Targeting the Ets binding site of the HER2/neu promoter with pyrrole-imidazole polyamides
Targeting the Ets binding site of the HER2/neu promoter with pyrrole-imidazole polyamides
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DOI：
10.1074/jbc.m000820200
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发表时间：
2000-08-11
影响因子：
4.8
通讯作者：
Beerman, TA
中科院分区：
文献类型：
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作者：
Chiang, SY;Bürli, RW;Beerman, TA
合成了三种DNA结合聚酰胺（1-3），其以高亲和力（K-a = 8.7）结合。10（9）m（-1）至1.4。10（10）m（-1））至两个7碱基对序列，其与HER 2/neu近端启动子的调控区内的Ets DNA结合位点（EBS; GAGGAA）重叠。如通过电泳迁移率变动测定所测量的，在抑制纯化的EBS蛋白质（丝氨酸盒限制的上皮（ESX））和HER 2/neu启动子探针之间的复合物形成方面，与EBS上游（1）或下游（2和3）的侧翼元件结合的聚酰胺比天然产物偏端霉素更有效一至两个数量级。在10 nm配体浓度下完全阻断Ets-DNA复合物的形成，而在紧邻HER 2/neu EBS上游的激活蛋白-2结合位点处激活蛋白-2-DNA复合物的形成不受影响，甚至在100 nm配体浓度下也是如此。在平衡时，聚酰胺1在抑制Ets/DNA结合方面同样有效，当在体外形成蛋白质-启动子复合物之前或之后加入时，证明其可用于破坏内源性Ets介导的HER 2/neu预起始复合物。聚酰胺2是电泳迁移率变动试验中Ets-DNA复合物形成的最有效抑制剂，也是使用ESX和HER 2/neu过表达人乳腺癌细胞系SKBR-3的核提取物在无细胞系统中测量的HER 2/neu启动子驱动转录的最有效抑制剂。
Three DNA binding polyamides (1-3) were synthesized that bind with high affinity (K-a = 8.7 . 10(9) m(-1) to 1.4 . 10(10) m(-1)) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream (1) or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe, One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nm ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nar ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3.