Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay
Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay
复制标题
DOI：
10.1158/1535-7163.mct-08-0878
复制
发表时间：
2009-01-01
影响因子：
5.7
通讯作者：
Simeonov, Anton
中科院分区：
文献类型：
--
作者：
Marchand, Christophe;Lea, Wendy A.;Simeonov, Anton
酪氨酰-DNA磷酸二酯酶I(Tdp1)是一种分解拓扑异构酶I(Top1)-DNA加合物的酶，这种DNA加合物是由天然DNA损伤和某些抗癌药物的作用而积累的。Tdp1催化拓扑异构酶I催化酪氨酸残基与DNA 3‘-磷酸之间的磷酸二酯键的水解。据报道，Tdp1的弱抑制剂数量有限，通过筛选化学文库来识别新的化学类型的需求尚未得到满足。在这里，我们提出了一种使用AlphaScreen技术的易于配置、高度小型化和健壮的Tdp1检测方法。不受抑制的酶反应与低信号有关，而抑制导致信号的增益，使目前的检测格式特别吸引自动化的大收集高通量筛选。我们报告了四个以前未报道的Tdp1抑制剂的鉴定和初步表征。其中苏拉明、NF449和甲基-3，4-去磷他汀是磷酸酪氨酸的模拟物，可能作为Tdp1底物的诱饵。我们还报道了一种利用SCAM Tdp1突变体来研究甲基-3，4-去磷他汀作用机制的新的生化方法。[摩尔癌症治疗2009；8(1)：240-8]
Tyrosyl-DNA phosphodiesterase I (Tdp1) resolves topoisomerase I (Top1)-DNA adducts accumulated from natural DNA damage as well as from the action of certain anticancer drugs. Tdp1 catalyzes the hydrolysis of the phosphodiester bond between the catalytic tyrosine residue of topoisomerase I and the DNA 3'-phosphate. Only a limited number of weak inhibitors have been reported for Tdp1, and there is an unmet need to identify novel chemotypes through screening of chemical libraries. Herein, we present an easily configured, highly miniaturized, and robust Tdp1 assay using the AlphaScreen technology. Uninhibited enzyme reaction is associated with low signal, whereas inhibition leads to a gain of signal, making the present assay format especially attractive for automated large-collection high-throughput screening. We report the identification and initial characterization of four previously unreported inhibitors of Tdp1. Among them, suramin, NF449, and methyl-3,4-dephostatin are phosphotyrosine mimetics; that may act as Tdp1 substrate decoys. We also report a novel biochemical assay using the SCAM Tdp1 mutant to study the mechanism of action of methyl-3,4-dephostatin. [Mol Cancer Ther 2009;8(1):240-8]