An immuno-dot blot assay for screening histidine kinase inhibitors

Emiko Kinoshita-Kikuta; Shino Maruta; Yoko Eguchi; Masayuki Igarashi; Toshihide Okajima; Ryutaro Utsumi; Eiji Kinoshita; Tohru Koike

doi:10.1016/j.ab.2020.113765

An immuno-dot blot assay for screening histidine kinase inhibitors

Anal Biochem. 2020 Jul 1:600:113765. doi: 10.1016/j.ab.2020.113765. Epub 2020 Apr 30.

Authors

Emiko Kinoshita-Kikuta¹, Shino Maruta¹, Yoko Eguchi², Masayuki Igarashi³, Toshihide Okajima⁴, Ryutaro Utsumi⁴, Eiji Kinoshita⁵, Tohru Koike¹

Affiliations

¹ Department of Functional Molecular Science, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
² Department of Science and Technology on Food Safety, Faculty of Biology-Oriented Science and Technology, Kindai University, Wakayama, Japan.
³ Institute of Microbial Chemistry, Tokyo, Japan.
⁴ Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.
⁵ Department of Functional Molecular Science, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. Electronic address: kinoeiji@hiroshima-u.ac.jp.

PMID: 32360198
DOI: 10.1016/j.ab.2020.113765

Abstract

Two-component signal transduction systems (TCSs), consisting of a histidine kinase (HK) and its cognate response regulator, are ubiquitous among bacteria and are associated with the virulence of pathogens. TCSs are potential targets for alternative antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in bacterial TCSs. Here, we describe an immuno-dot blot assay for the inhibition profiling of HKs using the anti-N3-phosphohistidine antibody. This simple method promises reliable detection of HK activity, and it is likely applicable in high-throughput screening of HK inhibitors.

Keywords: Anti-N3-phosphohistidine antibody; Histidine kinase inhibitor; Immuno-dot blotting; Inhibition profiling; Inhibitor screening; Two-component signal transduction system.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Dose-Response Relationship, Drug
High-Throughput Screening Assays
Histidine Kinase / antagonists & inhibitors*
Histidine Kinase / metabolism
Protein Kinase Inhibitors / pharmacology*
Quinones / pharmacology*
Signal Transduction / drug effects
Structure-Activity Relationship

Substances

Protein Kinase Inhibitors
Quinones
waldiomycin
Histidine Kinase