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BioAssay: AID 588716

Isothermal titration calorimetry (ITC) with Bcl-B and compounds active in primary screen

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(3)
 
 
Active(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(3)
 
 
AID: 588716
Data Source: NMMLSC (UNMCMD_BCLB_ITC_IC50_SET1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-27

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: bcl-2-like protein 10 [Homo sapiens]
Description ..   
Protein Family: Bcl-2_like

Gene:BCL2L10     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 3
Related Experiments
Show more
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
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
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
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

Screening Center/ PI: UNMCMD/ Larry Sklar Ph.D.
Lead Biologist at Screening Center: Peter Simons Ph.D.

Chemistry Center/ PI: Sanford-Burnham Center for Chemical Genomics / John C. Reed Ph.D.
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:

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. For the primary screen a multiplexed assay was run 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 subsequent followup assay of isothermal calorimetry was used to varify the results from the bead based assay. Isothermal calorimetry (ITC) uses a different physical principle to measure binding. ITC measurement of changes in temperature upon interaction between small molecule and a protein yields the binding affinity between the different binding partners. The ITC demonstration of binding is particularly instructive, for the flow cytometry and FP measurements are prone to similar optical artifacts.
Protocol
Isothermal titration calorimetry (ITC) was performed on an ITC200 calorimeter from Microcal. Two-microliter aliquots of solution containing 500 microM compound were injected into 200 microL cells containing 50 microM GST-Bcl-2 protein. Nineteen injections were made per titration. The experiments were performed at 23 degrees-C in buffer containing 20 milliM HEPES (pH 7.0). Experimental data were analyzed using Origin software provided by Microcal. A compound was described as active when it displayed a sigmoidal dose-response curve, which corresponds to IC50<10 microM at the protein concentration used.

PUBCHEM_ACTIVITY_SCORE was calculated based on the reported IC50 according to this equation:
PUBCHEM_ACTIVITY_SCORE = 100 *(1-IC50/10)
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: alternate confirmatory

BAO: bioassay specification: assay biosafety level: bsl2

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: meta target: molecular target: protein target: cytosolic protein

BAO: meta target: biological process target: cell death

BAO: meta target detail: binding reporter specification: interaction: protein-small molecule

BAO: detection technology: label free technology: isothermal titration calorimetry

BAO: bioassay specification: bioassay type: binding

BAO: bioassay specification: assay footprint: vial

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*Effective concentration of half maximal binding as estimated by curve fitFloatμM

* Activity Concentration.
Additional Information
Grant Number: 1X01 MH079850-01

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