功能蛋白与药物相互作用的亲和色谱法研究

【作者】 何媛；

【导师】 卫引茂；

【作者基本信息】 西北大学 ， 分析化学， 2012， 博士

【摘要】 小分子药物与蛋白质的相互作用研究对于许多生物过程具有非常重要的意义。蛋白质对药物的结合作用可影响进入循环系统药物的最终活性以及药物在体内的分布、排泄速率以及毒理性。此种结合作用一般包括一种药物与蛋白质的结合作用以及多种药物之间在同一种蛋白质上的直接或间接的竞争作用。高效亲和色谱法在药物与蛋白质相互作用研究中取得了长足进展,但在高活性蛋白质固定相的制备、药物与靶点蛋白相互作用研究和药物组分之间与蛋白质的竞争性研究方面存在一些有待完善和解决的问题,针对这些问题,作者选取受体蛋白中的β2-肾上腺素受体(β2-Adrenergic receptor,β2-AR),建立了一种β2-AR的定向固定化方法,并将该固定相应用于β2-AR与药物的相互作用研究中；选择人血清白蛋白(Human serum albumin, HSA)为模型蛋白,研究了中药活性组分之间与蛋白质的竞争性作用。全文包括以下五个部分：1.绪论介绍了药物与蛋白质相互作用的研究意义、内容及方法,评述了亲和色谱法的原理、固定相的制备方法、理论基础以及其在药物与蛋白质相互作用中的应用。2.定向固定化β2-肾上腺素受体制备及表征为制备高活性β2-AR色谱固定相,根据β2-AR的分子结构特点,建立了一种β2-AR定向固定化方法。以β2-AR键合量、特异性药物结合能力、药物结合量为活性表征参数,通过与随意固定化β2-AR色谱柱比较,证明了定向固定化方法的β2-AR键合量较高,且能够提高β2-AR的活性。定向固定化β2-AR方法的建立,为高活性蛋白质固定相制备方法研究提供了新方法。3.定向固定化β2-肾上腺素受体与药物的相互作用采用前沿色谱法和竞争置换法研究了定向固定化β2-AR与盐酸普萘洛尔、硫酸沙丁胺醇和盐酸氯丙那林的相互作用。在pH7.4,37℃时,前沿色谱法测定了三种药物在定向固定化β2-AR色谱柱上的吸附等温线,通过单、双朗格缪尔模型拟合,证明三种药物与β2-AR均存在一类结合位点,且盐酸普萘洛尔、硫酸沙丁胺醇和盐酸氯丙那林与β2-AR的结合常数分别为1.96×104L/mol、5.78×103L/mol和6.07×103L/mol。采用竞争置换法分别研究了硫酸沙丁胺醇、盐酸氯丙那林为竞争剂时与盐酸普萘洛尔在β2-AR分子上的竞争作用,说明药物之间与β2-AR存在直接竞争作用。竞争置换法测得硫酸沙丁胺醇、盐酸氯丙那林与β2-AR的结合常数分别为9.17×103L/mol和2.94×103L/mol,与前沿色谱法测定结果接近。本研究表明,定向固定化β2-AR可用于药物与β2-AR的相互作用研究中。4.丹参注射液活性组分与人血清白蛋白的竞争性作用研究本章采用亲和色谱法结合微透析-高效液相色谱法、分子对接法对丹参注射液中三种活性组分丹参素、原儿茶醛及咖啡酸与HSA的相互作用进行了深入的研究。在研究丹参注射液单组分与HSA结合作用时,采用前沿色谱法分别测定了原儿茶醛、咖啡酸在HSA上的吸附等温线,说明二者与HSA仅存在一类结合位点。在pH7.4,37℃的生理条件下,通过自我竞争法测得原儿茶醛、咖啡酸与HSA的结合常数分别为9.56×103L/mol和1.60×104L/mol。微透析-高效液相色谱法分别研究了单组分药物与HSA的结合作用,表明自由溶液中单组分与HSA之间存在一类结合位点,验证了亲和色谱法的测定结果,且该方法测得溶液中丹参素、原儿茶醛及咖啡酸与HSA的结合常数分别为1.27×105L/mo、4.03×104L/mol和2.75×104L/mol。在研究丹参注射液多组分之间与HSA的竞争性作用时,竞争置换法研究了三种组分之间与HSA竞争性作用,说明三种组分之间与HSA存在直接竞争作用,且丹参素、原儿茶醛及咖啡酸与HSA的结合常数分别为3.56×103L/mol、2.44×104L/mol和1.85×104L/mol。微透析-高效液相色谱法研究了组分之间与HSA竞争性作用,说明组分之间存在直接竞争,与竞争置换法结果相符。为进一步了解三种组分在HSA上结合域,采用亲和色谱法,通过与HSA四种标记物的竞争作用研究表明,丹参素、原儿茶醛及咖啡酸在HSA上的结合位点均为吲哚-苯二氮卓位点。热力学研究表明原儿茶醛、咖啡酸与HSA结合作用的驱动力分别为疏水作用和静电作用。5.一种药物-蛋白质结合常数的测定新方法将溶质进样量与保留值关系式用于药物与蛋白质结合常数的测定研究中,建立了一种研究药物与蛋白质结合常数的直接进样测定方法。采用该方法测定了盐酸普萘洛尔、硫酸沙丁胺醇和盐酸氯丙那林不同进样量在β2-AR色谱柱上的保留值,并测得盐酸普萘洛尔、硫酸沙丁胺醇和盐酸氯丙那林与β2-AR的结合常数分别为2.01×104L/mol、2.52×103L/mol和8.19×103L/mol。通过与亲和色谱法测定结果比较,表明直接进样测定方法是一种可应用于药物与蛋白质相互作用研究的简单、可靠的方法。更多还原

【Abstract】 The interaction of small molecules with proteins is important in many biological processes. Protein binding in blood is significant in determining the eventual activity and fate of drugs once they have entered the circulation. These interactions help control the distribution, rate of excretion, and toxicity of drugs in the body. They include the binding interaction between one drug and protein as well as direct or indirect competition between two drugs for the same binding protein. High-performance affinity chromatography is a powerful technique, which has received a great progress in studying the interactions between drugs and proteins. However, there are still many problems in this method to be solved. For instance, fewer methods in preparation of high activity protein stationary phase, insufficient study on the interaction between drugs and target proteins and less study on the drug-drug competition with one protein. To solve these problems, a method of oriented immobilization of β2-adrenergic receptor (β2-AR, one of the receptor proteins) was developed and applied in the study of the interaction between drugs and β2-AR. In addition, human serum albumin (HSA) was chosen as a model protein to investigate the competitive interactions between the bioactive components of Chinese herbal medicine and protein. This dissertation is divided into five chapters:1. IntroductionThe significance, content and methods for studying interaction between drugs and proteins were introduced. In addition, the principle of high-performance affinity chromatography (HPAC), methods of preparation high-performance affinity chromatography stationary phase, theories of high-performance affinity chromatography and their application in the interactions of drug-protein were reviewed.2. Preparation and characterization of oriented immobilization of β2-adrenergic receptorIn order to prepare highly active β2-AR stationary phase, a novel oriented immobilized method of β2-AR was developed according to the character of β2-AR molecular structure. In terms of the total β2-AR content, specific binding and binding capacity, it was proved that β2-AR column prepared by the oriented method had a better results than the β2-AR column immobilized to the silica through a random immobilization method. It provided a novel process to the preparation of highly active protein stationary phase.3. The interaction of drugs with oriented immobilized β2-adrenergic receptorThe interactions of propranolol, salbutamol sulphate and clorprenaline with oriented immobilized β2-AR were investigated respectively by the frontal analysis and the zonal elution. The binding isotherms of these three drugs on the β2-AR column were determined by frontal analysis at pH7.4and37C. It was showed that the three drugs had only one kind of binding site on the β2-AR molecular, and the association constants of propranolol, salbutamol sulphate and clorprenaline were1.96×104L/mol,5.78×103L/mol and6.07x103L/mol, respectively. Meanwhile, the competition interactions of these three drugs with β2-AR were determined by zonal elution. It was indicated that these three drugs competed the same binding site on β2-AR molecular and the association constants of salbutamol sulphate and clorprenaline with β2-AR were9.17×103L/mol and2.94×103L/mol, respectively. The association constants of three drug determined by zonal elution were similar with the results of frontal analysis. This research demonstrated that oriented immobilized β2-AR can be applied well in the interaction of drugs with β2-AR.4. The competitive interaction of three bioactive components in Danshen injection with human serum albuminThe interactions of three bioactive components (danshensu, caffeic acid and protocatechuic aldehyde) in Danshen injection with HSA had been studied by the HPAC, microdialysis-HPLC and molecular docking method.When the single component of Danshen injection with HSA was studied, the binding isotherms of protocatechuic aldehyde and caffeic acid were determined by frontal analysis. The results showed that both of the two components had only one kind of binding site on HSA. The association constants of protocatechuic aldehyde and caffeic acid with HSA were determined at pH7.4,37C by self-competition zonal elution, and the association constants were9.56×103L/mol and1.60×104L/mol, respectively. The binding interaction of single component with HSA was also investigated by microdialysis-HPLC. It was showed that the single component also had one kind of binding site on HSA in solution, corresponded with the results determined by HPAC. The association constants for danshensu, protocatechuic aldehyde and caffeic acid with HSA determined by microdialysis-HPLC were1.27×105L/mol,4.03×104L/mol and2.75×104L/mol, respectively.When the multicomponents of Danshen injection with HSA were studied, the results of zonal elution showed that the three components had a direct competition on HSA. The association constants for danshensu, protocatechuic aldehyde and caffeic acid determined by zonal elution were3.56×103L/mol,2.44×104L/mol and1.85×104L/mol, respectively. Microdialysis-HPLC was applied to study the competition of three components with HSA, and the results were consistent with zonal elution studies.Further study was performed to investigate the binding site of the three components on HSA molecular. Zonal elution studies with the probes of HSA as injected solutes showed that three components were binding to indole-benzodiazepine site on HSA molecular. Thermodynamic results indicated that the interactions between protocatechuic aldehyde and caffeic acid with HSA were mainly drived respectively by hydrophobic interaction and electrostatic force.5. A new method to determine association constants of protein-drug interactionAn equation describing the relationship between the injected amount of solutes and their retention values in liquid chromatography was introduced into drug-protein study. A new method to determine the association constant of drug-protein interaction was developed. This method was applied in studying the interaction of propranolol, salbutamol sulphate and clorprenaline with β2-AR. The association constants for propranolol, salbutamol sulphate and clorprenaline were2.01×104L/mol,2.52×103L/mol and8.19×103L/mol, respectively. Compared with the results of HPAC, it was indicated that this method is a simple and reliable method in drug-protein study.更多还原