【作者】 孙超；

【导师】 金英花；

【作者基本信息】 吉林大学 ， 细胞生物学， 2013， 博士

【摘要】 目前，癌症已经成为威胁人类健康的重大恶性疾病之一。美国癌症协会(American Cancer Society, ACS)公布的最新癌症统计数据(CA Cancer J Clin.2011;61:69)显示，2008年全球癌症新发病例为1270万，其中死亡率高达59.84%。细胞周期的亢进和细胞凋亡的受阻共同导致细胞癌变和肿瘤的发生。哺乳动物细胞通过细胞周期依赖性激酶(Cyclin-dependent kinases, Cdks)有序地激活驱动周期各个时相之间有条不紊的转换。Cdks活性异常上调是肿瘤细胞重要的生物学特征，如在乳腺癌、肺癌、结直肠癌、肝癌和恶性淋巴癌细胞中，频繁检测到极度活跃的Cdks活性。目前，Cdks已经成为癌症治疗的重要靶标。肿瘤细胞的另一个特征，即细胞凋亡通路的受损，也是肿瘤细胞对治疗产生耐药性的主要原因，例如：约80%的人肝癌细胞过表达XIAP蛋白质，以抑制Caspases的激活和进一步活化；50%以上人类肿瘤细胞增强表达抗凋亡蛋白质Bcl-2；肿瘤细胞通过表达诱饵受体DcR1和DcR2，抵制TRAIL诱导的细胞凋亡。以上机制直接或间接促成了肿瘤的发生和发展，也给肿瘤的治疗增加了难度。目前，治疗效果差、边缘效应严重以及多种药物获得性耐受，是肿瘤治疗面临的普遍难题。因此，研发强效且特异性高的抗癌药物是现今医药领域面临的巨大挑战。本论文中，我们以广谱Cdks抑制剂Xylocydine作为先导化合物，开发Cdks家族中掌控细胞周期G1/S期转换和S期进程的Cdk2特异性抑制剂。为此，我们解析了Cyclin A3-Cdk2-Xylocydine的晶体结构，进而确定了Xylocydine的改造位点，以此为基础设计、合成了一系列Xylocydine衍生物，并阐述了它们对肿瘤细胞的作用机制，获得了以下创新性研究成果：1)我们首次成功解析了Cyclin A3-Cdk2-Xylocydine三元复合物的晶体结构；2)晶体结构信息表明，Xylocydine C6位的Br原子是有效的修饰位点。通过对这一位置进行改造，我们合成了24个芳基和杂芳基取代的全新Xylocydine衍生物。利用体外Cdks激酶活性测定，获得了三个Cyclin A-Cdk2高选择性抑制剂，即3h、3i和3j；与Cyclin B-Cdk1相比，选择性至少提高了20倍。四甲基偶氮唑盐微量酶反应比色(3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazoniumbromide, MTT)结果显示，化合物3h-j对肿瘤细胞HeLa没有显著的细胞毒性，然而可以有效地阻滞HeLa细胞周期在G1/S期。这表明，3h-j在细胞内可能同样通过抑制Cdk2的活性，进而阻止肿瘤细胞的增殖。为了阐明Cdk2与抑制剂之间的构效关系，我们进行了分子对接分析。结果表明，Xylocydine6-Br位合理地修饰，确实能够对由Lys33、Glu51、Phe80和Asp145形成的Cdk2催化中心区域进行填充，且显著提高了抑制剂对Cdk2的选择性。此外，对Xylocydine改造过程中，其嘌呤环的空间方位、嘌呤环C4位-NH2与Cdk2Leu83残基以及晶体结构数据中的呋喃糖环C11位-OH与Cdk2Asp145残基之间形成的氢键，对抑制剂的活性可能有着至关重要地影响，应给予充分地重视。3)利用MTT法对Xylocydine衍生物进行细胞毒性分析，我们获得了一个具有肿瘤细胞特异性杀伤活性的新型化合物，将其命名为JRS-15。JRS-15对多种肿瘤细胞均展现出强烈的细胞毒性和促凋亡活性，其IC50值在12.42到28.25μM之间，比它的母体化合物Xylocydine更加强效。重要的是JRS-15对人源正常细胞LO2表现出较弱的细胞毒性。JRS-15作用机制研究表明，低浓度JRS-15(5.0μM)强烈阻滞HeLa细胞周期在G1/S期；较高剂量(25μM)时触发Bax和Bak转位到线粒体，导致线粒体膜电位去极化，随后释放促凋亡因子Cytochrome c和Smac到细胞浆中，从而激活起始Caspase-9，进而激活效应Caspase-3，最终诱导细胞凋亡。然而在整个过程中都没有检测到死亡受体通路介导的起始Caspase-8的激活。此外，JRS-15诱导的HeLa细胞凋亡伴随着抗凋亡蛋白XIAP和Bcl-xL的降解。进一步分析表明，Caspase-9和广谱Caspases抑制剂以及过表达抗凋亡蛋白XIAP或Bcl-xL均有效抑制JRS-15诱导的细胞凋亡。这些研究数据表明：JRS-15通过调节多种细胞凋亡调控蛋白，来诱导线粒体介导的內源型细胞凋亡。因此，我们合成的新型化合物JRS-15有望成为一个强效的抗癌候选药物。更多还原

【Abstract】 Cancer is among the most fatal diseases threatening humans worldwide. About12.7million cancer cases and7.6million deaths have occurred in2008according tothe latest global cancer statistics (CA Cancer J Clin.2011;61:69). Deregulation ofcell cycle and inactivation of apoptosis together lead to the malignant transformationand tumor development. In mammalian, the cell cycle is governed by the sequentialactivation of cyclin-dependent kinases (Cdks), thereby drives the orderly transitionof each phase. Abnormal upregulation of Cdk activity is the major biologicalcharacteristic in tumor cells, for example, in breast carcinoma, lung carcinoma,colorectal carcinoma, hepatocellular carcinoma and malignant lymphomas. Currently,Cdks have been proven to be important targets for anticancer therapy. Anotherhallmark of the tumor cells is the impaired apoptosis pathway, which is the mainreason for drug resistance, for instance, approximately80%of human hepatoma cellsoverexpress XIAP protein to inhibit the activation of caspases; over50%of humantumor cells enhance the expression of anti-apoptotic molecule Bcl-2; and in somecases, tumor cells resist TRAIL-induced apoptosis by expressing the decoy receptorsDcR1and DcR2. These mechanisms mentioned above directly or indirectlycontribute to the initiation and the development of cancer, and also increase thedifficulties for cancer treatment. Nowadays, poor therapeutic outcomes and seriousside effects, together with acquired resistance to multiple drugs, are commonproblems of cancer therapies. Therefore, there is an urgent need for novelcancer-targeted drugs with strong activity and high specificity, which has become thegreat challenge in the field of drug discovery and development.In this thesis, we choose Xylocydine, a broad-wide of Cdks inhibitor, as theleading compound to develop highly specific inhibitors of Cdk2, a critical regulatorof the G1/S phase transition and the S-phase progression in cell cycle. In this respect, we resolved the crystal structure of Cyclin A3-Cdk2-Xylocydine complex, anddetermined the modification site of Xylocydine basis on the crystal structureinformation. Then, we designed and synthesized a series of Xylocydine derivatives,and clarified their functional mechanisms against tumor cells.In this study, we have obtained three major innovative results:1) It is the first time that we have successfully resolved the crystal structure ofCyclin A3-Cdk2-Xylocydine complex (Resolution2.5);2) The information of Cyclin A3-Cdk2-Xylocydine structure suggested that theC6-Br position of Xylocydine is the potent modification stie. Then, wesynthesized twenty-four Xylocydine-derived compounds that substituted6-Brwith aryl and heteroaryl groups. The in vitro kinase assay showed that threecompounds,3h,3i and3j significantly inhibited Cyclin A-Cdk2activity, at leastincreasing20-folds selectivity compared to Cyclin B-Cdk1. The3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoniumbromide (MTT) assayshowed that3h-j exhibited inconspicuous cell killing effects, however, they allstrongly arrested the cell cycle at G1/S phase in HeLa cells. These data indicatethat3h-j may also inhibit the Cdk2activity in vivo, and thereby prevent theproliferation of tumor cells. To clarify the structure-activity relationship (SAR)between Cdk2and these inhibitors, we performed the molecular dockinganalysis, and the results showed that the region of the catalytic pocket in Cdk2formed by Lys33, Glu51, Phe80and Asp145amino acid residues confer a veryimportant opportunity for selectivity to Cdk2inhibitors. In addition, the spatialorientation of the purine ring, the hydrogen bonding between–NH2(XylocydineC4position) and Leu83(Cdk2) as well as-OH (Xylocydine C11position of thecrystal structure information) and Asp145(Cdk2) may play a crucial impact onthe activity of Cdk2inhibitors. These cases should be given full consideration inthe further work.3) By analyzing the cytotoxicity of Xylocydine derivatives using the MTT assay,we obtained an active compound, named JRS-15. JRS-15exhibited muchstronger cytotoxic and pro-apoptotic activity than its parent compound in various cancer cell lines, with IC50values ranging from12.42to28.25μM.Importantly, it is more potent for killing cancer than non-cancerous cells.Mechanistic studies showed that JRS-15treatment (5.0μM) dramaticallyarrested the cell cycle at G1/S phase, while under high concentration (25μM) itinduced apoptosis in HeLa cells. It triggered the translocation of both Bax andBak to the mitochondria, resulting in mitochondrial membrane potential (MMP)depolarization and the subsequent release of cytochrome c as well as the secondmitochondria-derived activator of caspase (Smac) into the cytosol. Thesequential activation of caspase-9and caspase-3eventually results in cellapoptosis. Caspase-8, an initiator caspase that is required to activate themembrane receptor-mediated extrinsic apoptosis pathway was not activated inJRS-15-treated cells. Further analysis showed that the levels of theanti-apoptotic proteins Bcl-xL and XIAP were significantly reduced uponJRS-15treatment. Furthermore, the caspase-9or pan-caspase inhibitor, andBcl-xL or XIAP overexpression all effectively prevented JRS-15-inducedapoptosis. Taken together, these results indicate that JRS-15induces cancer cellapoptosis by regulating multiple apoptotic proteins, and this compound maytherefore be a good candidate reagent for anticancer therapy.更多还原