【作者】 陈旭；

【导师】 李根喜；

【作者基本信息】 上海大学 ， 生物信息与系统生物学， 2020， 博士

【摘要】 生物活性小分子与DNA相互作用的研究是生物医学非常活跃的研究领域之一。对生物活性小分子与DNA相互作用机理的透彻揭示,不仅有利于理解细胞生理过程、解析代谢作用机制,而且可为高效的候选药物设计提供重要的指导,为有效控制不同疾病的靶向DNA药物研发提供重要依据。本论文以享有“亚洲人参”美誉的葛根中主要活性成分——葛根黄酮类化合物及其硒配合物为研究对象,探讨葛根黄酮及其硒配合物与单链DNA、双链DNA、四联体DNA、环状DNA等不同结构和功能DNA的相互作用,从不同角度揭示其互作的机制,并探索它们的相互作用对肿瘤细胞的影响。本论文的具体研究内容如下:(1)采用计算分子模拟对接方法评估了葛根黄酮类化合物与DNA的相互作用,预测了它们之间的亲和能力。由于黄酮类化合物具有C6-C3-C6双苯环联结的结构共性,因此运用适合芳香族分子的模拟计算研究,通过对接准确性较高的CDOCKER模块,首先研究了6种黄酮类化合物与双链DNA的结合情况。根据预测的结合能评分,6种黄酮类化合物与DNA亲和力的大小排序为:槲皮素>芒柄花素>大豆苷元>葛根素>4′-甲氧基葛根素>葛根素-6′′-O-木糖苷。然后,进一步研究了黄酮类化合物与艾滋病相关DNA和G-四联体DNA的亲和力,结果表明,槲皮素具有良好的DNA亲和能力,是一种潜在的先导化合物,为后续的实验研究提供了理论依据。(2)采用光谱法验证了葛根黄酮类化合物与DNA的相互作用,并验证了分子模拟对接的理论结果。我们用多种光谱法研究了葛根黄酮类化合物与两种不同双链DNA的结合亲和力,即人工合成的双链DNA(1BNA)和天然的小牛胸腺DNA(CT-DNA)。实验结果表明,6种黄酮类化合物与DNA的结合亲和力的大小顺序为:槲皮素>芒柄花素>大豆苷元>葛根素>4′-甲氧基葛根素>葛根素-6′′-O-木糖苷。光谱法实验研究结果与分子模拟对接预测的趋势结果一致,进一步说明槲皮素与DNA的亲和能力最佳。此外,通过这部分研究还获得了其相关作用的许多生物物理特征信息,为进一步明确化合物的构效关系打下了基础,并且为具有更好靶向性的天然活性先导化合物的筛选与开发创造了条件。(3)以滚环扩增的DNA为研究对象,进一步研究了葛根黄酮类化合物与DNA的相互作用。同时,建立了基于DNA滚环扩增的活性物质筛选平台,该平台可通过凝胶成像系统直接观测结果,赋予了活性物质筛选的简便性和可视化。研究发现,与DNA亲和力最强的槲皮素对DNA滚环扩增具有显著的抑制作用,而其它五种葛根黄酮类化合物没有明显的DNA抑制活性。(4)以与不同类型和功能DNA亲和力最强的槲皮素为先导化合物,并与硒配合形成活性更高的新型黄酮-硒配合物,且研究了槲皮素-硒配合物与DNA的相互作用。首先通过分子模拟对接设计并优化了槲皮素-硒配合物的结构;然后通过多种光谱分析并且在滚环扩增DNA的抑制活性筛选平台的辅助下,合成了目标槲皮素-硒配合物,并解析其分子结构;最后深入探讨了该槲皮素配合物与不同结构与功能DNA的相互作用。结果表明,该具有潜在抗癌性能的新型黄酮-硒配合物,汇聚了槲皮素和硒各自的优异生物活性。此研究为设计与开发靶向DNA的抗肿瘤候选药物提供了新的策略。(5)基于槲皮素及其硒配合物与不同结构和功能DNA的高亲和性,研究了它们与DNA相互作用对肿瘤细胞的影响。首先探讨了槲皮素及其硒配合物在细胞内的摄入情况,考察了两者对肿瘤细胞的细胞周期和细胞凋亡的影响,并分析它们能否通过细胞膜进入胞质甚至细胞核中发挥功能。结果表明,槲皮素-硒配合物的活性明显高于配体槲皮素,尤其是对MCF-7人乳腺癌细胞系的细胞毒性提高了8倍。该研究为靶向DNA的抗肿瘤预防或联合治疗药物的设计与开发提供了一种新的视角。更多还原

【Abstract】 The study of the interaction between bioactive small molecules and DNA has become one of the most active research area in biomedicine.A thorough elucidation of the interaction between bioactive small molecules and DNA not only will help to understand the physiological processes and metabolic mechanisms of cells,but also provide important guidance for designing of more efficient drug candidates,providing a strong basis to develop the targeted DNA drugs for the effective control of different diseases.In this paper,Puerariae Radix,a natural product known as "Asian ginseng",was used to study the interaction of Puerariae Radix flavonoids and the selenium complex with different DNA,including single-stranded DNA,double-stranded DNA,quadruplex DNA and circular DNA,to reveal their interaction mechanism from different perspectives,and explore their interaction effects on tumor cells.The specific research contents of this paper were as follows:(1)The interaction between pueraria flavonoids and DNA was evaluated by computational molecular docking,and their affinity was predicted.Since flavonoids had aromatic skeletons in common,the CDOCKER module with high accuracy of docking aromatic molecules was used to study the binding of six flavonoids with double-stranded DNA(ds DNA).According to the predicted score of binding energy,the affinity sequence of six flavonoids to ds DNA was as follows: quercetin >formononetin > daidzein > puerarin > 4’-methoxy puerarin > puerarin 6"-O-xyloside.In addition,the affinity of flavonoids to HIV DNA and G-quadruplex was further studied.The results showed that quercetin,with good DNA affinity,could be considered as a potential lead compound,providing a theoretical basis for subsequent studies.(2)The interaction between pueraria flavonoids and DNA was verified bymulti-spectral analyses,and the theoretical results of molecular simulation docking were confirmed.Several spectroscopic methods were discussed to study the binding affinity of pueraria flavonoids with two different double-stranded DNAs,i.e.synthetic double-stranded DNA(1BNA)and natural calf thymus DNA(CT-DNA).The results showed that the DNA binding affinity of six flavonoids followed the order: quercetin > formononetin > daidzein > puerarin > 4′-methoxy puerarin >puerarin 6″-O-xyloside.The experimental results were consistent with the trend predicted by molecular docking,indicating that quercetin had the best affinity with DNA.Moreover,this study was carried out to derive biophysical characteristics of the interactions,which provide an important basis for confirming the structure-activity correlation.These studies provided a new way for the screening and development of natural active lead compounds with better targeting.(3)The interaction between pueraria flavonoids and DNA was further studied with DNA amplified by rolling circle.Meanwhile,the bioactivity screening platform based on DNA rolling circle amplification was established,which could be directly observed by a simple visual gel imaging system.The results showed that quercetin,with the strongest affinity with DNA,had a significant inhibitory effect on DNA amplification,while the other five flavonoids had no obvious DNA inhibitory activity.(4)Quercetin,which has the strongest affinity with DNA of different types and functions,was used as a lead compound to form a new flavonoid-selenium complex combined with selenium ion,and the interaction between quercetin-selenium complex and DNA was studied.Firstly,the structure of the flavonoid-selenium complex was designed and optimized by molecular docking.With the assistance of multi-spectral analyses and the DNA inhibition screening platform,the aimed flavonoid-selenium complex was synthesized and its molecular structure was analyzed.Finally,the interaction between the novel flavonoid-selenium complex and DNA of different structures and functions was investigated.The results indicated thatvarious excellent biological activities of quercetin and selenium were respectively combined in the flavonoid complex.This study provided a new strategy for the design and development of anti-tumor drugs targeting DNA.(5)According to the high affinity between quercetin or its selenium complex and DNA of different structures and functions,the effect of their interaction with DNA on tumor cells was studied.The uptake of quercetin and its selenium complex in cells were firstly explored,then their effects on the cell cycle and apoptosis of tumor cells were investigated,and biological activities in the cytoplasm or even nucleus through the cell membrane were exhibited.The results showed that the bioactivity of the quercetin-selenium complex was significantly higher than that of the ligand quercetin,especially the cytotoxicity to the MCF-7 human breast cancer cell line increased by 8 times.These ideas gave a new perspective on the design and development of anti-tumor drugs targeting DNA for prevention or combination therapy.更多还原