| Project/Area Number |
10671985
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Chemical pharmacy
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
SHIMIZU Masato Tokyo Medical and Dental University, Institute of Biomaterials and Bioengineering, Associate Professor, 生体材料工学研究所, 助教授 (50126231)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Keiko Tokyo Medical and Dental University, Institute of Biomaterials and Bioengineering, Instructor, 生体材料工学研究所, 助手 (90147017)
YAMADA Sachiko Tokyo Medical and Dental University, Institute of Biomaterials and Bioengineering, Professor, 生体材料工学研究所, 教授 (10014078)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Active vitamin D / fluorovitamin D analog / Fluorescent-labeled vitamin D analog / Vitamin D receptor / Vitamin D-receptor complex / ィイD119ィエD1FNMR / Conformational analysis / ビタミンD-受容体複合体 / コンフォメーション / ビタミンD-受容体コンプレックス |
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| Research Abstract |
Biological responses mediated by the active vitamin D are regulated at the level of gene expression by binding of the vitamin D receptor (VDR)-ligand complex to the target gene. The key factor in this gene transactivation is the three-dimensional structure of VDR dependent on the ligand conformation. In this research project, to directly examine the vitamin D conformation binding to the VDR, we proposed the use of ィイD119ィエD1F-NMR with fluorinated vitamin D analogs as probe compounds. In addition, we investigate the intracellular interaction of vitamin D-VDR using fluorescent-labeled vitamin D analogs.
The low temperature ィイD119ィエD1F-NMR spectra of successfully synthesized 4,4-difluoro- and 19-fluorovitamin D analogs showed separated signals ascribed to two-A-ring conformations and those analogs were shown to bind to the VDR with high affinity (Kd= ca. 10ィイD1-9ィエD1M) indicating to be useful probe to analyze the VDR-bound A-ring conformation of vitamin D. The VDR protein was expressed in
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E. coli BL21(DE3) transfected with pET-14b plasmid vector containing rat cDNA insert. The A-ring of vitamin D adopts two chair conformations, the α- and β-form. The ィイD119ィエD1F-NMRspectrum of VDR-19-fluorovitamin D complex showed that the A-ring adopts a chair α-conformation. Recently, X-ray crystal structure of the active vitamin D-mutant VDR complex was determined and in the complex, the natural ligand occupies β-conformation . More detail investigations are required to determine the active form of the A-ring conformation associated with the expression of biological activities.
We designed and successfully synthesized two novel fluorescent-labeled vitamin D analogs. Though the binding affinity of those analogs for VDR was small (0.1%) relative to the natural ligand, the VDR-bound form is estimated to be still exclusive (B/F : ca. 10ィイD18ィエD1) on the basis of Kd of the natural ligand. Visual observation of the whole cell-ligand interaction is currently progressing through the use of the fluorescent analogs. Less
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