丙酮酸脱氢酶激酶抑制剂的筛选及抗肿瘤作用研究

发布时间：2018-02-08 20:30

本文关键词： 丙酮酸脱氢酶激酶肿瘤 Hsp90抑制剂高内涵分析二氯乙酸　出处：《南昌大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：不同类型的肿瘤可能有不同的遗传背景，但在大多数肿瘤细胞中，其共同特征表现为肿瘤代谢异常，主要体现为有氧糖酵解的增强和氧化磷酸化的抑制。异常的代谢特征为肿瘤细胞快速增殖及凋亡缺陷提供了内环境基础，使得肿瘤细胞在其相适应的环境中存活并发生快速增殖。因此，靶向肿瘤代谢成为目前国际上抗肿瘤治疗的新策略。丙酮酸脱氢酶激酶（PDHK）作为控制糖酵解和氧化磷酸化通路的关键节点激酶，其在肿瘤细胞的快速增殖、侵袭、迁移及凋亡抵抗等过程中发挥着重要的作用。PDHK在多种肿瘤组织中异常表达，且其异常表达与肿瘤患者对放化疗治疗的敏感性、耐药产生、预后以及存活期等密切相关。因此，PDHK已成为靶向肿瘤代谢抗肿瘤策略中极具潜质的靶点，但目前还未有比较理想的抑制剂。方法：本研究在前期已建立的PDHK抑制剂分子水平筛选模型（PDHK1~4）的基础上，建立了基于高内涵分析的细胞水平高通量筛选模型（人非小细胞肺癌NCI-H1299）。采用靶向PDHK的ATP结合口袋抑制剂发现策略，基于PDHK和Hsp90蛋白ATP口袋结构高度相似的研究基础，特定针对已知Hsp90抑制剂的母核进行定向设计，改造合成两类化合物，并在已建立的分子和细胞筛选模型上对获得的化合物进行靶向性筛选和药效学评价。结果：靶向性筛选首先在分子筛选模型上对基于Hsp90抑制剂定向设计的包括改造异恶唑侧链和苯环上取代基两类共300个化合物进行初步筛选，发现43个对PDHK1有良好抑制活性的化合物；对活性较好的化合物进一步采用高内涵分析和蛋白免疫印迹方法测定其细胞活性，发现5个对PDHK1有良好抑制活性的化合物。在此基础上，药效学研究上评价了5个活性化合物的亚型特异性，发现两类化合物的代表化合物MT-ACT-216和Yhhu-3803均对PDHK四个亚型有较好的抑制作用，，且MT-ACT-216能有效影响肿瘤细胞的代谢特征，包括明显减少乳酸的生成、增加肿瘤细胞的相对氧耗量、ATP和ROS的产生等。Yhhu-3803能明显增加肿瘤细胞ROS的产生。进一步，采用TMRM染色法检测化合物对细胞线粒体膜电位的作用，发现MT-ACT-216和Yhhu-3803均能降低线粒体膜电位，提示活性化合物有可能是通过降低线粒体膜电位这一机制诱导肿瘤细胞的凋亡。进一步采用磺酰罗丹明B（Sulforhodamine B，SRB）比色法检测化合物对肿瘤细胞的增殖效应，发现MT-ACT-216和Yhhu-3803对肿瘤细胞的增殖有抑制作用。结论：综上所述，本研究在已建立的筛选平台上筛选得到靶向PDHK的两个先导化合物MT-ACT-216和Yhhu-3803。通过对其药理学进行研究，揭示了靶向PDHK的化合物可能是通过改变肿瘤细胞代谢依赖性，抑制肿瘤细胞生长。以上研究结果为同类化合物的深入研究提供了详尽的实验证据，同时为靶向PDHK抑制剂的发现和开发提供了崭新的思路。
[Abstract]:Objective: different types of tumors may have different genetic backgrounds, but in most tumor cells, their common characteristics are abnormal metabolism. The abnormal metabolic characteristics provide the internal environmental basis for the rapid proliferation and apoptosis defects of tumor cells, which are mainly reflected in the enhancement of aerobic glycolysis and the inhibition of oxidative phosphorylation. That allows tumor cells to survive and proliferate rapidly in their adaptive environment. Targeted tumor metabolism has become a new strategy of antitumor therapy in the world. Pyruvate dehydrogenase kinase (PDHKK) is a key node kinase in the control of glycolysis and oxidative phosphorylation pathway, and its rapid proliferation and invasion in tumor cells. PDHK plays an important role in the process of migration and apoptosis resistance. The abnormal expression of PDHK in various tumor tissues is associated with the sensitivity and drug resistance of tumor patients to radiotherapy and chemotherapy. The prognosis and survival time are closely related, so PDHK has become a potential target in the anti-tumor strategy of tumor metabolism, but there is no ideal inhibitor at present. Methods: in this study, the molecular level screening model of PDHK inhibitors was established. A high throughput screening model at cell level (NCI-H1299A) was established based on high intension analysis (NCI-H1299A). The detection strategy of ATP combined with pocket inhibitor targeting PDHK was used. Based on the highly similar pocket structure of PDHK and Hsp90 protein ATP, a high throughput screening model was established for human non-small cell lung cancer (NCI-H1299A). Specific targeted designs were carried out for the mother nuclei of known Hsp90 inhibitors, two kinds of compounds were synthesized and targeted screening and pharmacodynamics evaluation were performed on the established molecular and cell screening models. Results: targeting screening was performed on a molecular screening model. Three hundred compounds including modified isooxazole side chain and substituted groups on benzene ring were selected based on Hsp90 inhibitor orientation design. It was found that 43 compounds with good inhibitory activity to PDHK1 and those with good activity were further determined by high intension analysis and Western blotting. Five compounds with good inhibitory activity to PDHK1 were found. On the basis of this, the specific subtypes of the five active compounds were evaluated in pharmacodynamic studies. It was found that both MT-ACT-216 and Yhhu-3803, the representative compounds of the two compounds, could inhibit the four subtypes of PDHK, and that MT-ACT-216 could effectively affect the metabolic characteristics of tumor cells, including significantly reducing the production of lactic acid. Yhhu-3803 could significantly increase the production of ROS in tumor cells by increasing the relative oxygen consumption of tumor cells and the production of ROS. Further, the effect of compounds on mitochondrial membrane potential was detected by TMRM staining. It was found that both MT-ACT-216 and Yhhu-3803 could decrease mitochondrial membrane potential. These results suggest that the active compounds may induce apoptosis of tumor cells by decreasing mitochondrial membrane potential, and the proliferative effect of these compounds on tumor cells was further examined by sulfonyl rhodamine (BX) Sulforhodamine BHSRB colorimetry. It was found that MT-ACT-216 and Yhhu-3803 could inhibit the proliferation of tumor cells. Conclusion: in conclusion, two leading compounds, MT-ACT-216 and Yhhu-3803, were screened on the established screening platform. The pharmacology of the two leading compounds MT-ACT-216 and Yhhu-3803 were studied. The results suggest that the compounds targeting PDHK may inhibit the growth of tumor cells by changing the metabolic dependence of tumor cells. These results provide detailed experimental evidence for the further study of similar compounds. At the same time, it provides a new idea for the discovery and development of targeted PDHK inhibitors.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965.1

【共引文献】