【作者】 王静；

【导师】 赵新锋；

【作者基本信息】 西北大学 ， 中药药物化学， 2019， 博士

【摘要】 呼吸系统疾病是重大慢性非传染性疾病,具有发病率高、药物依赖性大和反复发作等特点,迫切需要新技术和新方法,研发高效创新药物,以实现该类疾病的有效防治。中药作为呼吸系统疾病的主要防治手段,其活性成分筛选一直是中药创新药物研发的卡脖子瓶颈问题,已发展成为医药卫生领域正在新兴的难点和热点问题之一。本论文以三子养亲汤为例,针对其抗哮喘活性成分不明确和靶点不清晰等问题,重点开展活性成分固定化肾上腺素受体色谱筛选新方法的创建和应用研究。该研究有望实现三子养亲汤抗哮喘活性成分快速解析、解决药物活性成分筛选周期长和准确率低的瓶颈问题,能为其他中药活性成分筛选提供方法借鉴,对高效中药创新药物研发具有积极作用。全文共分四章,作者的主要贡献如下:1、建立了一步化β₂-AR色谱固定相制备新方法。以β₂-肾上腺素受体（β₂-Adrenoceptor,β₂-AR）为例,将O⁶-烷基鸟嘌呤DNA甲基转移酶融合至受体非活性碳末端,大肠杆菌表达获得融合受体,利用细胞裂解液中重组受体所含酶与苄基鸟嘌呤衍生物修饰微球间的特异性生物正交反应,将受体共价固定至微球表面,制备固定化β₂-AR色谱固定相。扫描电子显微镜等技术对其形貌进行表征,免疫扫描电子显微镜等技术对其抗体识别活性进行表征,液相色谱-质谱法对其脂质微环境进行表征。结果表明,该方法可通过一步反应实现β₂-AR单层均质定向固定化,具有特异性强,固定化受体稳定性好、活性高等特点,能为高活性受体色谱固定相的快速制备提供方法学借鉴。2、以β₂-AR特异性药物沙丁胺醇、甲氧那明和特布他林为工具,分别采用前沿分析法和扰动峰分析法研究了三种药物与固定化β₂-AR的相互作用,分子对接法对其进行探讨。结果表明,三种药物在固定化β₂-AR色谱固定相表面的吸附行为符合单朗格缪尔吸附模型,结合常数分别为1.63×10⁴ L/mol,3.03×10⁴ L/mol和0.76×10⁴ L/mol,氢键和范德华力为药物-受体相互作用的主要推动力;分子对接研究结果表明,沙丁胺醇等药物主要通过氨基酸残基Ser 203,Ser 204,Ser 207和Asp 113与受体发生特异性作用。上述结果提示:固定化β₂-AR可依据亲和力大小识别其特异性配体,能应用于受体-药物相互作用研究,为β₂-AR特异性靶向活性成分筛选提供色谱新方法。3、采用固定化β₂-AR色谱模型对中药复方三子养亲汤的抗哮喘活性成分进行了筛选,离体气管环张力法对其活性进行评价,分子对接法对其作用机制进行探讨。结果表明:迷迭香酸和芥子碱硫氰酸盐为三子养亲汤中β₂-AR特异性靶向活性成分,两种成分在10^-9-10^-4mol/L和10^-12-10^-7mol/L范围内对高钾预收缩大鼠主气管具有浓度依赖性舒张作用,β₂-AR特异性拮抗剂ICI 118551可显著抑制该作用,提示两种成分均通过特异性识别并结合β₂-AR发挥止咳平喘功效。分子对接研究结果表明,迷迭香酸和芥子碱主要作用于β₂-AR结构中的氨基酸残基Ser 203,Ser 204,Ser 207,Tyr 316和Asn 312。该研究为抗哮喘药物活性成分筛选和快速评价提供了技术支撑。更多还原

【Abstract】 Respiratory system disease is one of the major chronic non-infectious diseases with the characters of high morbidity,drug dependence,and recurrent episodes.New technologies and methods are urgently needed to develop highly effective and innovative drugs to achieve effective prevention and treatment of such diseases.It has become one of the emerging difficulties and hot issues in the field of medicine and health.Traditional Chinese medicine（TCM）is one of the major sources for defending respiratory diseases.The bioactive compounds screening from TCMs becomes a bottleneck problem in the research and development of innovative Chinese medicines and one of the emerging difficulties and hot issues in the field of medicine and health.Sanzi Yangqin Decoction（SYD）is a widely used recipe and usually prescribed for fighting against the respiratory system diseases.Focusing on the unclear anti-asthmatic active compounds and targets,we established a new chromatographic method based on the immobilizedβ₂-adrenoceptor（β₂-AR）and applied it to bioactive compounds screening in SYD in this thesis.This study is expected to achieve rapid analysis of the anti-asthma compounds and solve the bottleneck problem of a long research cycle and low accuracy from TCMs.The method is believed to provide a reference for the bioactive compounds screening from other TCMs and play a positive role in innovative drug development.The thesis is divided into four chapters,and among which the main contributions are as follows:1.A one-step immobilization method forβ₂-AR was established.Takingβ₂-AR as a probe,O⁶-alkylguanine DNA alkyltransferase（hAGT）was fused at the C terminus of the receptor.The fusion receptor was induced and expressed in E.coli BL21（DE3）.According to the specific bio-orthogonal reaction between hAGT and O⁶-benzylguanine derivatives modified gel,β₂-AR was captured and immobilized by a one-step method from the cell lysate through covalent bond.Scanning electron microscopy（SEM）,immuno-scanning electron microscopy（Immuno-SEM）,high-performance liquid chromatography coupled with mass spectroscopy（HPLC-MS）and other techniques were used for morphological,functional and micro-environmental characterization of the immobilizedβ₂-AR.The result illustrated thatβ₂-AR can be oriented immobilized on the gel surface as a monolayer with high specificity,high activity,and high stability.It can provide new method for the rapid preparation of receptor chromatography with high activity.2.Taking salbutamol,methyoxynamine,and terbutaline as probes,we used frontal analysis and perturbation peak method to investigate the binding interaction of the drugs and the immobilizedβ₂-AR.The results were also verified by molecular docking.The data showed that the three drugs had one type of common binding sites on the immobilizedβ₂-AR.Isothems of the three drugs were best fitted with Langmuir model.The binding constants of the three drugs were 1.63×10⁴L/mol,3.03×10⁴L/mol,and 0.76×10⁴L/mol.Hydrogen bond and Van der Waal’s force were the main driving forces of the interaction.The exposed amino acids were Ser 203,Ser 204,Ser 207 and Asp 113.It is proved that immobilizedβ₂-AR still preserves the ability to recognize its specific ligands.The established receptor chromatography can be applied in studying the binding interaction between the receptor and the ligand,which provides a new chromatographic method for targeted bioactive compounds screening.3.We used the established,immobilized receptor-based,chromatographic model to screen the anti-asthma compounds from SYD that targets toβ₂-AR.The pharmacological effect of the screened compounds was evaluated by the tension changes of the trachea ring in vitro.Molecular docking was used for validating the pharmacological effect.The result showed that rosmarinic acid and sinapine thiocyanate were the specific bioactive compounds targetedβ₂-AR in SYD.The two compounds exert the pharmacological effect through a concentration-dependent manner when the trachea was pre-treated with high potassium.And the concentration ranges were tested to be 10^-9-10^-44 mol/L and 10^-12-10^-77 mol/L,respectively.While when the trachea was pre-treated with ICI 118551,a specific antagonist ofβ₂-AR,the relaxation effect was inhibited.It proved that the antitussive and anti-asthmatic effects of the two compounds were exerted through specific identifying and binding withβ₂-AR.Molecular docking revealed that the two compounds mainly interacted with the Ser 203,Ser 204,Ser207,Tyr 316 and Asn 312 ofβ₂-AR.Our method is proved to be a powerful alternative in targeted screening and evaluating the bioactive compounds for anti-asthma effects.更多还原