β_2-肾上腺素受体色谱用于药物结合常数—结构参数间的关系研究
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摘要
中药等复杂体系是高效创新药物开发的重要源泉,其活性成分的筛选则是影响整个开发过程的关键因素。经典的色谱技术无疑是目前在活性成分筛选过程中广泛应用的技术之一。然而,由于该类色谱技术在筛选过程中不能对化合物的活性进行评估,因此建立新型具有活性识别能力的色谱技术就成为研究的必然趋势。β2-肾上腺素受体(β2-AR)色谱是实验室前期所建立的一种具有活性识别功能的亲和色谱技术,被证明可用于中药活性成分的筛选,但筛选的机制却不清晰。本论文围绕七种工具药与色谱柱上β2-AR的相互作用及其保留.结构关系来展开研究,旨在探讨受体亲和色谱筛选中药活性成分的具体机制。全文分为三章,作者的主要贡献如下：
1、在实验室前期β2-AR分离纯化的工艺基础上,采用细菌培养及亲和色谱等方法,获得了实验所需量的高纯度β2-AR,采用羰基二咪唑法将其温和地固载在大孔硅胶表面,得到了β2-肾上腺素受体色谱柱。采用甲氧那明、沙丁胺醇、妥布特罗等工具药对色谱柱的保留活性进行考察。结果表明,与从组织中纯化获得的受体相比,采用细菌培养法获得的受体同样具有生物活性,能特异性识别药物,证明此制备方法有望解决β2-AR来源难的问题。
2、基于已建立的受体色谱模型,采用前沿分析法研究了固载化的β2-AR与沙丁胺醇、氯丙那林、甲氧那明、妥布特罗、福莫特罗、克伦特罗、班布特罗的结合方式及结合常数。结果表明,这七种工具药只存在一类作用位点,结合常数分别为15.61×105 M-1、2.52×105M-1、2.33×105M-1、3.35×105M-1、18.87×105M-1、2.74×105M-1、10.57×105M-1,证明β2-AR色谱可用于研究药物与受体间的相互作用。
3、通过检索文献,获得上述七种工具药的结构参数,利用数据处理软件分析药物与受体的结合常数和结构参数之间的关系。发现：结构参数为logP、氢键数(Ha)、摩尔折射度及偶极距与结合常数有一定的相关性。其中,相关性较强的参数为logP和氢键数。结合常数与logP呈负相关,线性方程式为Ka=16982-5828logP；与氢键数呈正相关,线性方程式为Ka=-3488+1297Ha,且氢键数的影响力大于logP,提示受体色谱有望用于对未知成分的疏水性或结合常数等性质进行预测,从而初步评估其成药性。
Active components selecting of drugs in the complex system is a hot topic studied at Pharmaceutical Research, searching and discovering lead compounds with biological activity is a crucial aspect. In recent years, drug screening models that use receptor as a target have made great progress,β2-adrenergic receptor (β2-AR) Chosen in this paper is receptor in the surface of cell wall, Participating in many physiological and pathological process of body, and it is the primary target when asthma drugs effect on the body. We used the characteristic that receptor and ligand binding together Specifically, by researching the seven specific binding tool drugs, found the relationship between binding constant and molecular structure parameters, finally got the relationship of reserved-structure so as to provide guidance for Selecting and designing of drug molecules.There are three chapters in this paper, The author's main contributions are as follows:
1. Based on existent methods of Separatingβ2-AR, using bacterial culture and affinity chromatography, we obtained high-purityβ2-AR that the experimental needed, and then immobilized receptors on the surface of macro porous silica gel by chemical coupling method, finally got theβ2-AR chromatography column. Using tool drugs such as methoxyphenamine, salbutamol, botero inspect the bioactivity of the column,the result show that after the purified receptor bonded to the silica surface, it still retained biologically activity, and can be specifically combined with the tool drugs, herefore can be used for researching relationship between drug binding constants and structural parameters.
2. Based on the established model of receptor chromatography, we determined the association constant and the number of binding sites of tool drugs with the methods of frontal analysis.The result show that the association constants of salbutamo, clorprenaline, Methoxyphenamine, botero, formoterol, clenbuterol, bambuterol are 15.61×105M-1、2.52×105M-1、2.33×105M-1、3.35×105M-1、18.87×105 M-1、2.74×105M-1 and 10.57×105 M-1.
3.We got the structural parameters by insepcting the literature, and then can obtain the relationship between binding constant and structural parameters, finally we found that the structure parameters such as logP, hydrogen bond number (Ha), molar refractionand and dipole moment have a certain correlation with binding constants, which were negatively correlated with the logP, the linear equation was Ka=16982-5828logP; which were positively correlated with the number of hydrogen bonds, the linear equation was Ka=-3488+1297Ha, and the number of hydrogen bonds had more influence than logP.
1、在实验室前期β2-AR分离纯化的工艺基础上,采用细菌培养及亲和色谱等方法,获得了实验所需量的高纯度β2-AR,采用羰基二咪唑法将其温和地固载在大孔硅胶表面,得到了β2-肾上腺素受体色谱柱。采用甲氧那明、沙丁胺醇、妥布特罗等工具药对色谱柱的保留活性进行考察。结果表明,与从组织中纯化获得的受体相比,采用细菌培养法获得的受体同样具有生物活性,能特异性识别药物,证明此制备方法有望解决β2-AR来源难的问题。
2、基于已建立的受体色谱模型,采用前沿分析法研究了固载化的β2-AR与沙丁胺醇、氯丙那林、甲氧那明、妥布特罗、福莫特罗、克伦特罗、班布特罗的结合方式及结合常数。结果表明,这七种工具药只存在一类作用位点,结合常数分别为15.61×105 M-1、2.52×105M-1、2.33×105M-1、3.35×105M-1、18.87×105M-1、2.74×105M-1、10.57×105M-1,证明β2-AR色谱可用于研究药物与受体间的相互作用。
3、通过检索文献,获得上述七种工具药的结构参数,利用数据处理软件分析药物与受体的结合常数和结构参数之间的关系。发现：结构参数为logP、氢键数(Ha)、摩尔折射度及偶极距与结合常数有一定的相关性。其中,相关性较强的参数为logP和氢键数。结合常数与logP呈负相关,线性方程式为Ka=16982-5828logP；与氢键数呈正相关,线性方程式为Ka=-3488+1297Ha,且氢键数的影响力大于logP,提示受体色谱有望用于对未知成分的疏水性或结合常数等性质进行预测,从而初步评估其成药性。
Active components selecting of drugs in the complex system is a hot topic studied at Pharmaceutical Research, searching and discovering lead compounds with biological activity is a crucial aspect. In recent years, drug screening models that use receptor as a target have made great progress,β2-adrenergic receptor (β2-AR) Chosen in this paper is receptor in the surface of cell wall, Participating in many physiological and pathological process of body, and it is the primary target when asthma drugs effect on the body. We used the characteristic that receptor and ligand binding together Specifically, by researching the seven specific binding tool drugs, found the relationship between binding constant and molecular structure parameters, finally got the relationship of reserved-structure so as to provide guidance for Selecting and designing of drug molecules.There are three chapters in this paper, The author's main contributions are as follows:
1. Based on existent methods of Separatingβ2-AR, using bacterial culture and affinity chromatography, we obtained high-purityβ2-AR that the experimental needed, and then immobilized receptors on the surface of macro porous silica gel by chemical coupling method, finally got theβ2-AR chromatography column. Using tool drugs such as methoxyphenamine, salbutamol, botero inspect the bioactivity of the column,the result show that after the purified receptor bonded to the silica surface, it still retained biologically activity, and can be specifically combined with the tool drugs, herefore can be used for researching relationship between drug binding constants and structural parameters.
2. Based on the established model of receptor chromatography, we determined the association constant and the number of binding sites of tool drugs with the methods of frontal analysis.The result show that the association constants of salbutamo, clorprenaline, Methoxyphenamine, botero, formoterol, clenbuterol, bambuterol are 15.61×105M-1、2.52×105M-1、2.33×105M-1、3.35×105M-1、18.87×105 M-1、2.74×105M-1 and 10.57×105 M-1.
3.We got the structural parameters by insepcting the literature, and then can obtain the relationship between binding constant and structural parameters, finally we found that the structure parameters such as logP, hydrogen bond number (Ha), molar refractionand and dipole moment have a certain correlation with binding constants, which were negatively correlated with the logP, the linear equation was Ka=16982-5828logP; which were positively correlated with the number of hydrogen bonds, the linear equation was Ka=-3488+1297Ha, and the number of hydrogen bonds had more influence than logP.
引文
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