DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.
DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.
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DOI：
10.1111/cas.12420
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发表时间：
2014-07
期刊：
影响因子：
5.7
通讯作者：
Tahara H
中科院分区：
文献类型：
--
作者：
Miyagi T;Shiotani B;Miyoshi R;Yamamoto T;Oka T;Umezawa K;Ochiya T;Takano M;Tahara H
核因子-κB（NF-κB）是癌症进展和疾病炎症效应的关键调节因子。为了鉴定DNA与NF-κB结合的抑制剂，我们开发了一种新的均相检测序列特异性DNA结合蛋白的方法。这种方法，我们称之为DSE-FRET，是基于两种现象：蛋白质依赖性阻断自发DNA链交换（DSE）之间的部分双链DNA探针，和荧光共振能量转移（FRET）。如果用荧光团和猝灭剂标记的探针在不存在靶蛋白的情况下与未标记的探针混合，则在探针之间发生链交换并导致荧光升高。相反，通过靶蛋白阻断链交换导致较低的荧光强度。重组人NF-κB（p50）以浓度依赖性方式抑制特异性探针的荧光增强，但对非特异性探针无影响。携带NF-κB结合位点的竞争者恢复荧光，并且恢复程度与竞争者的NF-κB结合位点内的核苷酸取代的数量负相关。使用p50和p52（NF-κB的另一种形式）对两种NF-κB抑制剂伊文思蓝和去羟甲基表氧喹诺霉素（[−]-DHMEQ）进行评价，并获得IC 50值。DSE-FRET技术还检测到（-）-DHMEQ对p50和p52抑制的差异效应。这些数据表明，DSE-FRET可用于高通量筛选靶向DNA结合蛋白的抗癌药物。
Nuclear factor-κB (NF-κB) is a key regulator of cancer progression and the inflammatory effects of disease. To identify inhibitors of DNA binding to NF-κB, we developed a new homogeneous method for detection of sequence-specific DNA-binding proteins. This method, which we refer to as DSE-FRET, is based on two phenomena: protein-dependent blocking of spontaneous DNA strand exchange (DSE) between partially double-stranded DNA probes, and fluorescence resonance energy transfer (FRET). If a probe labeled with a fluorophore and quencher is mixed with a non-labeled probe in the absence of a target protein, strand exchange occurs between the probes and results in fluorescence elevation. In contrast, blocking of strand exchange by a target protein results in lower fluorescence intensity. Recombinant human NF-κB (p50) suppressed the fluorescence elevation of a specific probe in a concentration-dependent manner, but had no effect on a non-specific probe. Competitors bearing a NF-κB binding site restored fluorescence, and the degree of restoration was inversely correlated with the number of nucleotide substitutions within the NF-κB binding site of the competitor. Evaluation of two NF-κB inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([−]-DHMEQ), was carried out using p50 and p52 (another form of NF-κB), and IC50 values were obtained. The DSE-FRET technique also detected the differential effect of (−)-DHMEQ on p50 and p52 inhibition. These data indicate that DSE-FRET can be used for high throughput screening of anticancer drugs targeted to DNA-binding proteins.
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影响因子：
14.9
作者：
Wang J;Li T;Guo X;Lu Z
通讯作者：
Lu Z
影响因子：
2.7
作者：
Sharma, RK;Otsuka, M;Ramos, MJ
通讯作者：
Ramos, MJ
影响因子：
5.3
作者：
Grigoriev, M;Hsieh, P
通讯作者：
Hsieh, P
DOI：
10.1084/jem.187.2.143
发表时间：
1998-01-19
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Sha WC
通讯作者：
Sha WC
影响因子：
14.9
作者：
GARNER, MM;REVZIN, A
通讯作者：
REVZIN, A