定向固定化Beta2-肾上腺素受体构象调控色谱模型的建立及应用

发布时间：2018-01-25 13:41

本文关键词： 定向固定化 β_2-肾上腺素受体相互作用活性筛选蛋白质构象　出处：《西北大学》2015年博士论文　论文类型：学位论文

【摘要】：功能蛋白质与小分子配体的相互作用研究对于揭示生命活动过程所涉及的复杂信号传递和开发高效创新药物具有重要意义。以固定化功能蛋白质为核心的生物传感器、试剂盒、酶反应器和亲和色谱方法是实现上述研究的主要途径。然而,由于固定化界面蛋白质取向和构象杂乱不一,使得其在功能蛋白质与药物相互作用研究等方面的测定结果偏离生理状态。因此,固定化功能蛋白质的取向和构象调控方法研究就成为医药、化学和生物学领域的前沿研究问题之一。作者所在实验室在前期的工作中,建立了β2-肾上腺素受体色谱方法,证明方法可用于受体与药物的相互作用研究和药物活性成分筛选。基于此,本论文针对色谱固定相表面受体取向和构象杂乱不一的问题,围绕固定化β2-肾上腺素受体取向和构象诱导、调控方法的建立及应用展开系统研究。该研究有望为其他以固定化功能蛋白质为核心的高灵敏方法的建立提供依据,对高效创新药物的开发具有重要意义。全文共分4章,作者的主要贡献如下：(1)建立了一种可逆的β2-肾上腺素受体定向固定化新方法,构建了β2-肾上腺素受体取向及构象调控模型。利用镍离子与β2-肾上腺素受体结构中组氨酸标签所含高密度咪唑基之间的特异性作用,将该受体可逆的定向固定在大孔硅胶表面,制备了定向固定化的β2-肾上腺素受体色谱固定相。结果表明,该方法可获得取向统一的固定化受体。以工具药盐酸氯丙那林等为配体,对受体色谱模型的特异性和稳定性进行了表征。在此基础上,以盐酸麻黄碱和盐酸伪麻黄碱为构象依赖探针,通过改变色谱柱温度、流动相组成以及配体条件等对固定化受体的构象进行调控。结果表明：当色谱柱温度为25.0℃时,可获得固定化受体的最稳定构象态：当流动相磷酸盐缓冲溶液浓度为5.0mmol/L(pH=7.40)时,固定化β2-肾上腺素受体构象最优；以(S)-普萘洛尔为配体,考察了药物配体对固定化β2-肾上腺素受体构象的影响,证明药物配体可诱导固定化受体构象发生变化,且浓度为5.0nmol/L的(S)-普萘洛尔可使固定化β2-肾上腺素受体获得最优构象。这方面的研究为其他固定化功能蛋白质取向及构象的调控提供了方法学借鉴。(2)应用稳定构象态β2-肾上腺素受体模型研究了受体与硫酸特布他林等七种药物配体的相互作用,为明确药物的作用机制提供了依据。采用前沿色谱法测定了硫酸特布他林、盐酸甲氧那明、盐酸氯丙那林、盐酸妥布特罗、硫酸沙丁胺醇、盐酸克伦特罗和盐酸班布特罗与β2-肾上腺素受体相互作用的结合常数和结合位点数。结果表明：七种药物和β2-肾上腺素受体均存在一类结合位点,结合常数依次为：1.84x104 M-1,1.71×104 M-1,2.3×1O4M-1,2.09×104M-1,1.98×104 M-1,2.37×104M-1和9.43×104 M-1；结合位点数依次为：8.3×10-4M,3.88×10-5M, 1.02×10-5M,2.16×10-5M,7.5×10-5M,1.82×10-5M, 2.47×10-5M。采用微透析法对上述结果进行了验证,表明两种方法所得结合常数和结合位点数在数量级上相同,证明β2-肾上腺素受体色谱模型能用于研究药物分子与受体的相互作用,为在线蛋白质-药物相互作用高通量分析提供了方法学参考。(3)应用稳定构象态β2-肾上腺素受体色谱模型对延胡索和百部中的活性成分进行了筛选,采用前沿色谱和竞争置换色谱法对活性成分与受体的作用机制进行了探讨。加热回流法制备延胡索和百部的总提物,受体色谱进行分析,收集保留成分经反相液相色谱-质谱联用法进行结构鉴定。结果发现,四氢小檗碱、原阿片碱、紫堇碱和四氢非洲防己碱为延胡索中与β2-肾上腺素受体有特异性作用的活性成分；百部新碱为百部中作用于β2-肾上腺素受体的活性成分。进一步采用前沿色谱法测得四氢小檗碱和原阿片碱与β2-AR的结合常数分别为9.04×104M-1和4.30×104 M-1,结合位点数分别为6.67×10-4M和5.88×10-4M。采用竞争洗脱法研究了这两个成分在β2-AR上的作用位点,证明两个成分与β2-肾上腺素受体作用的位点是第三疏水区的天冬氨酸113,为复杂样品药物活性成分的筛选提供了方法,对高效创新药物的开发具有重要意义。
[Abstract]:Study on the interaction of functional protein and small molecule ligands plays an important role in the complex signal involved reveal the life activities of transfer and development of efficient and innovative drugs. Biosensor with immobilized protein functions as the core of the kit, enzyme reactor and affinity color spectrum method is the main way to achieve the above research. However, due to protein the orientation and conformation of Immobilized Mixed interface, making it in the functional interactions between protein and drug research results deviate from the physiological state. Therefore, the research orientation and conformation control method of immobilized protein function has become one of the frontier research in medicine, chemistry and biology fields. Author's laboratory in the early work the establishment of the beta adrenergic receptor, 2- method, prove the method can be used to study the interaction of drug and receptor and drug. Component selection. Based on this, this thesis focuses on the stationary phase surface receptor orientation and conformation of a messy problem on the orientation and conformation of immobilized beta adrenergic receptors induced by 2-, a systematic study of the establishment and application of control methods. The study is expected to establish other immobilized protein functions as the core of the high sensitive method to provide the basis, have important significance to development of efficient and innovative drugs. This thesis consists of 4 chapters, the author's main contributions are as follows: (1) the establishment of a new beta adrenergic receptor 2- directed immobilization method of reversible, constructs the beta 2- adrenergic receptor orientation and conformation of the regulation model. The use of nickel ion and histidine tag beta 2- adrenergic receptor structure contained in the specific action between high density imidazole, directed the receptor reversible immobilized on macroporous silica surface was prepared by directional immobilization of beta 2- kidney Adrenoceptor chromatographic stationary phase. The results showed that the immobilized receptor can be got by this method. The unity of drugs clorprenaline hydrochloride as ligand, receptor of chromatography model specificity and stability were investigated. On this basis, the ephedrine hydrochloride and pseudoephedrine hydrochloride as conformation dependent probe, by changing the column temperature, mobile phase composition and conformation of ligand conditions on the immobilized receptor regulation. The results show that when the column temperature is 25 degrees centigrade, the most stable conformation can be immobilized receptor: when the mobile phase of phosphate buffer solution concentration of 5.0mmol/L (pH=7.40), immobilized beta 2- adrenergic receptor conformation optimal; in (S) - propranolol as ligand, the effects of drugs on the immobilized ligand 2- beta adrenergic receptor conformation, that the drug can induce ligand immobilized receptor conformational change, and strong The degree of 5.0nmol/L (S) - propranolol can make the immobilized beta 2- adrenergic receptor to obtain optimal conformation. This study regulation for other immobilized protein function orientation and conformation provides methodological reference. (2) application of stable conformation of beta adrenergic receptor 2- model to investigate the interaction of receptors with sulfuric acid Tebu He Lin and other seven kinds of drug ligand, provide the basis for elucidating the mechanism of drugs. The determination of terbutaline sulfate by affinity chromatography, methoxyphenamine hydrochloride, clorprenaline hydrochloride, tulobuterol hydrochloride, salbutamol sulfate, the binding constants of clenbuterol and bambuterol hydrochloride and 2- beta adrenergic receptor interaction and the number of binding sites. The results show that there are seven kinds of binding sites for drug 2- and beta adrenergic receptor binding constants are as follows: 1.84x104 104 M-1,1.71 * M-1,2.3 * 1O4M-1,2.09 * 104M -1,1.98 * M-1,2.37 104 * 104M-1 and 9.43 * 104 M-1; the number of binding sites is as follows: 8.3 * 10-4M, 3.88 * 10-5M, 1.02 * 10-5M, 2.16 * 10-5M, 7.5 * 10-5M, 1.82 * 10-5M, 2.47 * 10-5M. using microdialysis of the above results are verified, show that the two methods combined with constant and the number of binding sites in the same order of magnitude, that beta adrenergic receptor 2- chromatography model can be used to study the interaction of drug molecules and receptors for protein analysis, online drug interactions, high-throughput provides methodological reference. (3) application of stable conformation of 2- beta adrenaline receptor chromatography models were screened for Corydalis and 100 in the active ingredient, the mechanism by frontal chromatography and competitive displacement chromatography of the active ingredient and the receptor are discussed. The heating reflux preparation of corydalis and Stemona extract, receptor chromatography analysis and collection Liucheng divided by reversed-phase liquid chromatography tandem mass spectrometry were identified. The results showed that four of tetrahydroprotoberberines, protopine, Corydaline and four hydrogen columbamine as active ingredients in Rhizoma Corydalis and beta 2- adrenergic receptor specific action; stemoninine for 100 active ingredients in beta 2- adrenergic receptor. Further by frontal chromatography measured four hydrogen binding constants of berberine and protopine and beta 2-AR were 9.04 * 104M-1 and 4.30 * 104 M-1, the number of binding sites were binding sites of 6.67 * 10-4M and 5.88 * 10-4M. by competitive research of these two components in the beta 2-AR elution method two, prove the composition and effect of beta 2- adrenergic receptor sites is the hydrophobic region of third aspartic acid 113, provides a method of screening for complex samples of active pharmaceutical ingredients, has important significance to development of efficient and innovative drugs.

【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R914

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