title: High-throughput screen for the chemical inhibitors of antiapoptotic bcl-2 family proteins by multiplex flow cytometry.
abstract: The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), 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.
authors.affiliation: Computational Chemistry Group, Romanian Academy Institute of Chemistry, Timisoara, Romania.
authors.forename: Ramona F
authors.initials: RF
authors.lastname: Curpan
authors.forename: Peter C
authors.initials: PC
authors.lastname: Simons
authors.forename: Dayong
authors.initials: D
authors.lastname: Zhai
authors.forename: Susan M
authors.initials: SM
authors.lastname: Young
authors.forename: Mark B
authors.initials: MB
authors.lastname: Carter
authors.forename: Cristian G
authors.initials: CG
authors.lastname: Bologa
authors.forename: Tudor I
authors.initials: TI
authors.lastname: Oprea
authors.forename: Arnold C
authors.initials: AC
authors.lastname: Satterthwait
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Edwards
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
mesh_terms: D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D001667:Binding, Competitive; D002151:Calorimetry / Q000379:methods; D002986:Clinical Trials as Topic; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005434:Flow Cytometry; D005454:Fluorescence Polarization / Q000379:methods; D005978:Glutathione / Q000378:metabolism; D049452:Green Fluorescent Proteins; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008956:Models, Chemical; D058990:Molecular Targeted Therapy; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000032:analysis*
chemical_list: D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D054852:Small Molecule Libraries; D049452:Green Fluorescent Proteins; D005978:Glutathione