High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1
| Autor: | Eduard Sergienko, John C. Reed, Paul Diaz, P.H.C. Godoi, Dayong Zhai, Brock Brown, Xochella Chan, Chaofang Jin, Russell Dahl, Ying Su, Justin Rascon, Thomas D.Y. Chung, Andrew Hurder |
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| Rok vydání: | 2011 |
| Předmět: |
Drug Evaluation
Preclinical Fluorescence Polarization Biochemistry Fluorescence Analytical Chemistry Protein–protein interaction Chemical library Maleimides Minor Histocompatibility Antigens Small Molecule Libraries chemistry.chemical_compound Neoplasms Minor histocompatibility antigen Fluorescence Resonance Energy Transfer Humans Chemistry Bcl-2 family Molecular biology High-Throughput Screening Assays Förster resonance energy transfer Pyrimidines Proto-Oncogene Proteins c-bcl-2 Apoptosis Molecular Medicine Fluorescence anisotropy Biotechnology HeLa Cells |
| Zdroj: | Journal of biomolecular screening. 17(3) |
| ISSN: | 1552-454X |
| Popis: | Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1. |
| Databáze: | OpenAIRE |
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