A high-throughput TNP-ATP displacement assay for screening inhibitors of ATP-binding in bacterial histidine kinases.
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.
Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, 80045, USA.
Michael T
MT
Guarnieri
Brian S J
BS
Blagg
Rui
R
Zhao
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D000071677:Histidine Kinase; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011494:Protein Kinases / Q000378:metabolism*; D011897:Random Allocation; D012486:Salmonella typhimurium / Q000201:enzymology
D004364:Pharmaceutical Preparations; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase