptotic protein NrH)

§ Panel component ID.
Protocol
Each component of the multiplex assay consists of a glutathione labeled bead, a GST Bcl-fusion protein target (six total, supplied by project collaborator), and a fluorescent peptide probe, F-Bim (FITC-Ahx-DMRPEIWIAQELRRIGDEFNAYYAR-OH; Commonwealth Biotech, USA). Bead sets are coated with individual GST-conjugated Bcl-2 proteins in HPSMTB buffer (30mM HEPES, 100mM KCl, 20mM NaCl, 1mM MgCl(2), 0.01% Tween-20, 0.1% BSA) and incubated overnight at 4 degrees C.

The multiplex is constructed by using beads for each protein target that have been labeled with varying intensities of red color, so that each assay is built on a unique bead set, and each bead set is associated with a unique optical address. Beads are first washed in HPSMTB buffer for 20 minutes before adding the appropriate GST-Bcl fusion protein. The bead sets (ThermoFisher Scientific product numbers XPR-1687-XPR-1696), have similar size (~ 4 micron diameter) and are distinguished by distinct emission characteristics at 665 +/-10 nm with excitation at 635 nm. Thus, GST-Bfl-1 might be non-covalently coated onto red level 1 beads, GST-Bcl-B onto red level 2 beads, etc. The bead sets (each with bound protein) and uncoated beads (see below) are first centrifuged separately, then combined and centrifuged again, and finally diluted just before loading into 384-well plates, to minimize bead-protein dissociation before the assay begins.

The HTS assay was conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 100 nM F-Bim in HPSMTB). Test compound concentration was 10 microM. Controls, which contained bead mixture and F-Bim but no test compound, were located in columns 1 and 2 on each plate. Plates were placed horizontal axis on rotators and incubated for 1-2 hours at 4 degrees C.

A glutathione-only bead set control (no associated GST-protein) was incorporated into each well as a fluorescence scavenger to determine inherent fluorescent properties (at 530 nm emission) of the test compounds. Specificity of F-Bim binding was determined with a Positive Control using a block of the F-Bim fluor with a non-fluoresceinated Bim peptide. The F-Bim blocking control was run daily as a separate single tube assay using Bim at 5 microM.

The primary screen yielded hit compounds that were evaluated in previous dose response screens. In the study reported here, a series of compounds were re-ordered in powder form and a confirmational dose response evaluation was done. Test compounds at 10 mM concentration in DMSO were serially diluted 1:3.16 eight times for a total of nine different test compound concentrations. Final compound dilutions in DMSO ranged from 1 microM to 10 mM. These dilutions were then diluted 1 to 100 to give an assay concentration range of 10 nanoM to 100 microM. The compounds were tested in either duplicate or triplicate dose response curves.

Sample acquisition and preliminary analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates [Kuckuck, et al. 2001]. The stream of particles is excited at 488 nm and 635 nM, and flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and emission at 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Analysis of the time-resolved acquisition data file uses IDLeQuery software to merge the flow cytometry data files with compound worklist files generated by HyperSip software. The raw data are parsed in IDLeQuery to produce annotated fluorescence summary data for each well. The parsed data are then processed through an Excel template file constructed specifically for the assay to segregate data for each target and the fluorescence scavenger in the multiplex. Gating based on forward scatter (FS) and side scatter (SS) parameters is used to identify singlet bead populations. Gating based on FL8 emission distinguishes the beads coated with different proteins, and the green median fluorescence intensity (MFI) per bead population (well) is calculated.


Calculations:

In dose response experiments, the assay was performed without compound and with nine different concentrations of compound, from 10 nanoM to 100 microM, to produce a series of 9 data points. HyperView calculates the median channel fluorescence (MCF) for each of these ligand concentrations, generating competition curves. The percent inhibition response was calculated by the following equation:
% response = 100 x (DMSO - SampleFL)/(DMSO -BlockCntrl)
where all variables are Median Fluorescence Intensity associated with the bead set bound with a specific protein. SampleFL is for beads in wells containing test compound, DMSO is the plate average of wells without test compounds, and BlockCntrl is for measurement in presence 5 micromolar non-fluorescent Bim. Baseline of % response is 0%.

These % response values were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent (EC50, microM), the low and high boundaries of the 95% confidence interval of the EC50 estimate, the Hill Slope, and the correlation coefficient (r squared) indicative of goodness-of-fit. Note that an compound eliciting inhibitory responses would have positive Hill Slope and a compound eliciting activating responses would have negative Hill Slope due to the data being fitted are the % inhibition response.

Fluorescence of the compounds could be green, which would increase the apparent amount of F-Bim bound (signal). None of such artifacts occurred with the large dynamic range of this assay, and no effort was made to annotate them. Fluorescence of the compounds could be red, which would shift the bead sets out of their gates, which had been set using whole plate data. Again, none of such artifacts occurred (bead numbers decreased at the upper end of a few dose response experiments), and no effort was made to annotate them.

Compounds with EC50 less than 10 microM and magnitude of response greater than 40% (i.e., Bottom of sigmoidal curve < 0.6 * Top of sigmoidal curve, listed as FIT_PERCENT_SPAN) were said to be "Active", and thus given a PUBCHEM_ACTIVITY_SCORE. The PUBCHEM_ACTIVITY_SCORE for each individual panel was based on the following equation;

PUBCHEM_ACTIVITY_SCORE = 50 * ( 1 - EC50/10 microM) + 50 * (Response - 40)/40

Overall PUBCHEM_ACTIVITY_SCORE is the sum of the activity scores from the individual panel targets.

In this assay active compounds have activity score greater than 0.

Keywords: NIH Roadmap, NMMLSC, high throughput flow cytometry, Bcl, Bim, Bfl-1, Bcl-B, multiplex, bead-based, screening, dose response
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Bfl1_SCOREOutcome for Bfl11bcl-2-related protein A1 isoform 1 [Homo sapiens]Integer
2Bfl1_OUTCOMEScore for Bfl11Outcome
3Bfl1_ACTIVITY_QUALIFIERQualifier for EC50 for Bfl11String
4Bfl1_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit for Bfl11FloatμM
5Bfl1_EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimate for Bfl11FloatμM
6Bfl1_EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimate for Bfl11FloatμM
7Bfl1_HILLSLOPEHill slope estimate for the fitted dose response curve for Bfl11Float
8Bfl1_FIT_PERCENT_SPANPercent response based on bottom and top span from EC50 curve fit estimate for Bfl11Float%
9Bfl1_RSQRCorrelation coefficient for the fitted dose response curve for Bfl11Float
10Bfl1_N_POINTSNumber of data points for each dose response curve for Bfl11Integer
11Bfl1_PERCENT_SPANPercent response based on data points for Bfl11Float%
12Bfl1_RESPONSE_0.01_MICROM (0.01μM**)Percent response measured with 0.01 micromolar concentration of test compound for Bfl11Float%
13Bfl1_RESPONSE_0.03_MICROM (0.03μM**)Percent response measured with 0.03 micromolar concentration of test compound for Bfl11Float%
14Bfl1_RESPONSE_0.10_MICROM (0.1μM**)Percent response measured with 0.10 micromolar concentration of test compound for Bfl11Float%
15Bfl1_RESPONSE_0.31_MICROM (0.31μM**)Percent response measured with 0.31 micromolar concentration of test compound for Bfl11Float%
16Bfl1_RESPONSE_1.00_MICROM (1μM**)Percent response measured with 1.00 micromolar concentration of test compound for Bfl11Float%
17Bfl1_RESPONSE_3.13_MICROM (3.13μM**)Percent response measured with 3.13 micromolar concentration of test compound for Bfl11Float%
18Bfl1_RESPONSE_9.93_MICROM (9.93μM**)Percent response measured with 9.93 micromolar concentration of test compound for Bfl11Float%
19Bfl1_RESPONSE_31.5_MICROM (31.5μM**)Percent response measured with 31.5 micromolar concentration of test compound for Bfl11Float%
20Bfl1_RESPONSE_100.0_MICROM (100μM**)Percent response measured with 100.0 micromolar concentration of test compound for Bfl11Float%
21DeterioratedBfl1_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit for Deteriorated Bfl11FloatμM
22DeterioratedBfl1_PERCENT_SPANPercent response based on data points for Deteriorated Bfl11Float%
23Bfl1CA_SCOREOutcome for Bfl1-CAmutant2bcl-2-related protein A1 isoform 1 [Homo sapiens]Integer
24Bfl1CA_OUTCOMEScore for Bfl1-CAmutant2Outcome
25Bfl1CA_ACTIVITY_QUALIFIERQualifier for EC50 for Bfl1-CAmutant2String
26Bfl1CA_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit for Bfl1-CAmutant2FloatμM
27Bfl1CA_EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimate for Bfl1-CAmutant2FloatμM
28Bfl1CA_EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimate for Bfl1-CAmutant2FloatμM
29Bfl1CA_HILLSLOPEHill slope estimate for the fitted dose response curve for Bfl1-CAmutant2Float
30Bfl1CA_FIT_PERCENT_SPANPercent response based on bottom and top span from EC50 curve fit estimate for Bfl1-CAmutant2Float%
31Bfl1CA_RSQRCorrelation coefficient for the fitted dose response curve for Bfl1-CAmutant2Float
32Bfl1CA_N_POINTSNumber of data points for each dose response curve for Bfl1-CAmutant2Integer
33Bfl1CA_PERCENT_SPANPercent response based on data points for Bfl1-CAmutant2Float%
34Bfl1CA_RESPONSE_0.01_MICROM (0.01μM**)Percent response measured with 0.01 micromolar concentration of test compound for Bfl1-CAmutant2Float%
35Bfl1CA_RESPONSE_0.03_MICROM (0.03μM**)Percent response measured with 0.03 micromolar concentration of test compound for Bfl1-CAmutant2Float%
36Bfl1CA_RESPONSE_0.10_MICROM (0.1μM**)Percent response measured with 0.10 micromolar concentration of test compound for Bfl1-CAmutant2Float%
37Bfl1CA_RESPONSE_0.31_MICROM (0.31μM**)Percent response measured with 0.31 micromolar concentration of test compound for Bfl1-CAmutant2Float%
38Bfl1CA_RESPONSE_1.00_MICROM (1μM**)Percent response measured with 1.00 micromolar concentration of test compound for Bfl1-CAmutant2Float%
39Bfl1CA_RESPONSE_3.13_MICROM (3.13μM**)Percent response measured with 3.13 micromolar concentration of test compound for Bfl1-CAmutant2Float%
40Bfl1CA_RESPONSE_9.93_MICROM (9.93μM**)Percent response measured with 9.93 micromolar concentration of test compound for Bfl1-CAmutant2Float%
41Bfl1CA_RESPONSE_31.5_MICROM (31.5μM**)Percent response measured with 31.5 micromolar concentration of test compound for Bfl1-CAmutant2Float%
42Bfl1CA_RESPONSE_100.0_MICROM (100μM**)Percent response measured with 100.0 micromolar concentration of test compound for Bfl1-CAmutant2Float%
43BclB_SCOREOutcome for BclB3RecName: Full=Bcl-2-like protein 10; Short=Bcl2-L-10; AltName: Full=Anti-apoptotic protein NrH; AltName: Full=Apoptosis regulator Bcl-BInteger
44BclB_OUTCOMEScore for BclB3Outcome
45BclB_ACTIVITY_QUALIFIERQualifier for EC50 for BclB3String
46BclB_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit for BclB3FloatμM
47BclB_EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimate for BclB3FloatμM
48BclB_EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimate for BclB3FloatμM
49BclB_HILLSLOPEHill slope estimate for the fitted dose response curve for BclB3Float
50BclB_FIT_PERCENT_SPANPercent response based on bottom and top span from EC50 curve fit estimate for BclB3Float%
51BclB_RSQRCorrelation coefficient for the fitted dose response curve for BclB3Float
52BclB_N_POINTSNumber of data points for each dose response curve for BclB3Integer
53BclB_PERCENT_SPANPercent response based on data points for BclB3Float%
54BclB_RESPONSE_0.01_MICROM (0.01μM**)Percent response measured with 0.01 micromolar concentration of test compound for BclB3Float%
55BclB_RESPONSE_0.03_MICROM (0.03μM**)Percent response measured with 0.03 micromolar concentration of test compound for BclB3Float%
56BclB_RESPONSE_0.10_MICROM (0.1μM**)Percent response measured with 0.10 micromolar concentration of test compound for BclB3Float%
57BclB_RESPONSE_0.31_MICROM (0.31μM**)Percent response measured with 0.31 micromolar concentration of test compound for BclB3Float%
58BclB_RESPONSE_1.00_MICROM (1μM**)Percent response measured with 1.00 micromolar concentration of test compound for BclB3Float%
59BclB_RESPONSE_3.13_MICROM (3.13μM**)Percent response measured with 3.13 micromolar concentration of test compound for BclB3Float%
60BclB_RESPONSE_9.93_MICROM (9.93μM**)Percent response measured with 9.93 micromolar concentration of test compound for BclB3Float%
61BclB_RESPONSE_31.5_MICROM (31.5μM**)Percent response measured with 31.5 micromolar concentration of test compound for BclB3Float%
62BclB_RESPONSE_100.0_MICROM (100μM**)Percent response measured with 100.0 micromolar concentration of test compound for BclB3Float%

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

Classification
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