A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A.
A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A.
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DOI：
10.1016/j.ab.2011.11.025
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发表时间：
2012-02-15
影响因子：
2.9
通讯作者：
Fesik SW
中科院分区：
文献类型：
--
作者：
Souza-Fagundes EM;Frank AO;Feldkamp MD;Dorset DC;Chazin WJ;Rossanese OW;Olejniczak ET;Fesik SW
复制蛋白A（RPA）与多个检查点蛋白相互作用，并通过ATR激酶促进信号传导，ATR激酶是哺乳动物对DNA损伤反应中检查点途径的关键调节因子。在癌细胞中，DNA修复活性的增加有助于对化疗的抵抗。因此，阻断检查点蛋白与RPA结合的小分子可以抑制DNA损伤反应，从而使癌细胞对DNA损伤剂敏感。在这里，我们报告了一个同质的，高通量的荧光偏振检测的发展，用于识别化合物，阻止关键的蛋白质-蛋白质相互作用位点的RPA 70 N的基本裂缝。一个FITC标记的肽来自ATR辅因子，ATRIP，被用作探针的结合试验。使用源自ATRIP、RAD 9、MRE 11和p53的肽证实了该测定准确检测相关配体的能力。使用2000种化合物的Spectrum收集，验证了该测定法用于高通量筛选。FPA试验在384孔板中进行，Z'因子≥0.76，并鉴定了几种能够抑制RPA 70 N结合界面的化合物。
Replication Protein A (RPA) interacts with multiple checkpoint proteins and promotes signaling through the ATR kinase, a key regulator of checkpoint pathways in the mammalian response to DNA damage. In cancer cells, increased DNA repair activity contributes to resistance to chemotherapy. Therefore, small molecules that block binding of checkpoint proteins to RPA may inhibit the DNA damage response and thus sensitize cancer cells to DNA-damaging agents. Here we report on the development of a homogenous, high-throughput fluorescence polarization assay for identifying compounds that block the critical protein-protein interaction site in the basic cleft of RPA70N. A FITC-labeled peptide derived from the ATR cofactor, ATRIP, was used as a probe in the binding assay. The ability of the assay to accurately detect relevant ligands was confirmed using peptides derived from ATRIP, RAD9, MRE11, and p53. The assay was validated for use in high-throughput screening using the Spectrum collection of 2000 compounds. The FPA assay performed with a Z’ factor of ≥0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface.
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影响因子：
64.8
作者：
Bartkova, J;Horejsi, Z;Bartek, J
通讯作者：
Bartek, J
影响因子：
3.5
作者：
Glanzer, Jason G.;Liu, Shengqin;Oakley, Gregory G.
通讯作者：
Oakley, Gregory G.
影响因子：
5.3
作者：
Xu, Xin;Vaithiyalingam, Sivaraja;Cortez, David
通讯作者：
Cortez, David
影响因子：
3.5
作者：
WANG, ZX
通讯作者：
WANG, ZX
影响因子：
56.9
作者：
Shuker, SB;Hajduk, PJ;Fesik, SW
通讯作者：
Fesik, SW