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2021 Fiscal Year Final Research Report

Development of high-throughput histidine kinase assay for drug discovery

Research Project

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Project/Area Number 19K07147
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 47050:Environmental and natural pharmaceutical resources-related
Research InstitutionHiroshima Bunkyo University (2021)
Hiroshima University (2019-2020)

Principal Investigator

Eiji Kinoshita  広島文教大学, 人間科学部, 教授 (80304418)

Co-Investigator(Kenkyū-buntansha) 江口 陽子  近畿大学, 生物理工学部, 准教授 (30757422)
Project Period (FY) 2019-04-01 – 2022-03-31
Keywordsフォスタグ / リン酸化タンパク質 / ヒスチジンキナーゼ / ヒスチジンキナーゼ阻害剤 / waldiomycin / Phos-tag Aqua / ドットブロット法
Outline of Final Research Achievements

The principal investigator newly synthesized several fluorophore-labeled Phos-tag derivatives and succeeded in phosphorylation profiling of histidine-phosphorylated proteins (histidine kinases) derived from bacteria by using the fluorophore-labeled Phos-tag derivatives as gel stains after SDS-PAGE analysis. This novel Phos-tag dye technology is suitable for the quantitative monitoring of histidine- and aspartate-phosphorylated proteins in bacterial signaling systems, and it can be applied directly on SDS-PAGE gels. Furthermore, as a result of attempting to detect phosphorylated proteins on the blot membrane using Phos-tag Aqua, which is one of the Phos-tag fluorescent gel stains, bacterial histidine kinases dot-blotted on the membrane were selectively visualized. This simple dot-blotting method using Phos-tag Aqua developed in this study has been demonstrated to be useful for high-throughput histidine kinase assays and high-throughput screening of bacterial histidine kinase inhibitors.

Free Research Field

人間栄養学

Academic Significance and Societal Importance of the Research Achievements

感染症治療薬について,病原菌特有のヒスチジンキナーゼ阻害薬が,次世代の抗生物質として注目されつつある。しかし,病原菌のヒスチジンキナーゼは化学的に不安定で短寿命なリン酸化タンパク質を生成する性質があり,阻害薬としての候補化合物を検証する方法が限られていた。本研究では,そのような不安定なリン酸化タンパク質を高感度に定量分析するための蛍光標識型分子デバイスを創出し,それを応用することで,病原菌特有のヒスチジンキナーゼ阻害薬を探索すべく多検体処理能力に秀でた高速スクリーニングシステム開発の可能性を証明できたことはとても意義深い。本手法が今後のこの分野におけるブレイクスルーになることを期待する。

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Published: 2023-01-30  

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