两亲性β-咔啉二聚体DNA嵌插剂的设计合成、抗肿瘤活性及作用机制

On account of malignant tumors doing harm to people's health, the development of antitumor drugs with high-efficiency and low toxicity based on the target is an important approach. Our group has discovered seven β-carbolines derived from Peganum harmala which significantly inhibited the growth of the human lung cancer cells. Then the biological activity of 10 dimers screened from the 103 bivalent β-carbolines in two and multi-point synthesis through 6 kinds of cells, was better than that of taxol. However, due to their poor fat/water-solubility and low bioavailability, the bioactivity was restricted and whether the bivalent β-carbolines strongly reacted with DNA resulted in unclear activity. To solve the key problem, considering DNA as the target, we will screen the skeleton of amphiphilic bivalent β-carbolines (N9-N2'/N9') using molecular docking to synthesize 100 dimers combining with the structure-activity relationship of β-carboline single/dimer. Then the activity in vitro will be tested by MTT method and 3D-QSAR model between the dimers and their antitumor activity will be built by the comparative molecular force field method (CoMFA, CoMSIA). The candidates strongly combining with DNA will be further studied the effect on DNA replication through the cell cycle arrest test. We will research the mode and mechanism of action between the dimers and intracellular and extracellular DNA utilizing various techniques, such as DNA fragmentation, fluorescence spectrum, self-imaging, isothermal titration calorimetry techniques, atomic force microscope, which will be be capable of revealing antitumor dominant mechanism and in favour of discovering new amphiphilic bivalent β-carbolines anti-tumor drugs with clinical prospects.

恶性肿瘤危害人类健康，基于靶标研发高效低毒药物是重要途径之一。课题组曾发现骆驼蓬中7个β-咔啉明显抑制人肺癌细胞生长，后利用6种胞系从β-咔啉骨架2位等多点合成的103个二聚体中筛出10个，优于紫杉醇。但它们脂/水溶性均差，生物利用低而活性受限，且是否经与DNA强作用而具活性仍不清晰。为解决该关键问题，以DNA为靶点，结合β-咔啉单/二聚体的构效关系，采用分子对接筛定两亲性β-咔啉二聚体（N9-N2′/N9′）骨架，据此合成二聚体100个，MTT法测试体外活性，用比较分子力场法（CoMFA、CoMSIA）建立他们与抗肿瘤活性的3D-QSAR模型。对与DNA结合较强候选物，通过细胞周期阻滞测试对DNA复制的影响；利用DNA片段化、荧光谱和自成像、等温滴定量热、原子力显微等5种技术研究他们与胞内、外DNA互作方式和机制，揭示抗肿瘤主要机理，发现新的具有临床前景的两亲性β-咔啉二聚体抗肿瘤药物。

β-咔啉结构经改造和二聚化，会表现出更强烈的抗癌活性。本课题在前一项目100多个二聚体基础上，在β-咔啉骨架2、6等4个位置新合成二聚体124个，对人乳腺癌细胞系（MCF-7）、人肺癌细胞（A549）等6种细胞系的抗肿瘤测试表明，有13个二聚体IC50值小于10 μM；N1-N1′、N2-N2′、 N9-N9′二聚化的抗肿瘤活性明显高于 N7-N7′、C3-C3；C3位酰胺类二聚体D4的抗肿瘤活性最强，对Hep-G2达2.84-3.54 μM；通过流式试验分析结合形态学观察表明，高活性二聚体6i和6u可通过调控S期细胞周期蛋白CCNA2，将肿瘤细胞阻滞于S期或G2/M期，显著诱导细胞凋亡。利用热变性实验等研究，二聚体与DNA发生嵌插结合，导致DNA构象改变，诱导DNA片段化，从而表现出很强的抗肿瘤活性。另外，合成的β-咔啉衍生物及二聚体具有显著的抗菌和抗病毒活性；β-咔啉可通过破坏细菌细胞壁、破坏细胞膜通透性和完整性及DNA促旋酶的功能等机制发挥抗菌作用。筛得有效抗新城疫病毒（NDA）化合物27种，且不同结构的β-咔啉衍生物可通过不同靶点或信号通路发挥抗病毒作用，活性最高化合物11可通过抑制HSP90β从而抑制NDV L蛋白；或通过抑制GSK-3β，激活β-catenin介导的Wnt通路，从而上调干扰素及干扰素刺激基因的表达，主要是通过促进宿主细胞的免疫反应以及抑制病毒蛋白质的合成这两种途径。这两种途径均有效发挥抗新城疫病毒（NDV）作用。上述结果不仅丰富了β-咔啉二聚体结构多样性，且筛选出高活性先导化合物，发现二聚体抗肿瘤效果是通过调节肿瘤微环境中多种途径和多靶点所实现的，有利于对多种抗肿瘤机制之间的关联和网络构架分析，对后续从分子和功能层次理解β-咔啉单体和二聚体抗肿瘤、抗病毒作用机制具有重要价值，为研发相关新药提供了新思路。