基于CDK2和STAT3蛋白结构的抗癌小分子的筛
发布时间：2018-04-03 14:12
本文选题：细胞周期 切入点：CDK2 出处：《大连医科大学》2017年硕士论文
【摘要】：研究背景:随着分子生物学和结构生物学的发展,作为受体的生物大分子的三维结构被越来越多的发现和测定。根据已知生物大分子的结构信息,利用计算化学和计算生物学方法筛选与其相互作用的小分子,即基于结构的药物设计方法(structure-based durg design,SBDD)。这个方法能够大大降低新药研发的成本和周期,因此越来越多的应用在目前的药物研发领域。肿瘤作为一种严重威胁人类健康的疾病,一直是科学家们致力于攻克的目标。细胞周期蛋白依赖性激酶(cyclin dependent kinase,CDK),是丝/苏氨酸蛋白激酶家族中的一员,是参与细胞周期调控的关键酶。CDK2作为其中一个亚型,其与cyclinE和cyclin A结合后,能够促进细胞G1到S期的转变,同时参与调控S期进程。有研究发现,超过80%的癌症都会出现CDK2过表达,如肝癌,乳腺癌等,因此发现靶向作用于CDK2的抑制剂有利于发现新型抗癌药物;信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)参与JAK-STAT3信号通路的作用,JAK-STAT3信号通路是由细胞因子刺激而激活的信号通路,参与细胞核基因转录调控,对细胞正常生理功能的维持起着不可或缺的作用。然而STAT3的持续激活会诱导一系列基因高表达,促进细胞增殖,恶性转化,抑制细胞凋亡,从而导致癌症的发生,促进癌症进程。近年来也陆续发现STAT3在多种癌症中高表达,使得STAT3成为发现抗癌药物重要靶标。目前CDK2-cyclinA复合物和STAT3的三维结构都已发现并研究报道,这也为研究针对特异蛋白的小分子抑制剂提供了基础。研究目的:利用CDK2-cyclinA复合物晶体结构设计发现作用于CDK2蛋白ATP口袋的新的母核结构的化合物;利用STAT3晶体结构设计靶向作用于其SH2结构的抑制剂。研究方法:本文以CDK2-cyclinA和STAT3蛋白三维结构为基础,首先运用计算机辅助药物设计方法进行分子对接,筛选商业化合物库,对小分子进行打分和排序,购买其中打分靠前的小分子。对购买的CDK2潜在小分子抑制剂进行体外活性初筛,挑选活性最好的进行药物化学改造,以提高小分子的生物活性。将改造后具有相同母核、不同取代基的小分子进行体外活性检测,得到最好的小分子在癌细胞中进行CCK8实验,接着运用米氏方程验证小分子抑制剂的竞争性。运用FP实验对购买的STAT3潜在小分子抑制剂进行体外活性初筛,将活性较好的几个在MDA-MB-468细胞中进行CCK8实验,最终筛出活性最好的一个在多种癌细胞中进行CCK8验证。使用Biacore实验验证其作用位点。实验结果:CDK2抑制剂:初筛得到81号化合物,半数抑制浓度IC50是30.34μM。经过药物化学改造,得到CDK2抑制剂WZ-26,其IC50是3.81μM,较初筛得到的化合物活性提高了近10倍。在人源肝癌细胞HepG2中进行细胞增殖抑制检测,其IC50是30.58μM;经实验验证WZ-026是ATP竞争性抑制剂。STAT3抑制剂:FP实验初筛后挑选几个对STAT3具有最佳结合力的小分子进行细胞增殖抑制检测,最终得到作用于STAT3的抑制剂B9,在MDA-MB-468细胞中,IC50为5.1μM;在MDA-MB-231和DU145细胞中的IC50为18.87μM和63.77μM,表现出了明显的细胞增殖抑制活性,同时还利用Biacore的方法验证了小分子的作用区域是STAT3的SH2结构域。结论:运用基于结构的药物设计方法得到了:(1)具有抑制CDK2活性的小分子81,对其进行药物化学改造后,得到了小分子WZ-26,它是一个ATP竞争性抑制剂,在人源肝癌细胞中表现出增殖抑制活性;(2)具有抑制STAT3活性的小分子B9,在乳腺癌和前列腺癌细胞系中都表现出了增值抑制活性。其作用区域位于STAT3蛋白的SH2结构域。
[Abstract]:Background: with the development of molecular biology and structural biology, as the three-dimensional structure of biological macromolecules and the receptor was determined. According to the discovery of more and more structural information of known biological macromolecules and small molecules using the calculation method of chemistry and computational biology screening and interaction, namely the structure based drug design approach (structure-based durg design, SBDD). This method can greatly reduce the cost and cycle of new drug research and development, so more and more used in current drug research and development. As a serious threat to human health and disease cancer, scientists are working to capture the target. Cyclin dependent kinase (cyclin dependent, kinase, CDK) that is a threonine protein kinase family member / wire, is a key enzyme of.CDK2 in cell cycle regulation as one subtype, with cyclinE and cyc Lin combined with A, can promote cell G1 to S transition period, also involved in the regulation of S progression. Studies have found that more than 80% of all cancers occur over expression of CDK2, such as liver cancer, breast cancer, so that targeted inhibitors on CDK2 for the discovery of new anticancer drugs; signal transduction and transcription the activation of factor 3 (signal transducer and activator of transcription 3, STAT3) in the role of JAK-STAT3 signaling pathway, JAK-STAT3 signaling pathway is activated by cytokines, involved in gene transcription regulation, maintain the normal physiological function of cell plays an integral role. However, STAT3 can induce the high expression of a sustained activation a series of genes that promote cell proliferation, malignant transformation, inhibition of apoptosis, which leads to the occurrence of cancer, promote cancer progression. In recent years have found that STAT3 was highly expressed in many cancers To make STAT3 become the important target of anticancer drugs found. At present the three-dimensional structure of CDK2-cyclinA complex and STAT3 have been found and reported, which also provide a basis for the study of small molecule inhibitors of specific proteins. Objective: the structure design of nulei structure of new compounds found in CDK2 protein using ATP pocket CDK2-cyclinA composite crystal; crystal structure design using STAT3 inhibitors targeting its SH2 structure. Methods: on the basis of CDK2-cyclinA and STAT3 protein three-dimensional structure, molecular docking firstly by using the method of computer aided drug design, commercial screening of compound libraries, marking and sorting of small molecules, which marks on the purchase small molecules. The CDK2 potential of small molecule inhibitors for in vitro activity screening, selection of active transformation of pharmaceutical chemistry best, in primary school The molecular biological activity. After the transformation, with the same parent nucleus, small molecules with different substituents were in vitro, obtained the best CCK8 experiment of small molecules in cancer cells, then using the Michaelis equation verification competition of small molecule inhibitors. The use of FP in vitro experiment on STAT3 potential of small molecule inhibitors to buy at the beginning the screen, some good activity for the CCK8 experiment in MDA-MB-468 cells, finally screened the best activity in a variety of cancer cells. Using Biacore CCK8 validation experiments to verify its site. Results: CDK2 inhibitors: preliminary screening of 81 compounds, half inhibitory concentration IC50 is 30.34 M. after transformation medicinal chemistry, CDK2 inhibitor WZ-26, the IC50 is 3.81 M, a preliminary screening of compound activity increased nearly 10 times. The cell proliferation inhibition test in human hepatocellular carcinoma cell line HepG2, IC The 50 is 30.58 M; the experiment WZ-026 is a competitive inhibitor of ATP.STAT3 inhibitors: FP experiment after the screening of STAT3 to select a few small molecules with the best combination of force detecting inhibition of cell proliferation, resulting in the role of STAT3 inhibitor B9 in MDA-MB-468 cells, IC50 5.1 M in MDA-MB-231 and DU145; cells in the IC50 was 18.87 M and 63.77 M, showed obvious inhibitory activity of cell proliferation, but also the use of Biacore method to verify the interaction region of small molecules is the SH2 domain of STAT3. Conclusion: the use of drug design method based on the structure are: (1) is a small molecule that inhibits CDK2 the activity of 81, after the reform of medicinal chemistry, the small molecule WZ-26, which is a competitive inhibitor of ATP, on human hepatocellular carcinoma cells showed inhibition of proliferation activity; (2) with a small molecular B9 inhibited the activity of STAT3, and the former in breast cancer The adenocarcinoma cell lines of the adenocarcinoma show a value added inhibitory activity. The area of action is located in the SH2 domain of STAT3 protein.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91
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