多重耐药蛋白OptrA的耐药机制及其抑制剂研究

发布时间：2018-05-01 04:30

本文选题：肠球菌 + 抑制剂　；参考：《吉林大学》2017年硕士论文

【摘要】：ABC转运蛋白(ATP-binding cassette transporter)存在于哺乳动物、细菌、真菌等多种细胞中,且与多种细胞的耐药有关。耐药相关(ARE)蛋白,作为ABC蛋白的亚家族,介导细菌对靶向核糖体的一系列抗生素的抗性,在细菌的耐药过程中有着重要的作用。恶唑烷酮类药物(利奈唑胺和泰地唑胺),被认为是治疗VRE、MASA引起感染的最有效药物,而在2015年新发现的ARE蛋白OptrA可介导肠球菌、MASA对恶唑烷酮类抗生素的耐药性,使得恶唑烷酮类抗生素面临着失效的危险。以ARE蛋白为靶点的抑制剂的研究越来越受到人们的重视,因此研究OptrA的耐药机制和抑制剂显得非常重要。本实验通过膜蛋白交联实验和金黄色葡萄球菌体外翻译实验分别确定OptrA与膜蛋白的相互作用及对核糖体的保护情况,并通过ATP水解酶活性实验和最低抑菌浓度实验确定OptrA的ATP水解酶活性中心的关键残基及其介导细菌耐药性的活性中心。使用基于片段的药物筛选方法筛选了OptrA的抑制剂,并在体外验证了该抑制剂对OptrA蛋白的ATP水解酶活性的抑制效果。同时,我们试图解析OptrA的蛋白质晶体结构。虽然并未得到很好的分辨率的晶体结构,但是我们使用同源建模和分子对接初步确定了抑制剂和蛋白质相互作用的位点。结果显示,在金黄色葡萄球菌RN4220的膜蛋白上并没有检测到OptrA蛋白,说明OptrA蛋白不与膜蛋白相互作用。OptrA蛋白可以剂量依赖性的保护核糖体对m RNA的翻译不被抗生素抑制。使用谷氨酰胺替换OptrA全长氨基酸序列的208和488位置的谷氨酸,不但能使OptrA失去其ATP水解酶活性,而且能使其宿主细胞RN4220失去对氟苯尼考的耐药性。基于片段的药物筛选的抑制剂-CP 1可以抑制30%的OptrA的ATP水解酶活性。同源建模和分子对接结果显示CP 1与OptrA蛋白的结合位点为Ile-496、Ile-292、Ile-289和Lys-271。综上所述,OptrA通过保护核糖体,从而介导细菌对抗生素的耐药性。OptrA全长氨基酸序列的208和488位置的谷氨酸为OptrA的ATP水解酶活性中心的关键残基,OptrA的ATP水解酶活性中心为其介导细菌耐药的活性中心。CP 1可以作为OptrA的ATP水解酶抑制剂的先导化合物,为进一步通过结构优化获得更高效抑制剂的研究奠定了重要的实验依据和理论基础。
[Abstract]:ABC transporter ATP-binding cassette transporter exists in mammalian, bacteria, fungi and other cells, and is related to drug resistance of many kinds of cells. As a subfamily of ABC proteins, resistance of bacteria to a series of antibiotics targeting ribosomes plays an important role in the process of drug resistance. Oxazolidinones (linazolidomide and tazolidomide) are considered to be the most effective agents in the treatment of VREMASA infection, and the newly discovered ARE protein OptrA in 2015 mediates the resistance of Enterococcus to oxazolidinone antibiotics. This puts oxazolidinone antibiotics at risk of failure. More and more attention has been paid to the study of inhibitors targeting ARE protein, so it is very important to study the mechanism of drug resistance and inhibitors of OptrA. The interaction between OptrA and membrane protein and the protection of ribosome were determined by membrane protein cross-linking test and staphylococcus aureus translation test in vitro, respectively. The key residues of ATP hydrolase active center of OptrA and the active center of bacterial resistance were determined by ATP hydrolase activity test and minimum inhibitory concentration test. A fragment based drug screening method was used to screen the inhibitor of OptrA, and the inhibitory effect of the inhibitor on the ATP hydrolase activity of OptrA protein was verified in vitro. At the same time, we try to analyze the protein crystal structure of OptrA. Although the crystal structure with good resolution has not been obtained, we use homologous modeling and molecular docking to preliminarily identify the sites for the interaction between inhibitors and proteins. The results showed that no OptrA protein was detected on the membrane protein of Staphylococcus aureus RN4220, indicating that the OptrA protein could not interact with the membrane protein. Optra protein could protect the ribosomal translation of m RNA from antibiotic inhibition in a dose-dependent manner. Using glutamine to replace the glutamate at 208 and 488 positions of the OptrA amino acid sequence can not only make OptrA lose its ATP hydrolase activity, but also make its host cell RN4220 lose its resistance to florfenicol. Fragment-based drug screening inhibitor-CP 1 could inhibit the ATP hydrolase activity of 30% of OptrA. The results of homology modeling and molecular docking showed that the binding sites of CP1 to OptrA protein were Ile-496, Ile-292, Ile-289 and Lys-271. To sum up, OptrA protects ribosomes, Thus, the glutamic acid at 208 and 488 positions of the full-length amino acid sequence of OptrA is the key residue of ATP hydrolase active center of OptrA, and the ATP hydrolase activity center of OptrA is involved in the activity of mediated bacterial drug resistance. Heart. CP1 can be used as a leading compound of ATP hydrolase inhibitor of OptrA. It lays an important experimental and theoretical basis for the further study of obtaining more efficient inhibitors through structural optimization.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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