新型拓扑异构酶抑制剂RiccardinD选择性靶向TopoIIα的抗肿瘤作用分子机理研究

The incidence and mortality rate in malignant tumor increased recent years. The adverse effects caused by targeting both TopoIIα and TopoIIβ limited the clinical application of chemotherapy agents.It is significnat to develop inhibitors selectively targeting TopoIIα.Riccardin D(RD), a novel macrocyclic bisbibenzyl compounds extracted from the Chinese liverwort plant, and its analogues were found to be effective in vivo and vitro studies on inhibiting the growth of a variety of human tumors including non-small cell lung cancer, intestinal adenomaoral squamous carcinoma,hepatocellular carcinoma andleukemia. However,the accurate target of RD inhibiting tumor growth is not clear. Pre-test confirmed that RD induced apoptosis by selectively targeting TOPOIIα. Based on this, the homology modeling, molecular docking,SPR, transfected technology was used to explore two problems: 1. The specific binding sites of RD targeting TOPOIIα? 2.The regulation effect of HMGB1-RAGE-TLRs-NF-κB signaling pathway on the TOPOIIα inhibition produced by RD? To clarify the aboved issues will be helpful to modificate the structure of RD and its analogs.It is of great significance to develop the drug targeting TopoIIα after identifying the molecular mechanisms of such compounds.

恶性肿瘤发病率及死亡率逐年升高，以TopoII为靶点的化疗药物因双靶向引起的不良反应临床应用受限，开发选择性靶向TopoIIα拓扑异构酶抑制剂意义重大。Riccardin D(RD)是从苔类植物分离所得结构全新的大环双联卞类化合物，实验发现RD及其类似物对肺癌、结肠癌、肝细胞癌、白血病及口腔癌有较好疗效，但RD发挥抗肿瘤作用的靶点尚不明确。前期证实RD可选择性靶向TOPOIIα诱导癌细胞凋亡。在此基础上，采用同源模建、分子对接、SPR、细胞转染等技术深入探讨：1. RD与TOPOIIα靶酶的具体结合位点？2. RD是否通过调控HMGB1-RAGE-NF-κB信号通路对TOPOIIα产生抑制作用？阐明上述问题有利于以RD为代表的双联卞类化合物结构的进一步修饰优化，深入解析此类化合物抗肿瘤的分子机制对选择性靶向TOPOIIα的药物研发有着重要意义。

恶性肿瘤发病率逐年升高，以拓扑异构酶II为靶点的化疗药物在临床取得一定疗效，被美国国立癌症研究所列为重点研究的六大类抗肿瘤药物之一。但临床应用的拓扑异构酶II抑制剂多为TopoIIα和TopoIIβ双靶向制剂，可诱发继发性急性髓样白血病、产生心脏毒性、多药耐药等副作用。因此，开发靶点专一、高效低毒的选择性拓扑异构酶II抑制剂意义重大。课题前期研究发现，大环双联卞类化合物Riccardin D可选择性抑制TopoIIα催化活性，两者作用机理未明确。本课题研究发现，Riccardin D与TopoIIα催化核心区域中催化活性位点的结合能力较弱，Riccardin D与TopoIIα ATPase结构域的相互作用试验结果显示无显著结合。根据课题第二部分设计内容，试验结果显示，与未处理组比较，Riccardin D处理组的K562细胞内及培养上清HMGB1蛋白和mRNA表达水平明显降低，具有统计学意义。同时也检测了Riccardin D对HMGB1-RAGE-NF-κB信号通路的调控研究，结果显示Riccardin D可显著降低该信号通路中关键蛋白NF-κB、磷酸化NF-κB以及下游信号分子的TNFα的表达，提示Riccardin D可能通过对HMGB1-RAGE-NF-κB信号通路的调控发挥抗肿瘤作用。此外应用AutoDock3.0.3将Riccardin D与NF-κB的活性区域进行对接模拟，经过分子对接后，发现Riccardin D与NF-κB的活性区域间可以结合。

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