Identification of small molecule inhibitors of ERCC1-XPF that inhibit DNA repair and potentiate cisplatin efficacy in cancer cells.
Identification of small molecule inhibitors of ERCC1-XPF that inhibit DNA repair and potentiate cisplatin efficacy in cancer cells.
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DOI：
10.18632/oncotarget.12072
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发表时间：
2016-11-15
期刊：
影响因子：
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通讯作者：
Patrick SM
中科院分区：
文献类型：
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作者：
Arora S;Heyza J;Zhang H;Kalman-Maltese V;Tillison K;Floyd AM;Chalfin EM;Bepler G;Patrick SM
ERCC1-XPF异源二聚体是一种5‘-3’结构特异性内切酶，在哺乳动物细胞的多种DNA修复途径中起重要作用。ERCC1-XPF(ERCC1-ERCC4)修复顺铂-DNA链内加合物和链间交联物，其特异性抑制已被证明增强顺铂对癌细胞的细胞毒作用。在这项研究中，我们描述了一种高通量筛选(HTS)，用于识别抑制ERCC1-XPF内切酶活性的小分子。初步筛选确定了两种在纳摩尔范围内抑制ERCC1-XPF活性的化合物。这些化合物在二次筛选中与另外两个不相关的核酸内切酶进行了验证，以确保特异性。这些筛选的结果用体外凝胶核酸酶试验进行了验证。电泳迁移率改变分析(EMSA)进一步表明，这些化合物不抑制纯化的ERCC1-XPF与DNA的结合。其次，在肺癌细胞中，这些化合物增强了顺铂的细胞毒性，并抑制了DNA修复。构效关系(SAR)研究确定了其中一种原始HITS的相关化合物，这也增强了顺铂对癌细胞的细胞毒性。令人兴奋的是，在肺癌异种移植模型中，给予NSC16168化合物可增强顺铂的抗肿瘤活性。ERCC1-XPF DNA修复抑制剂的进一步开发有望使癌细胞对基于DNA损伤的化疗敏感。
ERCC1-XPF heterodimer is a 5′-3′ structure-specific endonuclease which is essential in multiple DNA repair pathways in mammalian cells. ERCC1-XPF (ERCC1-ERCC4) repairs cisplatin-DNA intrastrand adducts and interstrand crosslinks and its specific inhibition has been shown to enhance cisplatin cytotoxicity in cancer cells. In this study, we describe a high throughput screen (HTS) used to identify small molecules that inhibit the endonuclease activity of ERCC1-XPF. Primary screens identified two compounds that inhibit ERCC1-XPF activity in the nanomolar range. These compounds were validated in secondary screens against two other non-related endonucleases to ensure specificity. Results from these screens were validated using an in vitro gel-based nuclease assay. Electrophoretic mobility shift assays (EMSAs) further show that these compounds do not inhibit the binding of purified ERCC1-XPF to DNA. Next, in lung cancer cells these compounds potentiated cisplatin cytotoxicity and inhibited DNA repair. Structure activity relationship (SAR) studies identified related compounds for one of the original Hits, which also potentiated cisplatin cytotoxicity in cancer cells. Excitingly, dosing with NSC16168 compound potentiated cisplatin antitumor activity in a lung cancer xenograft model. Further development of ERCC1-XPF DNA repair inhibitors is expected to sensitize cancer cells to DNA damage-based chemotherapy.