Assay development and high-throughput screening of small molecular c-Abl kinase activators
| Autor: | Da-Yuan Wang, Brett Siegfried, Kyung O. Johanson, Hong Zhang, Chad Quinn, Robert S. Ames, Jingsong Yang, Thau F. Ho, Tracey Yi, Hu Li, Glenn A. Hofmann, Josh Cottom |
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| Rok vydání: | 2010 |
| Předmět: |
High-throughput screening
Genetic Vectors Enzyme Activators BacMam Biology Transfection Biochemistry Analytical Chemistry Small Molecule Libraries Enzyme activator chemistry.chemical_compound Adenosine Triphosphate hemic and lymphatic diseases Drug Discovery Humans Phosphorylation Proto-Oncogene Proteins c-abl ABL Kinase Molecular biology High-Throughput Screening Assays HEK293 Cells chemistry Molecular Medicine Biological Assay Tyrosine kinase Adenosine triphosphate Baculoviridae Biotechnology |
| Zdroj: | Journal of biomolecular screening. 16(1) |
| ISSN: | 1552-454X |
| Popis: | A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur. The assay was calibrated such that inactive c-Abl protein was activated by ATP alone to a degree that it not only demonstrated a measurable c-Abl activity but also maintained a robust assay window for screening. The screen resulted in 8624 primary hits with30% response. Further analysis showed that 1024 had EC(50)10 µM with a max % response of50%. These hits were structurally and chemically diverse with possibly different mechanisms for activating c-Abl. In addition, selective hits were shown to be cell permeable and were able to induce c-Abl activation as determined by In-Cell Western (ICW) analysis of HEK-MSRII cells transduced with BacMam virus expressing full-length c-Abl. |
| Databáze: | OpenAIRE |
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