High-throughput screen for the chemical inhibitors of antiapoptotic bcl-2 family proteins by multiplex flow cytometry.
Assay Drug Dev Technol
; 9(5): 465-74, 2011 Oct.
Article
en En
| MEDLINE
| ID: mdl-21561376
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 (â¼195,000 compounds) using high-throughput flow cytometry.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Proto-Oncogénicas
/
Proteínas Proto-Oncogénicas c-bcl-2
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Proteínas Reguladoras de la Apoptosis
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Bibliotecas de Moléculas Pequeñas
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Descubrimiento de Drogas
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Ensayos Analíticos de Alto Rendimiento
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Proteínas de la Membrana
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Assay Drug Dev Technol
Asunto de la revista:
FARMACOLOGIA
Año:
2011
Tipo del documento:
Article
País de afiliación:
Rumanía