Identification of novel PARP inhibitors using a cell-based TDP1 inhibitory assay in a quantitative high-throughput screening platform.
Identification of novel PARP inhibitors using a cell-based TDP1 inhibitory assay in a quantitative high-throughput screening platform.
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DOI：
10.1016/j.dnarep.2014.03.006
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发表时间：
2014-09
期刊：
影响因子：
3.8
通讯作者：
Pommier, Yves
中科院分区：
文献类型：
--
作者：
Murai, Junko;Marchand, Christophe;Shahane, Sampada A.;Sun, Hongmao;Huang, Ruili;Zhang, Yiping;Chergui, Adel;Ji, Jiuping;Doroshow, James H.;Jadhav, Ajit;Takeda, Shunichi;Xia, Menghang;Pommier, Yves
抗癌拓扑异构酶I（Top1）抑制剂（喜树碱及其临床衍生物伊立替康和拓扑替康，以及茚并异喹啉）通过稳定Top1-DNA切割复合物（Top1 cc）诱导致死性DNA损伤。这些损伤通过平行的修复途径修复，包括酪氨酰-DNA磷酸二酯酶1（TDP 1）相关途径和同源重组。由于脊椎动物中的TDP 1缺陷细胞对Top1抑制剂高度敏感，因此抑制TDP 1的小分子应增强Top1抑制剂的细胞毒性。我们开发了一种基于细胞的高通量筛选方法，用于使用与人TDP 1（hTDP 1）互补的TDP 1缺陷型鸡DT 40细胞系（TDP 1-/-）发现人TDP 1的抑制剂。在hTDP 1细胞中显示出与Top1抑制剂喜树碱（CPT）协同作用的任何化合物应该是TDP 1抑制剂或Top1 cc的替代修复途径的抑制剂。我们在CPT存在或不存在的情况下针对hTDP 1细胞筛选了400，000-化合物小分子文库储存库（SMLR，NIH Molecular Libraries）。在不存在或存在CPT的情况下使用hTDP 1和TDP 1-/-细胞的二次筛选中确认后，五种化合物被确认为潜在的TDP 1途径抑制剂。所有五种化合物在hTDP 1细胞中显示出与CPT的协同作用，但在TDP 1-/-细胞中没有，表明化合物抑制TDP 1相关的修复途径。然而，在体外凝胶为基础的测定显示，五个化合物不直接抑制TDP 1催化活性。我们测试了化合物抑制聚（ADP-核糖）聚合酶（PARP）的能力，因为已知PARP抑制剂通过抑制TDP 1向Top1 cc的募集来增强CPT的细胞毒性。因此，我们通过ELISA和Western印迹发现五种化合物抑制PARP活性。我们鉴定了最有效的化合物（Cpd 1），其提供接近维利帕尼（一种领先的临床PARP抑制剂）的特征。Cpd 1可能代表一种新的支架用于开发PARP抑制剂。
Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its clinical derivatives irinotecan and topotecan, and the indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1-/-) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1 inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1-/- cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1-/- cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit PARP activity by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.
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影响因子：
14.9
作者：
Dexheimer TS;Kozekova A;Rizzo CJ;Stone MP;Pommier Y
通讯作者：
Pommier Y
影响因子：
16.8
作者：
Berti, Matteo;Chaudhuri, Arnab Ray;Thangavel, Saravanabhavan;Gomathinayagam, Shivasankari;Kenig, Sasa;Vujanovic, Marko;Odreman, Federico;Glatter, Timo;Graziano, Simona;Mendoza-Maldonado, Ramiro;Marino, Francesca;Lucic, Bojana;Biasin, Valentina;Gstaiger, Matthias;Aebersold, Ruedi;Sidorova, Julia M.;Monnat, Raymond J., Jr.;Lopes, Massimo;Vindigni, Alessandro
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Vindigni, Alessandro
影响因子：
14.9
作者：
Alagoz M;Wells OS;El-Khamisy SF
通讯作者：
El-Khamisy SF
影响因子：
5.7
作者：
Marchand, Christophe;Lea, Wendy A.;Simeonov, Anton
通讯作者：
Simeonov, Anton
影响因子：
4.8
作者：
Interthal, H;Chen, HJ;Champoux, JJ
通讯作者：
Champoux, JJ