Design and Synthesis of Highly Sensitive Fluorogenic Substrates for Glutathione S-Transferase and Application for Activity Imaging in Living Cells
Design and Synthesis of Highly Sensitive Fluorogenic Substrates for Glutathione S-Transferase and Application for Activity Imaging in Living Cells
复制标题
DOI：
10.1021/ja802423n
复制
发表时间：
2008-11-05
影响因子：
15
通讯作者：
Nagano, Tetsuo
中科院分区：
文献类型：
--
作者：
Fujikawa, Yuuta;Urano, Yasuteru;Nagano, Tetsuo
在这里，我们报告的荧光底物谷胱甘肽S-转移酶（GST），多基因家族的酶主要参与内源性和外源性化合物的解毒，包括药物代谢的发展。GST通常在多种恶性肿瘤中过表达，并且参与对多种抗癌药物的耐药性的发展。尽管这种酶的医学意义，没有实用的荧光底物的GST活性的荧光成像或高通量筛选GST抑制剂。因此，我们着手开发GST的新荧光底物。在前期研究中，我们发现3，4-二硝基苯甲酰苯胺（NNBA）是GST的特异性底物，并建立了其谷胱甘肽化和还原的机制。使用这些结果作为基础上的关/开控制的荧光，我们设计和合成新的荧光底物，DNAFs，和细胞膜可渗透的变体，DNAT-Me。这些荧光底物提供了一个显着的荧光增加GST催化谷胱甘肽化，并具有良好的动力学参数为目前的目的。我们利用DNAT-Me检测了HuCCT 1细胞系中GSH/GST活性的核定位，这些结果表明新开发的荧光底物不仅可用于高通量GST抑制剂的筛选，而且可用于肿瘤耐药机制的研究。
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.