Assay development and high-throughput screening of small molecular c-Abl kinase activators.
J Biomol Screen
; 16(1): 53-64, 2011 Jan.
Article
in En
| MEDLINE
| ID: mdl-20938045
ABSTRACT
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 with >30% response. Further analysis showed that 1024 had EC(50) <10 µM with a max % response of >50%. 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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Proto-Oncogene Proteins c-abl
/
Enzyme Activators
/
High-Throughput Screening Assays
Type of study:
Diagnostic_studies
/
Screening_studies
Limits:
Humans
Language:
En
Journal:
J Biomol Screen
Journal subject:
BIOLOGIA MOLECULAR
Year:
2011
Type:
Article
Affiliation country:
United States