| Project/Area Number |
17H07004
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| Research Category |
Grant-in-Aid for Research Activity Start-up
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| Allocation Type | Single-year Grants |
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| Research Field |
Environmental and hygienic pharmacy
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| Research Institution | University of Shizuoka |
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Principal Investigator |
Shizu Ryota 静岡県立大学, 薬学部, 助教 (50803912)
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| Research Collaborator |
Negishi Masahiko 米国国立環境衛生科学研究所
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| Project Period (FY) |
2017-08-25 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
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| Keywords | 核内受容体 / インビトロ評価系 / 組換えタンパク質 / 蛍光偏光解消法 / CAR / PXR / in vitro評価系 / 結晶化 / 転写因子 / 蛋白質 / 結晶構造 |
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| Outline of Final Research Achievements |
Hepatic nuclear receptor CAR and PXR play central roles in the xenobiotics-induced induction of drug metabolizing enzymes. The receptors are activated by lots of chemicals and the activation represents the basis for several clinically important drug-drug interactions. In this study, we have attempted to establish an in vitro evaluation systems for CAR and PXR activation to estimate drug-drug interaction of new chemicals.
Recombinant CAR and PXR proteins were purified form E. coli as an 6x His and SUMO fusion proteins. These proteins were incubated with their ligands and subjected to fluorescence polarization assay with FITC-labeled oligo DNA which contains nuclear receptor binding motif. We observed the fluorescence polarization was induced by the ligand binding. By modifying this method, we will be able to establish the in vitro evaluation system for the receptors activation and drug-drug interaction.
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| Academic Significance and Societal Importance of the Research Achievements |
組換えタンパク質を利用した生物物理学的解析による受容体の活性化評価系は、動物個体や培養細胞を用いた解析では観察が困難な詳細な活性化機序解析を可能にするが、機能的な核内受容体全長の組換えタンパク質を精製した報告は少なく、CAR及びPXR全長の組換えタンパク質の報告は皆無であった。したがって、全長の核内受容体組換えタンパク質の調製法を確立し、それらを利用してDNA結合やリガンド応答性の解析を行った本研究成果は、CARやPXRの異物応答性核内受容体の研究領域にとどまらず、全ての核内受容体の研究に有益な共通した情報を提供でき、その学術的意義は大きいと期待される。
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