A High-Throughput TNP-ATP Displacement Assay for Screening Inhibitors of ATP-Binding in Bacterial Histidine Kinases
A High-Throughput TNP-ATP Displacement Assay for Screening Inhibitors of ATP-Binding in Bacterial Histidine Kinases
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DOI：
10.1089/adt.2010.0289
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发表时间：
2011-04-01
影响因子：
1.8
通讯作者：
Zhao, Rui
中科院分区：
文献类型：
--
作者：
Guarnieri, Michael T.;Blagg, Brian S. J.;Zhao, Rui
细菌组氨酸激酶（HK）是GHKL超家族的成员，其共享独特的三磷酸腺苷（ATP）结合Bergerat折叠。我们的前期研究表明，Gyrase、Hsp 90、MutL（GHL）抑制剂与HK的ATP结合口袋结合，可能为设计针对这些激酶的新型抗生素提供先导化合物。在这篇文章中，我们开发了一种竞争分析使用荧光ATP类似物，2 '，3'-O-（2，4，6-三硝基苯基）腺苷5 '-三磷酸。该方法可用于靶向HK或其他ATP结合蛋白的化合物文库的高通量筛选。我们利用该测定筛选靶向细菌HK PhoQ的GHL抑制剂库，并讨论了2 '，3'-0-（2，4，6-trinitrophenyl）adenosine 5-triphosphate竞争测定在GHKL抑制剂筛选之外的应用。
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'-0-(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'-0-(2,4,6-trinitrophenyl) adenosine 5-triphosphate competition assay beyond GHKL inhibitor screening.