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BioAssay: AID 1693

Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions via Bim (BCL2-like 11)

This report summarizes the series of assays used to identify small molecule regulators of Bcl-2 family protein interactions. One arm of apoptosis is regulated by the balance of anti-apoptotic and pro-apoptotic Bcl-2 family members. The binding of fluorochrome-conjugated BH3 peptides (in this case, Bim (also known as BCL2-like 11)) to Bcl-2 family members provides the basis for construction of a more ..
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AID: 1693
Data Source: NMMLSC (UNM_Bcl_Multiplex_Summary)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-04-23
Modify Date: 2013-10-30

Data Table ( Complete ):           Active    All
Targets
Sequence: bcl-2-like protein 11 isoform 18 [Homo sapiens]
Description ..   
Protein Family: Bim protein N-terminus

Gene:BCL2L11          More BioActivity Data..


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BioActive Compound: Chemical Probe: 1    Active: 1
Depositor Specified Assays
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AIDNameTypeComment
621TR-FRET secondary assay for HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1confirmatoryTR-FRET secondary assay for HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1
432HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1confirmatoryHTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1
748High Throughput Fluorescence Polarization Screen for Bcl-B Phenotype ConvertersconfirmatoryHigh Throughput Fluorescence Polarization Screen for Bcl-B Phenotype Converters
950Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2.screeningHTS Bim-Bcl-2
951Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-B.screeningHTS Bim-Bcl-B
952Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-W.screeningHTS Bim-Bcl-w
1007Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XL.screeningHTS Bim-Bcl-xL
1008Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1screeningHTS Bim-Bfl-1
1009Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1screeningHTS Bim-Mcl-1
1330Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-W.confirmatoryDose Response Bim_Bcl-w
1327Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-B protein.confirmatoryDose Response Bim_Bcl-b
1320Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1.confirmatoryDose Response Bim_Bfl-1
1322Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XL.confirmatoryDose Response Bim_Bcl-x
1328Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2confirmatoryDose Response Bim_Bcl-2
1329Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1.confirmatoryDose Response Bim_Mcl-1
1324Profiling Assay to determine GST-GSH interactions in multiplex bead-based assays (HPSMTB buffer)confirmatoryProfiling Assay for GST-GSH interaction in multiplex bead-based assays
1776Profiling compound fluorescence on GSH Beads with 488 nm excitation and 530 nm emissionotherProfiling compound fluorescence on GSH Beads with 488 nm excitation and 530 nm emission##
2081Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-WconfirmatoryDose Response Bim_Bcl-w compound powder confirmation
2075Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2confirmatoryDose Response Bim_Bcl-2 compound powder confirmation
2084Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XLconfirmatoryDose Response Bim_Bcl-XL compound powder confirmation
2080Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1confirmatoryDose Response Bim_Bfl-1 compound powder confirmation
2086Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1confirmatoryDose Response Bim_Mcl-1 compound powder confirmation
2077Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-BconfirmatoryDose Response Bim_Bcl-B compound powder confirmation
504627Bcl-2 family members Fluorescence polarization assay with Set1 of powder compoundsotherOnHold
720677SAR analysis of selective Bcl-B inhibitors using fluorescence polarization assay, set 2confirmatorySAR analysis of selective Bcl-B inhibitors using fluorescence polarization assay, set 2
588716Isothermal titration calorimetry (ITC) with Bcl-B and compounds active in primary screenconfirmatory
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
588575SAR analysis of selective Bcl-B inhibitors using fluorescence polarization assayconfirmatory
588578SAR analysis of selective Bcl-B inhibitors using a Fluorescence Polarization Bcl-XL/Bim-BH3 Assayconfirmatory
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 Implementatiion: Peter Simons Ph.D, Susan Young MS, Anna Waller Ph.D, Mark Carter MS

Assay Background and Significance:

This report summarizes the series of assays used to identify small molecule regulators of Bcl-2 family protein interactions. One arm of apoptosis is regulated by the balance of anti-apoptotic and pro-apoptotic Bcl-2 family members. The binding of fluorochrome-conjugated BH3 peptides (in this case, Bim (also known as BCL2-like 11)) to Bcl-2 family members provides the basis for construction of a fluorescence-based assay amenable to flow cytometry high throughput screening for small molecule regulators of these interactions. This 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).

The present report assembles the list of compounds that showed activity across all Bcl protein assays.

This project lead to a probe for Bcl-B:
(SID / CID / ML#)
124756688 / 53301938 / ML258
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-Axh-DMRPEIWIAQELRRIGDEFNAYYAR-OH; Commonwealth Biotech, USA). Bead sets are coated with individual GST-conjugated Bcl-2 proteins in HPSMTB buffer (30milliM HEPES, 100milliM KCl, 20milliM NaCl, 1milliM MgCl(2), 0.01% Tween-20, 0.1% BSA) and incubated overnight at 4 degrees C.

The mulitplex 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 noncovalently coated onto red level 1 beads, GST-Bcl-XL onto red level 2 beads, etc. The 6 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 assay is conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microL of bead mixture, 0.1 microL of test compound, and 5 microL of 100nM F-Bim in HPSMTB). Test compound concentration is 10 microM. Controls, which contain bead mixture and F-Bim but no test compound, are located in columns 1 and 2 on each plate. Plates are 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) is 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 is determined with a Positive Control using a block of the F-Bim Fluor with a non-fluoresceinated F-Bim peptide. The F-Bim blocking control is run daily as a separate single tube assay using F-Bim at 5 microM.

Sample 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. Flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Analysis is performed using time-resolved acquisition into a single data file and analysis using 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 median fluorescence per bead population is calculated.

Summary and Sequence of Assays Performed

HTS Bim-Bcl-2 AID # 950
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 116

HTS Bim-Bcl-B AID # 951
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 142

HTS Bim-Bcl-w AID # 952
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 47

HTS Bim-Bcl-xL AID # 1007
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 82

HTS Bim-Bfl-1 AID # 1008
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 237

HTS Bim-Mcl-1 AID # 1009
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 196

Dose Response Bim_Bcl-w AID # 1330
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 18

Dose Response Bim_Bcl-b AID # 1327
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 6

Dose Response Bim_Bfl-1 AID # 1320
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 97

Dose Response Bim_Bcl-x AID # 1322
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 6

Dose Response Bim_Bcl-2 AID # 1328
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 9

Dose Response Bim_Mcl-1 AID # 1329
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 3

Profiling Assay for GST-GSH interaction in multiplex bead-based assays AID # 1324
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 20%
# compounds evaluated: 834
# active compounds: 19

Dose Response Bim_Bcl-w compound powder confirmation AID # 2081
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 5

Dose Response Bim_Bcl-B compound powder confirmation AID # 2077
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 9

Dose Response Bim_Bfl-1 compound powder confirmation AID # 2080
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 23

Dose Response compound powder confirmation Bim_Bcl-xl AID # 2084
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 0

Dose Response Bim_Bcl-2 compound powder confirmation AID # 2075
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 6

Dose Response Bim_Mcl-1 compound powder confirmation AID # 2086
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 0

Abbreviations: nm for nanometer, nanoM for nanomolar, microM for micromolar, milliM for millimolar, microL for microliter
Comment
The Gene ID utilized to describe the target covers the actual fluorescently tagged Bim protein used as the ligand for this multiplex project: F-Bim (FITC-Axh-DMRPEIWIAQELRRIGDEFNAYYAR-OH). There is no protein GI for this short peptide sequence.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML#ML Probe numberString
2EC50EC50 reported in Probe report for probe compound against specific targetFloatμM
Additional Information
Grant Number: 1X01 MH079850-01

Data Table (Concise)
Classification
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