新烟碱类杀虫剂与乙酰胆碱受体相互作用机制的分子模拟研究

发布时间：2018-08-24 21:06

【摘要】：新烟碱杀虫剂是作用在乙酰胆碱受体(nAChRs)的杀虫剂,其在农业和动物杀虫方面使用的非常广泛。但是由于它们的广泛应用,导致了昆虫对其耐药性的出现。为了开发出新的更有效的杀虫剂,探究新烟碱类杀虫剂耐药以及不耐药发生的分子机理变得十分重要。其中靶标位点的突变是产生耐药的一个重要原因,但是耐药性发生的具体机理尚不明确。针对这一问题,本论文采用多种分子模拟方法如分子对接、分子动力学模拟、MM/GBSA计算,PCA分析和RIN分析等阐明由于乙酰胆碱受体关键氨基酸突变导致其对新烟碱类杀虫剂耐药性发生的分子机理。具体研究内容包括以下三部分:论文第一部分研究的是第一代乙酰胆碱受体激动剂Imidacloprid(IMI)由于靶标的突变而引发耐药性的分子机制。nAChRs的靶标位点突变(Y151S和R81T)是引起昆虫对IMI耐药的重要原因。然而昆虫的nAChRs结构未知,在这里我们采用与其结构相似的蜗牛的Ls-ACh BP代替昆虫的nAChRs的结构研究耐药性发生的分子机制。通过序列比对发现,nAChRs中的残基Y151和残基R81在位置上分别对应于Ls-AChBP中的残基H145和残基Q55。由于Ls-AChBP与IMI复合物的晶体结构已知,我们通过突变Ls-AChBP两个相应位置的氨基酸残基得到IMI与两个突变体(H145S和Q55T)复合物的结构,以研究乙酰胆碱受体对IMI耐药性发生的分子机制。结合自由能计算的结果表明,两个突变都降低了IMI和结合口袋之间的相互作用。从进一步的残基能量分解分析,我们发现突变前后结合口袋残基的能量贡献发生了改变。此外,残基相互作用网络(RIN)分析结合PCA分析显示两个突变残基通过改变与突变残基直接和间接相互作用残基的构象而改变了IMI结合口袋的构象和残基柔性。Q55T突变体中M114与T55形成氢键相互作用造成M114的构象改变,而H145S突变体中S145与Y192、T144的直接相互作用导致Y192和T144也随之发生构象变化。M114、Y192、T144这些结合口袋周围的残基构象变化造成了IMI与靶标结合作用的减弱。这些结果很好地解释了两个突变体会对IMI产生耐药性的原因,将有助于设计更有效的抑制剂。论文的第二部分主要研究了当昆虫的乙酰胆碱受体发生R81T突变时,Sulfoxaflor和Dinotefuran对突变体具有不同亲和力的原因。首先通过分子对接的方法将Sulfoxaflor和Dinotefuran对接到Ls-AChBP,得到其与Ls-AChBP复合物的结构。结合口袋溶剂可极化表面积的计算发现Sulfoxaflor与Ls-AChBP结合的体系结合口袋的体积变小,而使得配体与受体之间的结合更紧密。因此虽然Sulfoxaflor与直接突变残基作用减弱,但它与结合口袋依然保持较强的结合能力。而Dinotefuran的耐药性是由于结合口袋残基柔性和构象发生了较大的改变,结合口袋变得松散导致W143、T144、Y185、A103、L112的能量贡献减小。论文的第三部分根据第二部分得到的Sulfoxaflor不耐药的结论,通过对Sulfoxaflor进行结构改造以期得到不耐药且活性更好的候选小分子。首先是通过薛定谔软件对Sulfoxaflor进行结构改造,然后再运用类药性五规则、分子对接等技术对设计的小分子进行评价,并分别计算了筛选出来的小分子与结合受体的结合自由能。最后筛选出43个具有合适的类药性同时和野生型以及Q55T突变的Ls-AChBP都具有较强结合能力的小分子。本论文从分子水平上解释了乙酰胆碱受体发生突变的昆虫对新烟碱类杀虫剂Imidacloprid,Dinotefuran,Sulfoxaflor具有不同程度的耐药性的机理,并基于Sulfoxaflor的骨架结构进行了小分子抑制剂的设计和改造。所得结果将有助于进一步发展更加有效的杀虫剂。
[Abstract]:Neo-nicotine insecticides are insecticides that act on acetylcholine receptors (nAChRs). They are widely used in agriculture and animal insecticides. However, their wide application has led to the emergence of insect resistance. In order to develop new and more effective insecticides, to explore the occurrence of resistance and non-resistance to new nicotine insecticides Molecular mechanism becomes very important. The mutation of target site is an important reason for drug resistance, but the specific mechanism of drug resistance is still unclear. To solve this problem, many molecular simulation methods, such as molecular docking, molecular dynamics simulation, MM/GBSA calculation, PCA analysis and RIN analysis, are used to clarify the acetylcholine. The molecular mechanism of resistance to new nicotine insecticides induced by mutations in key amino acids of alkaline receptors is discussed in this paper. The main contents are as follows: In the first part of this paper, the molecular mechanism of resistance induced by mutations in target sites of the first generation acetylcholine receptor agonist Imidacloprid (IMI) is studied. (Y151S and R81T) are important reasons for insect resistance to IMI. However, the structure of nAChRs in insects is unknown. Here, we use the structure of snails with similar structure to replace the structure of nAChRs in insects to study the molecular mechanism of drug resistance. The residues H145 and Q55 in Ls-AChBP were identified. Since the crystal structure of the Ls-AChBP-IMI complex is known, the structure of the IMI-IMI complex with two mutants (H145S and Q55T) was obtained by mutating the residues of the corresponding amino acids in Ls-AChBP to study the molecular mechanism of the resistance of acetylcholine receptors to IMI. The results show that both mutations reduce the interaction between IMI and binding pockets. Further energy decomposition analysis showed that the energy contribution of binding pocket residues changed before and after mutation. The conformation of IMI binding pocket and residue flexibility were changed by the direct and indirect interaction of residues. The hydrogen bond interaction between M114 and T55 in Q55T mutant resulted in the conformation change of M114, while the direct interaction between S145 and Y192, T144 in H145S mutant resulted in the conformation change of Y192 and T144. These results provide a good explanation for the resistance of the two mutants to IMI and will help to design more effective inhibitors. The second part of this paper focuses on Sulfoxaflor when the R81T mutation occurs in the acetylcholine receptor of insects. Sulfoxaflor and Dinotefuran were first docked to Ls-AChBP by molecular docking to obtain the structure of the Ls-AChBP complex. Sulfoxaflor maintained a strong binding capacity with the binding pocket, although the interaction between Sulfoxaflor and the direct mutant residue was weakened. In the third part of the paper, according to the conclusion that Sulfoxaflor is not drug-resistant, Sulfoxaflor is reconstructed to obtain the candidate molecules which are not drug-resistant and have better activity. The binding free energies of the selected small molecules with binding receptors were calculated by docking and other techniques. Finally, 43 small molecules with suitable drug-like properties and strong binding ability to both wild-type and Q55T mutant LS-AChBP were screened out. Insects with mutations in alkali receptors exhibited different resistance mechanisms to new nicotine insecticides Imidacloprid, Dinotefuran and Sulfoxaflor. Small molecule inhibitors were designed and modified based on the skeleton structure of Sulfoxxaflor. The results will be helpful for further development of more effective insecticides.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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