Document Detail

Development and validation of a high-throughput intrinsic ATPase activity assay for the discovery of MEKK2 inhibitors.
MedLine Citation:
PMID:  23134735     Owner:  NLM     Status:  MEDLINE    
The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. To identify inhibitors of MEKK2 kinase activity, we have developed a novel activity assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase activity. This MEKK2 ATPase assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase assay IC50 data for multiple inhibitors to literature values and to IC50 data generated using MEKK2 binding and transphosphorylation assays. Taken together, these data indicated that genuine MEKK2 activity was being measured in this assay and no other ATPases contributed to the signal. A miniaturized version of the assay was validated for high-throughput screening, and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding, and transphosphorylation assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 activity with IC50 values of <100 nM. Thus, this assay has utility for the discovery of small-molecule inhibitors of MEKK2 activity.
Syed Ahmad; Mark A Hughes; Gary L Johnson; John E Scott
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Validation Studies     Date:  2012-11-07
Journal Detail:
Title:  Journal of biomolecular screening     Volume:  18     ISSN:  1552-454X     ISO Abbreviation:  J Biomol Screen     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-03-25     Completed Date:  2013-09-26     Revised Date:  2014-03-07    
Medline Journal Info:
Nlm Unique ID:  9612112     Medline TA:  J Biomol Screen     Country:  United States    
Other Details:
Languages:  eng     Pagination:  388-99     Citation Subset:  IM    
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MeSH Terms
Adenosine Triphosphatases / metabolism*
Drug Discovery*
Enzyme Assays / methods*
High-Throughput Screening Assays / methods*
Inhibitory Concentration 50
MAP Kinase Kinase Kinase 2 / antagonists & inhibitors*,  metabolism
Phosphorylation / drug effects
Protein Kinase Inhibitors / pharmacology*
Grant Support
U54 CA156733/CA/NCI NIH HHS; U54 CA156735/CA/NCI NIH HHS; U54CA156735/CA/NCI NIH HHS
Reg. No./Substance:
0/Protein Kinase Inhibitors; EC Kinase Kinase Kinase 2; EC 3.6.1.-/Adenosine Triphosphatases

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