【作者】 朱晓磊；

【导师】 杨光富； 湛昌国；

【作者基本信息】 华中师范大学 ， 有机化学， 2008， 博士

【摘要】 自20世纪40年代以来,除草剂就一直被用于农田防除杂草,但是随着长期和大面积的使用,杂草对除草剂的抗性也随之而来,特别是单一靶标的除草剂。抗性杂草的出现,特别是交互抗性,给常规的防除方法带了巨大的困难。同时也使得许多除草剂的使用寿命缩短。例如,澳大利亚麦田抗禾草灵的瑞士黑麦草（A.myosuroides）产生代谢交互抗性,为害面积达0.5×108hm²,并为害其他作物。因此,了解和研究杂草抗药性的发生和形成机理是现代农药开发和研究中的一项重要任务,基于抗性机理的反抗性除草剂设计也显得尤为重要。ACCase是植物代谢过程中催化植物脂肪酸合成的关键酶,它包含有三个亚基,分别为BC、CT和BCCP,其中CT亚基为本论文的研究范畴。CT的生物功能主要为催化乙酰辅酶A形成丙二酸单酰-辅酶A,然后在脂肪酸合成酶作用下形成脂肪酸。APP类和CHD类为CT的两类主要的抑制剂类型,早在80年代这两类抑制剂就被广泛的应用于防除禾本科杂草,但是由于连续使用最终导致了抗性杂草的出现。到目前为止,已经发现有35种禾本科杂草对该类除草剂产生了抗性,主要分布在美国、加拿大、澳大利亚等14个国家。抗性机制主要分为两类:靶酶突变和代谢加速,其中靶酶突变为主要的抗性机制,文献中已报道了多个抗性突变位点。但是对于高等植物中CT的三维结构及其与抑制剂的结合方式仍然未知,分子水平上的抗性机制的研究无法进行。直到2003年,Tong.L等首次报道了酵母中CT的游离型和复合物型的晶体结构,这就为研究高等植物中CT的性质奠定了基础。本论文的主要研究内容如下:首先,通过文献查阅和CT一级序列的网络搜索,选取狗尾草中CT抗性型和敏感型的氨基酸序列,以酵母中CT为模板进行同源模建,经过二聚体构建和分子动力学模拟得到了二者的稳定构象。对比二者的构象,我们发现第695位（等同于另一个单体的142位）氨基酸残基的空间取向是不一致的,在敏感性靶酶（foxACC-2S）中,Ile-695的支链是伸向活性腔外部的,而在抗性型靶酶（foxACC-2R）中,Leu-695的支链是伸向活性腔内部的,从而干扰了蛋白质与抑制剂的结合,产生抗性。这是从分子水平上阐述I695L突变产生抗性的分子机制。同时,我们还采用分子对接的方法研究了敏感型CT与APP类抑制剂的结合方式,发现了两个对抑制剂结合起重要作用的氨基酸残基（Ser-698和Tyr-728）,为合理药物设计奠定了基础。其次,根据2005年Délye等的报道,选取看麦娘中CT的氨基酸序列,以酵母中CT为模板进行同源模建,得到了来源于看麦娘的游离型CT（AJ_free）和抑制剂复合型CT（AJ_com）的三维结构。通过对比发现,两者的构象变化与晶体结构一致。同时,AJ_com模型中受体与配体的结合方式也与晶体复合物类似,从而证明我们所得模型的可靠性。在此基础上,进一步采用手动对接和分子动力学的方法研究了CT与四种APP类抑制剂的相互作用,对每个体系分别进行MM/PBSA和熵的计算,计算得到的结合自由能的定性趋势与实验值一致,进一步从另一侧面证明了模型的可靠性,同时也说明了结合方式是可信的以及采用手动对接研究相似骨架化合物结合方式的方法是可行的。四种APP类抑制剂的结合方式用于后续的抗性机制研究。再次,针对上面的结合方式,采用直接进行氨基酸突变和分子动力学模拟相结合的方法,研究了CT中W374C、I388N、D425G和G443A四个抗性位点的抗性机制。一共进行了16个体系的分子动力学模拟和MM/PBSA及熵的计算。结果显示,与野生型靶酶相比,抗性靶酶对APP类抑制剂的结合力发生了不同程度的下降,从而使得结合自由能减小,最终表现出抗性。对于D425G和G443A两个远距离抗性位点,其抗性机制是通过改变或破坏了388-446段氨基酸残基链的Hbond网络,进而带动了活性腔内重要的氨基酸残基发生构象变化,降低了受体与配体之间的范德华或者Hbond相互作用,从而产生抗性。以上抗性机制研究为进一步的反抗性分子设计提供了良好基础。最后,在上述的16个突变体系中随机选择了hal₃88和dicl₄25两个体系开展了反抗性分子设计研究。在不改变抑制剂骨架的基础上,通过改变取代基,调节受体与配体之间的焓变或者熵变,目的就是使得体系的结合自由能增大,克服抗性。结果显示,在haloxyfop分子上把吡啶环邻位-Cl原子取代为-CN,可以增大体系的焓变,而在diclofop分子上把苯环邻位-Cl原子取代为-CH₃,可以增大体系的熵变。以上分子设计为进一步开展反抗性除草剂的合成奠定了理论依据。更多还原

【Abstract】 Since the 1940s,herbicide has been used for control of weeds in the farmland.But along with the long-term and big area use,the weed’s resistance appeared,especially for herbicide only having the single target.And then,it did not easy to control weed by using the general method.At the same time, the service live of many herbicides was reduced.For example,A.myosuroides,which is resistant to diclofop in the Australian wheat field,shows cross-resistance to many herbicides,damages the area to reach 0.5×108hm²,and damages other crops.Therefore,studying resistant mechanism becomes very important.And then designing anti-resistance inhibitor would be an arduous task for researchers.ACCase is the key enzyme in the plant metabolism process catalyzing the plant fatty acid biosynthesis.It contains three subunits:BC,CT and BCCP.The CT subunit is our research category, whose biological function is mainly to catalyze acetyl-CoA to form malonyl-CoA,and then forms the fatty acid under the Fattyacidsynthetase.APPs and CHDs are the two kind of mainly inhibitor type for CT.As early as in 1980s,they had been widely used for control gramineous weed,and they had caused the resistant weed’s appearance finally.So far,already discovered that some 35 kind of gramineous weed,mainly distributing in the US,Canada,Australia and so on 14 countries,have showed resistance to APPs and CHDs.The resistant mechanism mainly contains two types:target enzyme mutation and metabolism acceleration.And the former is the main resistant mechanism. Many resistant sites had been reported in the literatures.As to higher plant,the 3D-structure of CT and its binding model with inhibitor were unknown,and the resistant mechanism research in molecular level is unable to carry out.Until 2003,Tong.L et al reported for the first time the crystal structure of CT from yeast in the form of free enzyme and complex with inhibitors,which has laid the foundation for the research in the higher plant.This dissertation may be summarized as follows:Firstly,through the literature consult and the network search for CT’s primary sequence,we select the sensitive and resistant CT sequence from foxtail millet to carry on the homology modeling. The two sable conformations were obtained followed by the dimer construction and the molecular dynamics simulation.Comparing the conformation of these two models,we found that the spatial orientation of residue 695 is different from each other.In foxACC-2S,the Ile-695 extends its side chain outside the active site,while Leu-695 in foxACC-2R extended its side chain toward the cavity of the active site,inhibiting the entrance of ligand.It is the first time we elaborated from the molecular level the resistant mechanism of I695L.Simultaneously,using molecular docking,we study the interaction between the sensitive CT and APPs inhibitors,and discover two important amino acid residues（Ser-698 and Tyr-728）,which would laid the foundation for the reasonable medicine design.Secondly,according to Délye et al reports in 2005,selects the sequence of CT from A. myosuroides to be used for homology modeling.The two models（AJ_free and AJ_com） were obtained alter molecular dynamics simulation.The difference between AJ_free and AJ_com in conformation is consistent with the difference between the crystal structures.The binding model in AJ_com is similar to the crystal complex structure.These results had proven the models we obtained are credible.And then,based on manual docking and molecular dynamics methods,the binding model between CT and four APPs inhibitors were obtained.We carried on the MM/PBSA and entropy computations for every system.The tendency obtained from computing binding free energy is consistent with the experiment value.So,from another point of view,these results had proven the model accuracy and the binding model’s accuracy,simultaneously,also showed the method of manual docking for similar skeleton compound was feasible.And the binding models of four APPs inhibitors were used in the following resistance mechanism research.Thirdly,combining with the amino acid mutation directly and molecular dynamics simulation, the resistant mechanism of four resistant sites（W374C,I388N,D425G and the G443A） for each compound were studied.There are all sixteen mutated systems.We carried on the MM/PBSA and entropy computation for each system.The results show that the binging free energy was reduced by enthalpy and entropy for each mutated system.The enthalpy mainly manifests for VDW interaction or the Hbond interaction between acceptor and ligand,the entropy mainly represents by the conformation entropy.Meanwhile,as to D425G and G443A,belonging to the non-active site,they firstly changed or broken the Hbond network of 388-446,then led the residues in the active site to have the conformation change.Therefore,the binding free energy was reduced by VDW or Hbond interaction between receptor and ligand.The resistant mechanism provides the theory instruction for anti-resistant design.Finally,we randomly selected two mutated system for re-designing anti-resistant compound. Based on not changing the inhibitor’s skeleton,we substituted different groups in order to increasing the binding free energy between receptor and ligand.The results show that the enthalpy would increase by substituting o-Cl to-CN for the haloxyfop,and the entropy would increase by substituting o-Cl to -CH₃ for diclofop.When the binding free energy increased,the resistance would be overcome. All these results would be helpful for synthesis anti-resistant herbicide.更多还原