AID	Name	Type	Comment
1693	Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions via Bim (BCL2-like 11)	summary	Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions via Bim (BCL2-like 11).

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.

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)

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

Grant Number: 1X01 MH079850-01