神经氨酸酶突变H1N1流感病毒的抗药性预测研究

发布时间：2018-06-26 14:43

本文选题：H1N1神经氨酸酶 + 突变　；参考：《郑州大学》2011年硕士论文

【摘要】：从2009年四月份以来,H1N1流感病毒的爆发引起了全世界的关注。世界卫生组织和疾病预防与控制中心一致认为奥司他韦是治疗感染流感病毒病人最有效的抗病毒药物。但是,在2010年二月份世界卫生组织宣布已经发现200多个病例出现了针对奥司他韦出抗药性。奥司他韦(达菲)是神经氨酸酶(NA)的抑制剂,神经氨酸酶在流感病毒复制,释放和致病过程中起着非常重要的作用。奥司他韦口服后经肝脏和肠道酯酶催化迅速转化为其活性代谢物奥司他韦羧酸,奥司他韦羧酸的构型与神经氨酸的过渡态相似,能够竞争性地与流感病毒神经氨酸酶的活性位点结合。流感病毒NA有9个亚型,根据遗传和结构特性分了两类,第一类NA包括N1,N4,N5,N8,;另一类包括N2, N3, N6, N7和N9。第一类和第二类的不同在于150-loop(残基147-152)活性位点附近是否有一个大的空腔。但是,催化位点在所有类中都是保守的。催化位点(R118、D151、D152、R224、E276, R292、R371和Y406)被发现和唾液酸相互作用,而一些辅助位点(E119、R156、W178、S179、D198、1222、E227、H274、E277、N294和E425)被认为支持催化位点的结构。在流感病毒NA的N1亚型中,已证实H274Y和N294S流感病毒株对达菲表现出了强烈的抗药性。在N2和N9亚型中,感染E119V、I222V和R292K突变株的病人对达菲表现出了抗性。在2009年爆发的H1N1流感病毒株中,H274Y突变株对达菲的敏感性平均降低了1466倍。所以,流感病毒NA活性位点的突变有可能影响到流感病毒株对达菲的敏感性的改变。本论文,我们从NCBI数据库中发现了6种新的H1N1流感病毒NA活性位点改变的突变株(E119K、D151G、S179P、D198G、R292W和Y406H),通过同源建模、分子对接、分子动力学模拟和自由能计算研究了这些突变株对达菲的抗药性,并且对有抗药性的突变株进行了残基能量分解计算,分析了抗药性机制。和WT-OTV复合物比较,突变S179P-OTV和R292W-OTV分别使结合自由能下降了-9.5 kcal/mol和-11.88 kcal/mol,这表明了S179P和R292W突变对达菲产生了抗药性。在突变S179P中,非极性氨基酸脯氨酸取代了极性氨基酸丝氨酸,所以原来起主导作用的极性相互作用降低了2.28kcal/mol。另外Glu227形成的氢键数目的减少也驱使S179P对达菲产生抗药性。在R292W突变中,色氨酸取代精氨酸缩小了侧链的大小,这有可能增大了结合腔的空间,降低了抑制剂与蛋白酶间的亲和能。
[Abstract]:The outbreak of the H1N1 influenza virus since April 2009 has attracted worldwide attention. The World Health Organization and the Centers for Disease Control and Prevention agree that oseltamivir is the most effective antiviral drug for influenza patients. However, in February 2010, the World Health Organization announced that more than 200 cases had been found to be resistant to oseltamivir. Oseltamivir (Tamiflu) is an inhibitor of neuraminidase (na), which plays an important role in influenza virus replication, release and pathogenesis. After oral administration, oseltamivir was rapidly transformed into oseltamivir carboxylic acid, a metabolite of oseltamivir carboxylic acid, which was catalyzed by esterase from liver and intestine. The configuration of oseltamivir carboxylic acid was similar to that of neuraminic acid. To be able to competitively bind to the active sites of influenza virus neuraminidase. There are nine subtypes of influenza virus na, which can be classified into two groups according to their genetic and structural characteristics. The first type of na includes N1N4N5N8, and the other includes N2, N3, N6, N7 and N9. The difference between the first and the second is whether there is a large cavity near the active site of 150-loop (residue 147-152). However, the catalytic sites are conserved in all classes. The catalytic sites (R118D151D152H276, R292OR371 and Y406) were found to interact with sialic acid, while some of the auxiliary sites (E119ON156W178S179C D198 1222E227H274E277N294 and E425) were considered to support the structure of the catalytic sites. In the N1 subtype of influenza virus na, H274Y and N294S influenza strains showed strong resistance to Tamiflu. In N2 and N9 subtypes, patients infected with E119VFV I222V and R292K mutants showed resistance to Tamiflu. The sensitivity of the H 274Y mutant to Tamiflu decreased by an average of 1466 times in the 2009 H1N1 influenza virus strain. Therefore, the mutation of na activity site of influenza virus may affect the sensitivity of influenza virus strain to Tamiflu. In this paper, we have found six new H1N1 influenza virus mutants with altered na activity sites (E119KTX D151GN S179PU D198GFR292W and Y406H) from the NCBI database, and linked to each other by homologous modeling and molecular docking. Molecular dynamics simulation and free energy calculation were used to study the resistance of these mutants to Tamiflu, and the residue energy decomposition of resistant mutants was carried out, and the mechanism of drug resistance was analyzed. Compared with WT-OTV complex, mutation S179P-OTV and R292W-OTV reduced binding free energy by -9.5 kcal/mol and -11.88 kcal / mol, respectively, which indicated that S179P and R292W mutations were resistant to Tamiflu. In the mutant S179P, the non-polar amino acid proline replaced the polar amino acid serine, so the dominant polar interaction decreased by 2.28 kcal / mol. In addition, the decrease in the number of hydrogen bonds formed by Glu227 also drives S 179P to develop resistance to Tamiflu. In R292W mutation, tryptophan replaces arginine reduces the size of side chain, which may increase the space of binding cavity and decrease the affinity between inhibitor and protease.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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