基于蛋白质结构的抗糖尿病天然产物作用靶点和机制研究

发布时间：2018-09-18 20:25

【摘要】：本文共包括两部分,第一章详细论述了金线莲苷衍生物C6对血管内皮生长因子B (VEGFb)-血管内皮生长因子受体1(VEGFR1)通路的抑制作用,综合评价C6对自发性2型糖尿病db/db小鼠模型的治疗作用,探讨其降糖作用机制：第二章则论述了新型11β-HSD1天然小分子抑制剂虚拟筛选及活性评价的研究过程。第一章本课题组前期对具有降糖降脂活性天然产物金线莲苷进行结构改造和修饰,通过化学合成法合成一系列衍生物C1-6,前期活性初筛显示C6可显著控制STZ诱导2型糖尿病大鼠血糖水平,且其治疗效果显著优于其他衍生物。本文通过计算机反向虚拟筛选技术发现其潜在的作用靶点为VEGFR1。为验证反向虚拟筛选的结果,本文成功表达并纯化VEGFb及其受体VEGFR1结合部分结构域,微量热泳动研究发现二者结合力极高,解离常数(Kd)为757±67.5 nM, C6可显著抑制VEGFb与VEGFR1的结合,EC50为5.37±0.79 μM,远低于其他衍生物。体内实验证实C6具有良好的体内抗糖尿病活性,能治疗并改善自发性2型糖尿病db/db小鼠的高血糖、胰岛素抵抗和高血脂等症状,提高糖尿病小鼠的糖耐量；降低肝脏及肾脏损伤程度,保护胰腺形态,抑制胰岛增生,维护胰岛细胞功能稳态；同时C6能降低心肌组织内、/EGFb-VEGFR1信号通路下游蛋白FATP3和FATP4的表达水平；说明其降糖机制为控制心脏和骨骼肌中脂肪酸的内皮摄取和运输,阻止异常的脂质沉积,增加肌肉葡萄糖摄取,最终达到治疗2型糖尿病的目的。综上所述,金线莲苷衍生物C6是一个新型小分子VEGFb-VEGFR1信号通路抑制剂,可作为潜在的2型糖尿病治疗候选药物。第二章糖皮质激素(Glucocorticoids, GCs)具有调节糖和脂肪的代谢、免疫反应、细胞增殖等作用,糖皮质激素的蓄积往往会导致代谢综合征的出现,包括肥胖、胰岛素抵抗、高血脂、高血压和内脏的脂肪堆积等。11p羟基类固醇脱氢酶1(11β-HSD1)是调节人体代谢的关键酶,其在NADPH辅助下催化无活性可的松还原成有活性氢化可的松,放大局部活性糖皮质激素水平,而抑制11β-HSD1可恢复外周组织对胰岛素的敏感性,因此11β-HSD1成为当今治疗糖尿病研究热点。天然产物因其骨架结构丰富、药理作用广泛及相对低毒性等优势,成为当今最大的药物来源。本文通过计算机虚拟筛选技术,从ZINC天然产物数据库及本课题组324个天然产物中筛选潜在的11β-HSD1抑制剂,然后通过微量热泳动技术评价课题组评分前20的化合物与11β-HSD1的结合力,并采用HPLC-MS/MS方法评价其体外抑制活性,最后采用蛋白质晶体学技术尝试得到活性天然产物与11β-HSD1的共结晶。本文发现一个天然活性11β-HSD1抑制剂,其1C50为1.86μM且可与重组人11β-HSD1显著结合(Kd=256±11μM),虽然暂时未获得分辨率较高的晶体结构,但计算机对接发现该化合物可有效占据11β-HSD1的活性位点,是极具有潜力的先导化合物,为11β-HSD1抑制剂的研发提供理论和实验基础。
[Abstract]:The first chapter discusses the inhibitory effect of Clematis glycoside derivative C6 on vascular endothelial growth factor B (VEGFb) - vascular endothelial growth factor receptor 1 (VEGFR1) pathway in detail, evaluates the therapeutic effect of C6 on spontaneous type 2 diabetic db / DB mice, and explores its hypoglycemic mechanism. The second chapter discusses the novel mechanism of action. Chapter 1: The structure modification and modification of the natural product of eleanosine, which has hypoglycemic and lipid-lowering activity, were carried out in the early stage. A series of derivatives C1-6 were synthesized by chemical synthesis. The preliminary screening showed that C6 could significantly control STZ-induced type 2 diabetes mellitus. In order to verify the results of the reverse virtual screening, we successfully expressed and purified some of the binding domains of VEGFb and its receptor, VEGFR1, and found that the binding force of the two domains was detected by microthermophoresis. The dissociation constant (Kd) was 757 65507 Glucose tolerance in mice with urinary disease; decreased liver and kidney damage, protected pancreatic morphology, inhibited islet proliferation, and maintained islet function homeostasis; C6 also decreased the expression of FATP3 and FATP4 downstream proteins of / EGFb-VEGFR1 signaling pathway in myocardial tissue; indicating that the mechanism of lowering blood glucose was to control the fatty acids in heart and skeletal muscle. In summary, Clematis glycoside derivative C6 is a novel small molecule inhibitor of VEGFb-VEGFR1 signaling pathway and can be used as a potential candidate drug for the treatment of type 2 diabetes. Chapter 2 Glucocorticoid Glucocorticoid accumulation often leads to metabolic syndrome, including obesity, insulin resistance, hyperlipidemia, hypertension and visceral fat accumulation. 11p-hydroxysteroid dehydrogenase 1 (11beta-HSD1) is the key enzyme in regulating human metabolism, and its accumulation in NADP. H-assisted reduction of inactive cortisone to active hydrocortisone enlarges locally active glucocorticoid levels, and inhibition of 11-beta-HSD1 restores insulin sensitivity in peripheral tissues. Therefore, 11-beta-HSD1 has become a hot topic in the treatment of diabetes mellitus. In this paper, the potential 11 beta-HSD1 inhibitors were screened from ZINC natural product database and 324 natural products by computer virtual screening technology, and then the binding force between the compounds in the first 20 scores and 11 beta-HSD1 was evaluated by microcalorimetric swimming technique, and the binding force between the compounds in the first 20 scores and 11 beta-HSD1 was evaluated by HPLC-MS/M. S method was used to evaluate its inhibitory activity in vitro, and protein crystallography was used to try to obtain the co-crystallization of the active natural product with 11 beta-HSD1. A natural active 11 beta-HSD1 inhibitor, whose 1C50 was 1.86 mu M, was found to bind significantly to recombinant human 11 beta-HSD1 (Kd = 256 + 11 mu M), although no high resolution crystal structure was obtained for the time being. However, computer docking showed that the compound could occupy the active site of 11 beta-HSD1 effectively, and it was a promising lead compound, providing theoretical and experimental basis for the research and development of 11 beta-HSD1 inhibitors.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R914;R965

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