通过二氨基对甲氧基苯并咪唑（Hx）聚酰胺靶向抑制 NF-Y：DNA 结合来调节拓扑异构酶 IIα 的表达和化学敏感性。
Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide.
机构信息
Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, London, WC1E 6BT, UK.
Department of Chemistry, Hope College, Holland, MI 49423, United States.
出版信息
Biochim Biophys Acta Gene Regul Mech. 2017 May;1860(5):617-629. doi: 10.1016/j.bbagrm.2016.10.005. Epub 2016 Oct 24.
BACKGROUND
Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxIP 2 and HxIP 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole.
METHODS
DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®.
RESULTS
Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxIP 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP 3 stimulated dose-dependent (0.1-2μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage.
CONCLUSIONS
Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents.
GENERAL SIGNIFICANCE
Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.
背景
靶向拓扑异构酶 IIα（topo IIα）启动子上的倒 CCAAT 盒 2（ICB2）的序列特异性多聚酰胺 HxIP1 可以抑制 NF-Y 结合，重新诱导基因表达，并增加依托泊苷的敏感性。为了增强生物活性，合成了含二氨基的衍生物（HxIP2 和 HxIP3），在咪唑/吡咯的 N1-杂环位置引入了一个烷基氨基。
方法
使用 DNase I 足迹法评估二氨基 Hx-多聚酰胺的 DNA 结合能力，并使用 EMSA 评估其破坏 NF-Y:ICB2 相互作用的能力。用 HxIP*3 预处理后，用 Cell-Titer Glo®测定细胞活力，用 RT-PCR 和免疫印迹法测定 A549 细胞中 18 小时多聚酰胺处理后 topo IIαmRNA 和蛋白（免疫印迹）水平。γH2AX 用作依托泊苷诱导的 DNA 损伤的标志物。
结果
在 topo IIα 启动子上引入 N1-烷基氨基基团降低了对靶序列 5'-TACGAT-3'的选择性，但增加了 DNA 结合亲和力。共聚焦显微镜显示两种荧光二氨基多聚酰胺均定位于细胞核内，但 HxIP2 无法破坏 NF-Y:ICB2 相互作用，对 topo IIα 的下调也没有影响。相比之下，HxIP3 抑制 NF-Y 结合刺激 topo IIα 的剂量依赖性（0.1-2μM）重新诱导，并通过增强 DNA 损伤增强拓扑异构酶 II 毒物的细胞毒性，从而增强拓扑异构酶 II 毒物的细胞毒性。
结论
N1-位的多聚酰胺官能化提供了一种设计策略，可以改善药物样性质。二价阳离子 HxIP*3 增加了 topo IIα 的表达，并增强了对拓扑异构酶靶向药物的化学敏感性。
一般意义
拓扑异构酶 IIα 表达的药理学调节有可能提高细胞对临床使用的抗癌治疗药物的敏感性。本文是由 Roberto Mantovani 教授编辑的特刊“核因子 Y 在发育和疾病中的作用”的一部分。
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