奥司他韦羧酸衍生物抗N2型禽流感病毒的活性研究

发布时间：2018-03-14 12:47

本文选题：N2型禽流感病毒　切入点：神经氨酸酶　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：禽流感(avian influenza,AI)是由A型禽流感病毒(avian influenza virus,AIV)引起以禽类发病为主的一种急性、高度接触性、致死性的人畜共患传染病。神经氨酸酶(neuraminidase)是禽流感病毒表面的主要蛋白之一,对禽流感病毒的复制和传播发挥着重要的作用,因此以神经氨酸酶为靶点设计抗禽流感病毒抑制剂成为目前抗禽流感病毒药物研究的热点方向之一。到目前为止,上市的神经氨酸酶抑制剂主要有扎那米韦、奥司他韦、帕拉米韦。有研究显示,禽流感病毒对现有药物都陆续出现不同程度的耐药性。因此,开发基于新靶点和新结构的抗禽流感药物迫在眉睫。2013年,高福院士课题组在解析N2型神经氨酸酶的结构时,发现高浓度奥司他韦羧酸能诱导刚性关闭的N2型150-loop打开。本课题试图利用配体与N2的相互作用,诱导150-loop打开,从而为设计开发高活性NA抑制剂指明方向。本论文主要包括三部分内容。第一,根据神经氨酸酶的蛋白结构,在奥司他韦羧酸C-5氨基上引入芳甲基、噻吩甲基、酰基和磺酰基等基团,设计了19种目标化合物:Lu17A1~Lu17A19。第二,进行目标化合物在CEF细胞上抗H5N2的活性检测。根据每一化合物的IC_(50)值,比较目标化合物的抑制活性。实验结果显示,Lu17A16、Lu17A17、Lu17A18、Lu17A19抑制活性较差,Lu17A7、Lu17A9、Lu17A10、Lu17A11、Lu17A13抑制活性较好,其它化合物表现出了中等强度的抑制活性。为证实目标化合物确实是作用于N2型神经氨酸酶,本论文选取了活性最佳的目标化合物(Lu17A9)进行了神经氨酸酶活性评价。结果显示,以Lu17A9为代表的目标化合物确实作用于N2,可以作为N2型神经氨酸酶抑制剂。第三,筛选出5种抑制活性较好的化合物(Lu17A7、Lu17A9、Lu17A10、Lu17A11、Lu17A13),检测其在鸡胚上抗H9N2的活性。收取鸡胚尿囊液,进行血凝检测及荧光定量PCR检测,分别得出HA和CP值。结果显示,HA及CP值所对应的化合物所表现出的抑制活性一致,这与其在细胞上抗H5N2所表现的抑制活性也是一致的。通过上述实验结果分析得出化合物结构特点对其抑制活性具有一定的影响。结论如下:第一,奥司他韦羧酸分子上C-5氨基碱性的保持及疏水性取代基的引入有利于提高化合物的抑制活性。芳甲基及噻吩甲基的引入有利于提高化合物抑制活性,酰基和磺酰基的引入则不利于提高化合物抑制活性。第二,芳基上取代基的相对位置、取代基的数目、大小以及杂原子的存在对化合物的抑制活性也有一定的影响。取代基的芳甲基上存在对位取代基时抑制活性好于间位取代基;芳基上有较大体积取代基或多余两个取代基时活性较低;芳甲基芳基中存在杂原子时活性相对较好。
[Abstract]:Avian influenza virus (Avian Influenza) is an acute, highly contact and fatal zoonotic disease caused by avian influenza virus A (AIV). Neuraminidase is one of the main proteins on the surface of avian influenza virus. It plays an important role in the replication and transmission of avian influenza virus. Therefore, the design of anti-avian influenza virus inhibitors using neuraminidase as a target has become one of the hot research directions of anti-avian influenza virus drugs. The main inhibitors of neuraminidase on the market are zanamivir, oseltamivir and paramivir. Studies have shown that the avian influenza virus has shown varying degrees of resistance to existing drugs. It is urgent to develop anti-avian influenza drugs based on new targets and structures. In 2013, the research group of Gaofu studied the structure of N2 neuraminidase. It was found that high concentration of oseltamivir carboxylic acid could induce the opening of rigid closed N2 150-loop. This paper mainly includes three parts. Firstly, according to the protein structure of neuraminidase, aryl methyl and thiophene methyl were introduced into the C-5 amino group of oseltamivir carboxylic acid. Acyl group and sulfonyl group were used to design 19 target compounds:: Lu17A1, Lu17A19. second, the activity of the target compounds in CEF cells was determined. According to the ICSP 50 value of each compound, The results showed that Lu17A16, Lu17A18, Lu17A18, Lu17A19, Lu17A7, Lu17A9, Lu17A10, Lu17A11, Lu17A13, and other compounds showed moderate inhibitory activity. In this paper, the target compound Lu17A9 was selected to evaluate the activity of neuraminidase. The results showed that the target compound represented by Lu17A9 acted on N _ 2 and could be used as N _ 2 type neuraminidase inhibitor. Five compounds, Lu17A7, Lu17A9, Lu17A10, Lu17A11, Lu17A13, were screened for their anti-H9N2 activity on chicken embryo. Chicken embryo allantoic fluid was collected for hemagglutination detection and fluorescence quantitative PCR assay. Ha and CP values were obtained respectively. The results showed that the compounds corresponding to HA and CP values showed the same inhibitory activity. Through the analysis of the above experimental results, it is concluded that the structural characteristics of the compounds have a certain effect on the inhibitory activity. The conclusions are as follows: first, The retention of C-5 amino base and the introduction of hydrophobic substituents on oseltamivir carboxylic acid molecules can improve the inhibitory activity of the compounds, and the introduction of aryl methyl and thiophene methyl can improve the inhibitory activity of the compounds. The introduction of acyl and sulfonyl groups is not conducive to improving the inhibitory activity of the compounds. Second, the relative position of the substituents on the aryl group, the number of substituents, The size and the existence of heteratomic atoms also have a certain influence on the inhibitory activity of the compounds. The suppressive activity of the substituted aromatic methyl group is better than that of the meso-substituted group in the presence of the p-substituent group. The activity of aryl group is lower when there are larger volume substituents or superfluous substituents, and the activity of aryl methyl aryl group is better when there are hetero atoms in the aryl group.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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