| RRC ID |
43480
|
| 著者 |
Fujikawa Y, Urano Y, Komatsu T, Hanaoka K, Kojima H, Terai T, Inoue H, Nagano T.
|
| タイトル |
Design and synthesis of highly sensitive fluorogenic substrates for glutathione S-transferase and application for activity imaging in living cells.
|
| ジャーナル |
J Am Chem Soc
|
| Abstract |
Here we report the development of fluorogenic substrates for glutathione S-transferase (GST), a multigene-family enzyme mainly involved in detoxification of endogenous and exogenous compounds, including drug metabolism. GST is often overexpressed in a variety of malignancies and is involved in the development of resistance to various anticancer drugs. Despite the medical significance of this enzyme, no practical fluorogenic substrates for fluorescence imaging of GST activity or for high-throughput screening of GST inhibitors are yet available. So, we set out to develop new fluorogenic substrates for GST. In preliminary studies, we found that 3,4-dinitrobenzanilide (NNBA) is a specific substrate for GST and established the mechanisms of its glutathionylation and denitration. Using these results as a basis for off/on control of fluorescence, we designed and synthesized new fluorogenic substrates, DNAFs, and a cell membrane-permeable variant, DNAT-Me. These fluorogenic substrates provide a dramatic fluorescence increase upon GST-catalyzed glutathionylation and have excellent kinetic parameters for the present purpose. We were able to detect nuclear localization of GSH/GST activity in HuCCT1 cell lines with the use of DNAT-Me. These results indicate that the newly developed fluorogenic substrates should be useful not only for high-throughput GST-inhibitor screening but also for studies on the mechanisms of drug resistance in cancer cells.
|
| 巻・号 |
130(44)
|
| ページ |
14533-43
|
| 公開日 |
2008-11-5
|
| DOI |
10.1021/ja802423n
|
| PMID |
18841967
|
| MeSH |
Anilides / chemistry
Anilides / metabolism
Catalysis
Cell Line, Tumor
Drug Design
Fluorescent Dyes / analysis
Fluorescent Dyes / chemical synthesis
Fluorescent Dyes / chemistry*
Fluorescent Dyes / metabolism
Glutathione / chemistry
Glutathione / metabolism
Glutathione S-Transferase pi / chemistry
Glutathione S-Transferase pi / metabolism
Glutathione Transferase / analysis
Glutathione Transferase / chemistry*
Glutathione Transferase / metabolism
HeLa Cells
Humans
Kinetics
Microscopy, Fluorescence / methods
Models, Molecular
Spectrometry, Fluorescence / methods
Substrate Specificity
|
| IF |
14.612
|
| 引用数 |
117
|
|
WOS 分野
|
CHEMISTRY, MULTIDISCIPLINARY
|
| リソース情報 |
| ヒト・動物細胞 |
HT1080(RCB1956) |
