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Assay Drug Dev Technol. 2011 Apr;9(2):174-83. doi: 10.1089/adt.2010.0289. Epub 2010 Nov 4.

A high-throughput TNP-ATP displacement assay for screening inhibitors of ATP-binding in bacterial histidine kinases.

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1
Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, 80045, USA.

Abstract

Bacterial histidine kinases (HK) are members of the GHKL superfamily, which share a unique adenosine triphosphate (ATP)-binding Bergerat fold. Our previous studies have shown that Gyrase, Hsp90, MutL (GHL) inhibitors bind to the ATP-binding pocket of HK and may provide lead compounds for the design of novel antibiotics targeting these kinases. In this article, we developed a competition assay using the fluorescent ATP analog, 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate. The method can be used for high-throughput screening of compound libraries targeting HKs or other ATP-binding proteins. We utilized the assay to screen a library of GHL inhibitors targeting the bacterial HK PhoQ, and discuss the applications of the 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate competition assay beyond GHKL inhibitor screening.

PMID:
21050069
PMCID:
PMC3065726
DOI:
10.1089/adt.2010.0289
[Indexed for MEDLINE]
Free PMC Article
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