【作者】 祝静静；

【导师】 夏钢；

【作者基本信息】 华东师范大学 ， 基因组学， 2013， 博士

【摘要】 高通量筛选是新药发现和研发的重要手段,有效的提高了药物研发的效率和缩短药物研发周期。一个完整的高通量筛选平台包括配套的仪器设备,经济、快捷的生物活性检测方法,以及结构丰富多样的化合物库。天然产物库是新药发现和研发的重要资源,具有资源丰富,种类多样等特点。本论文中所用的天然产物库是包括10,000个醇提物组分和50,000个高效液相组分分别来自500种和171种中草药。通过整合自动液体处理站,微孔板自动分液器,多功能酶标仪以及384pin tool建立了自动化的384格式的高通量筛选平台。在高通量平台构建过程中,建立了8种用来检测酪氨酸激酶活性的酪氨酸激酶模型,2种用来检测胰高血糖素样肽受体(GLP1R)激动剂的细胞模型和10种检测细胞毒性的模型经实践验证,本论文研究所构建的高通量筛选平台能够达到每天5万样次的筛选通量。白血病和淋巴瘤是循环系统来源的恶性血液病,血液系统来源的肿瘤和实体瘤在机制和病理上有很多不同,因此开发特异性针对血液系统来源的肿瘤治疗的药物,对于实现肿瘤治疗的“个体化”具有重要的意义。而在构建的细胞模型中,BaF3是一种小鼠原B细胞具有生长周期短的特点,因此以该细胞模型为基础从天然产物库中筛选治疗白血病的活性分子。经过前期的高通量筛选,发现了一种活性组分对BaF3细胞具有很强的细胞增殖抑制能力,但是对于实体瘤细胞A549, SNU16, SNU638等的细胞增殖抑制能力要弱50倍以上。通过对活性组分进行分离纯化及结构鉴定,活性跟踪,最终鉴定活性组分为人参环氧炔醇。用流式细胞仪检测人参环氧炔醇对实体瘤细胞A549和BaF3细胞的细胞凋亡和周期指标的影响,检测结果表明人参环氧炔醇对BaF3细胞在细胞凋亡和细胞周期的影响明显高于实体瘤细胞A549。人参环氧炔醇对于其他血液瘤细胞,T细胞白血病Jurkat和淋巴瘤细胞Raji也有较高的细胞增殖抑制能力。这一现象说明人参环氧炔醇对白血病和淋巴瘤细胞具有选择性的杀伤作用,是治疗白血病和淋巴瘤的活性分子。GLP1受体(GLP1R)是2型糖尿病治疗的一个重要靶点。在体内,GLP1结合细胞上的GLP1R之后激活受体,然后使得葡萄糖依赖性的胰岛素基因表达升高,促进胰岛素的分泌。机体自身分泌的GLP1在体内的半衰期很短,易被二肽基肽酶(DPP-IV)降解而失去活性,因此寻找长效的GLP1R的激动剂是治疗2型糖尿病的一个药物研发方向。本论文研究建立了用于高通量筛选的检测GLP1R激动剂活性的-细胞模型,对来源于中草药的天然产物库进行筛选。发现了一个来源于脆蛇具有GLP1R激动剂的活性组分。和目前治疗2型糖尿病的一个临床药物Exenatide很像,该活性组分是一个具有DPP-IV的耐受性的肽类化合物。活体脆蛇的各个器官的蛋白活性检测显示活性组分主要集中在胃肠道,这与其它物种的GLP1高表达区域相吻合。本论文通过转录组测序鉴定出了脆蛇的GLP1序列。用该序列化学合成的脆蛇GLP1仍然具有GLP1R激动剂活性,但是丧失了部分DPP-IV的耐受性。说明天然脆蛇GLP1的DPP-IV耐受性可能与其二级结构或者翻译后的修饰有关。更多还原

【Abstract】 High throughput screening (HTS) is an important tool in current drug discovery. A complete HTS platform is consisted of a set of compatible equipments which is capable of precision liquid handling and highly sensitive signal acquisition, a chemical compound library which includes compounds with variety chemical scaffolds and biological activities, and a collection of HTS-compatible methods to measure biological activities. Natural product library is a good source for the drug discovery because of its abaundant and diversity. The chemical library used in this study includes10,000flash fractions and50,000HPLC fractions extracted from respectively500and171different herbs widely used in Traditional Chinese Medicine (TCM). This study combined liquid handling, dispenser, molecular device M5and384pin tool to esdablish a384format HTS platform. During the construction of the HTS system, eight assays to measure the cellular kinase activity of different tyrosine kinase, two assays to measure different GLP1R agonist activity, and10assays to masure cytotoxcicity were established. A throughput of50,000wells/day was routinely achieved in our subsequent screens using this HTS platform.Leukemia and lymphoma are hematologic malignancies, which may have different mechanisms and pathology from solid tumors. Specific drug discovery for leukemia treatment is crucial for personal cancer treatment. Because BaF3is rat pro-B cells, which expand rapidly it was used to as a cellular model to screen for inhibitors against leukemia. After the screening, one active fraction was found with high potency against the proliferation of BaF3. This fraction has little inhibitory effect on the growth of solid tumor cells, such as A549, SNU16and SNU638. After further extraction and purification procedures, the structure of active component is identified as panaxydol. Using the flow cytometry analysis, panaxydol was found to induce the apoptosis of BaF3/, but not of A549cells. Cytotoxic activity of panaxydol is also confirmed in Jurkat and Raji cells which are originally isolated from patients with leukemia and lymphoma.Glucagon-like peptide1receptor (GLP1R) is a validated target for type2diabetes treatment. In vivo, GLP1R can be activated by binding with GLP1peptide and subsequently increase glucose-dependent insulin gene expression and insulin secretion in L cells. Due to the activity DPP-IV, the endogenous GLP1peptide is cleaved and lost its activity, so a longer half-life GLP1R agonist is in great interest as a candidate in treating type2diabetes. A HTS to screen for GLP1R agonists from a natural product compound library derived from Traditional Chinese Medicine (TCM) was conducted. One fraction extracted from dry Ophisaurus harti was found to have specific GLP1agonist activity. Similar to Exenatide, a clinical-proven GLP1R agonist in treating type2diabetes, active fraction also showed sensitivity to trypsin digestion and resistant to DPP-Ⅳ cleavage, revealing its peptidic nature and DPP-Ⅳ resistance. Interestingly, Exenatide was originally identified from Heloderma Suspectum, a lizard species that belong to the same suborder of Anguimorpha as the host animals of active fraction. The active ingredient was later identified as O. harti GLP1through transcriptome analysis. The O. harti GLP1peptide was chemically synthesized based on its sequence, and was demonstrated to preserve its GLP1R agonist activity. However, chemically synthesized GLP1peptide is not DPP-Ⅳ resistant, and we suspect this was due to lack of special secondary structure, or post-translational modification which only presented in biologically generated GLP1.更多还原