High-Throughput Screen for the Chemical Inhibitors of Antiapoptotic Bcl-2 Family Proteins by Multiplex Flow Cytometry
- 1 October 2011
- journal article
- research article
- Published by Mary Ann Liebert Inc in ASSAY and Drug Development Technologies
- Vol. 9 (5), 465-474
- https://doi.org/10.1089/adt.2010.0363
Abstract
The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-XL, Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose–response series, resulting in three “active” compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.Keywords
This publication has 20 references indexed in Scilit:
- Bcl-B Expression in Human Epithelial and Nonepithelial MalignanciesClinical Cancer Research, 2008
- Nur77 converts phenotype of Bcl-B, an antiapoptotic protein expressed in plasma cells and myelomaBlood, 2007
- Biomolecular screening of formylpeptide receptor ligands with a sensitive, quantitative, high-throughput flow cytometry platformNature Protocols, 2006
- Comparison of chemical inhibitors of antiapoptotic Bcl-2-family proteinsCell Death & Differentiation, 2006
- Glutathione‐S‐transferase‐green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSHCytometry Part A, 2006
- Apoptosis-based therapies for hematologic malignanciesBlood, 2005
- Characterization of the anti-apoptotic mechanism of Bcl-BBiochemical Journal, 2003
- Development of a high-throughput fluorescence polarization assay for Bcl-xLAnalytical Biochemistry, 2002
- Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assayJournal of Immunological Methods, 1999
- A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening AssaysSLAS Discovery, 1999