Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms
| Autor: | Shi-Chung Ng, Jie Qian, Haichao Zhang, Kenneth M. Comess, Michael L. Coen, Andrew M. Petros, Jeffrey R. Huth, Martin J. Voorbach, Saul H. Rosenberg, David J. Burns, Usha Warrior |
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| Rok vydání: | 2003 |
| Předmět: |
Magnetic Resonance Spectroscopy
High-throughput screening Biophysics bcl-X Protein Peptide Bcl-xL Apoptosis Fluorescence Polarization Mass spectrometry Biochemistry Binding Competitive Mass Spectrometry Inhibitory Concentration 50 Combinatorial Chemistry Techniques Dimethyl Sulfoxide Molecular Biology chemistry.chemical_classification Chromatography biology Titrimetry Cell Biology Nuclear magnetic resonance spectroscopy chemistry Pharmaceutical Preparations Proto-Oncogene Proteins c-bcl-2 biology.protein Carrier Proteins Fluorescence anisotropy Function (biology) Plate reader |
| Zdroj: | Analytical biochemistry. 328(2) |
| ISSN: | 0003-2697 |
| Popis: | Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented. An activity assay utilized fluorescence polarization, based on the binding of fluorescein-labeled peptide [the BH3 domain of BAD protein (F-Bad 6)] to Bcl-xL. A 384-well plate assay with mixtures of 10 drug compounds per well, combined with a fast plate reader, resulted in a throughput of 46,080 data points/day. Utilizing this screening format, 370,400 compounds were screened in duplicate and 425 inhibitors with an IC 50 below 100 μM were identified. The second assay format, affinity selection/mass spectrometry (ASMS), used ultrafiltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by flow injection electrospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with affinities below 100 μM were identified. Two novel classes of Bcl-xL inhibitors were identified by both methods and confirmed to bind 13 C-labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy. |
| Databáze: | OpenAIRE |
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