HCV相关靶标与抑制剂相互作用的分子模拟研究
发布时间：2019-06-20 23:53
【摘要】：发现高效专一地抑制在丙型肝炎病毒(hepatitis C virus,HCV)生命周期中起关键作用的蛋白的抑制剂是抗HCV药物研发的重要环节。非结构蛋白3/4A(nonsturcture protein 3/4A,NS3/4A)、非结构蛋白3解旋酶(nonsturcture protein3 helicase,NS3解旋酶)和非结构蛋白5B(nonsturcture protein 5B,NS5B)是抗HCV药物的重要靶标,它们在HCV复制和翻译过程中担负着重要的角色,如对核酸的解旋和易位等。目前,一系列能够有效地抑制这些蛋白的药物已经被发现,如已批准上市的针对NS3/4A的药物波普瑞韦、特拉瑞韦和司美匹韦等。但是HCV耐药病毒株的出现使得对现有药物的耐药越来越严重,耐药性的产生严重影响了药物的疗效。因此寻找新颖的能够有效地抗HCV耐药病毒株的抑制剂迫在眉睫。随着计算机技术的不断进步,分子模拟方法作为寻找新型抑制剂的重要工具已被广泛应用于靶标与药物的作用机制、耐药机制等研究中。如分子动力学模拟、拉伸分子动力学模拟、自适应偏置力模拟和Metadynamics模拟等分子模拟方法都已经广泛应用于HCV相关靶标结构和功能的研究当中。本论文将从结构和能量的角度详细地阐释相关抑制剂与HCV靶标NS5B、NS3解旋酶和NS3/4A的相互作用机制和抑制剂的解离机制。这些实验结果将为设计全新的抗HCV药物提供一定的理论基础。本论文首先简述了HCV生命周期的各个阶段、主要的HCV药物作用靶标的结构和功能以及目前针对这些靶标上市或在研的药物。并总结了分子模拟方法在HCV相关靶标与抑制剂之间的相互作用,靶标对抑制剂的耐药机制等方面的运用情况。然后简单介绍了本论文用到的几种分子模拟方法:自适应偏置力模拟、拉伸分子动力学模拟和Metadynamics模拟等。基于这些模拟方法,本论文的研究内容包括4个部分。论文第一部分阐释了NS5B突变对抑制剂BMS-791325的耐药机制。我们运用分子动力学模拟、结合自由能计算、氨基酸残基能量分解和自适应偏置力模拟等方法探讨了BMS-791325与NS5B蛋白的野生型(WT)、突变型A421V、L392I和P495L的作用机制。模拟结果表明NS5B与BMS-791325结合的关键作用能为疏水相互作用能,NS5B中氨基酸残基L392、A393、A396、T399、H428、V494、P495和W500的能量贡献值超过1 kcal/mol。BMS-791325从NS5B解离的第一步为氨基酸残基R503与BMS-791325之间的亲水作用能降低,第二步为拇指区Ⅰ变构位点与BMS-791325之间疏水作用的消失促使BMS-791325最终逃离ns5b。氨基酸残基突变(a421v、l392i和p495l)导致bms-791325与ns5b结合亲和力和bms-791325从ns5b解离的平均力势降低。发生p495l之后,495位氨基酸残基骨架环结构消失,使其周围的蛋白柔性增大,蛋白不能很好地锚定抑制剂。在a421v和l392i突变型ns5b体系中,突变后氨基酸残基与抑制剂之间疏水相互作用能的降低是其产生耐药的主要原因。论文第二部分研究了药物索菲布韦的三磷酸活性代谢产物gs-461203及底物utp与ns5b靶标的结合与解离机制。我们从晶体结构出发分别构建了三元复合物ns5b-rna-gs-461203和ns5b-rna-utp。gs-461203和utp分别与ns5b-rna的分子动力学模拟结果表明:极性和非极性相互作用能对gs-461203与ns5b和utp与ns5b的结合是有利的;与utp相比,gs-461203的2’-氟-2’-碳甲基核糖能够与氨基酸残基s282和i160形成很强的作用,使gs-461203能够竞争性地结合到ns5b-rna结合位点中。运用随机加速分子动力学模拟的方法预测得到utp和gs-461203从ns5b-rna结合位点的解离路径是从ns5b手掌区的背面解离。通过拉伸分子动力学模拟gs-461203和utp的解离过程发现,它们的解离过程大致分为3步:小分子的平移,小分子碱基和核糖的翻转和小分子与靶标完全分开。s282t对utp与ns5b-rna的结合影响较小,但是对gs-461203与ns5b-rna结合影响较大。论文第三部分研究了ns3解旋酶与其3个吲哚环类抑制剂之间的相互作用。我们运用metadynamics模拟方法研究了抑制剂在ns3解旋酶活性口袋中的作用机制及解离过程,并构建了抑制剂解离过程的自由能表面变化图。抑制剂的解离过程大致分为吲哚环1位连接的疏水基团构象发生变化之后离开疏水空腔、吲哚环3位的乙羧基与ns3解旋酶之间的氢键断裂和抑制剂调节到利于从ns3解旋酶结构域Ⅰ和Ⅲ之间的裂缝往外逃离的构象而完全解离3步。该类抑制剂的吲哚环与活性口袋之间有很好的几何匹配,3位的乙羧基能够与氨基酸残基g255和t269形成很强的氢键作用,使其构成了吲哚环类抑制剂的基本骨架。吲哚环1位引入疏水基团能够插入到活性口袋的疏水空腔而阻碍抑制剂的解离;吲哚环6位引入体积庞大的基团能够增加抑制剂逃离所要克服的空间位阻。论文第四部分研究了ns3/4a突变对bms-650032的耐药机制。分子动力学模拟和结合自由能计算的结果显示非极性相互作用能在bms-650032与ns3/4a结合过程中起到关键作用。根据残基能量分解结果定义了bms-650032与ns3/4a相互作用的11个关键的氨基酸残基。metadynamics模拟bms-650032逃离ns3/4a的活性口袋表明bms-650032的p2’和p4部分先离开疏水平面,接着p1和p1’部分逃离活性口袋,最后bms-650032整体从活性口袋中解离出来。a156t、r155k和d168a突变导致bms-650032与ns3/4a的结合亲和力下降以及BMS-650032更加容易从活性口袋中解离。其中A156T突变,扰乱了NS3/4A的疏水口袋,使BMS-650032与NS3/4A的结合能力降低。而R155K和D168A突变通过破坏R123-D168-R155-D81之间的盐桥来削弱NS3/4A与BMS-650032之间的结合能力。
[Abstract]:It is found that the inhibitor of protein that plays a key role in the life cycle of hepatitis C virus (HCV) is an important link to the development of anti-HCV drugs. Non-structural protein 3/ 4A (NS3/ 4A), nonstructural protein 3 (nonstructural protein 3 help, NS3 helicase) and nonstructural protein 5B (NS5B) are important targets for anti-HCV drugs. They play an important role in the process of HCV replication and translation, such as the derotation and translocation of nucleic acids. Currently, a series of drugs that are able to effectively inhibit these proteins have been found, such as those that have been approved to be listed for NS3/ 4A, Pooprius, Trarebet, and the Division. But the emergence of the HCV-resistant virus strain makes the drug resistance of the existing medicine more and more serious, and the production of the drug resistance seriously affects the curative effect of the medicine. Therefore, it is urgent to find novel inhibitors capable of effectively resisting the HCV-resistant strains. With the development of computer technology, the molecular simulation method, as an important tool for finding new inhibitors, has been widely used in the research of target and drug action mechanism, drug resistance mechanism and so on. Molecular simulation methods such as molecular dynamics simulation, tensile molecular dynamics simulation, self-adaptive bias force simulation and Metadynamics simulation have been widely used in the research of HCV-related target structures and functions. The mechanism of the interaction between the inhibitor and the HCV target NS5B, the NS3 helicase and the NS3/ 4A and the dissociation mechanism of the inhibitor will be explained in detail from the structure and energy. These results will provide a theoretical basis for designing new anti-HCV drugs. This paper first describes the various stages of the HCV life cycle, the structure and function of the main HCV drug action targets, as well as the currently marketed or otherwise developed drugs for these targets. The application of the molecular simulation method to the interaction between the target and the inhibitor of HCV and the mechanism of drug resistance of the target to the inhibitor are also summarized. In this paper, several molecular simulation methods, such as self-adaptive bias force simulation, tensile molecular dynamics simulation and Metadnics simulation, are briefly introduced. Based on these simulation methods, the research content of this thesis includes four parts. The first part of the paper illustrates the resistance mechanism of the NS5B mutation to the inhibitor BMS-791325. The effects of wild-type (WT), mutant A421V, L392I and P495L of BMS-791325 and NS5B were discussed by molecular dynamics simulation, free energy calculation, amino acid residue energy decomposition and self-adaptive bias force simulation. The simulation results show that the key role of the combination of NS5B and BMS-791325 is that the energy contribution of the amino acid residues L392, A393, A396, T399, H428, V494, P495 and W500 in the NS5B exceeds 1 kcal/ mol. The first step of the dissociation of the BMS-791325 from the NS5B is that the hydrophilic action between the amino acid residue R503 and the BMS-791325 can be reduced, The second step is that the disappearance of the hydrophobic interaction between the allosteric site of the thumb region and the BMS-791325 causes the BMS-791325 to finally escape the ns5b. The amino acid residue mutations (a421v, l392i, and p495l) resulted in a decrease in the average force potential for the binding affinity of bms-791325 and ns5b and the dissociation of bms-791325 from ns5b. After the occurrence of p495l, the structure of the 495-position amino acid residue skeleton ring disappeared, so that the protein in the periphery of the 495-position amino acid residue was increased flexibly, and the protein could not be well anchored to the inhibitor. In that mutant n5b system of a421v and l392i, the decrease in the hydrophobic interaction between the amino acid residue and the inhibitor after the mutation is the main cause of the drug resistance. The second part of the paper studies the binding and dissociation mechanism of the triphosphate active metabolite gs-461203 and the substrate utp and the ns5b target of the drug. The results of the molecular dynamics simulation of ns5b-rna-gs-461203 and ns5-rnab-utp. g-461203 and utp, respectively, with ns5b-rna show that the polarity and non-polar interaction can be advantageous for the combination of gs-461203 and ns5b and utp and ns5b. The 2 '-fluoro-2'-carboxyribose of the gs-461203 is capable of forming a strong effect with the amino acid residues s282 and i160, allowing the gs-461203 to be competitively bound to the ns5b-rna binding site. The dissociation path of utp and gs-461203 from the ns5b-rna binding site was predicted from the back of the ns5b palmar region using a method of random accelerated molecular dynamics simulation. The dissociation process of gs-461203 and utp, which is simulated by the stretching of the molecular dynamics, is found to be roughly divided into three steps: the translation of small molecules, the inversion of the small molecular bases and the ribose, and the complete separation of the small molecules from the target. S282t has a smaller binding effect on utp and ns5b-rna, but the combination of gs-461203 and ns5b-rna is significant. The third part of the paper deals with the interaction between the ns3 helicase and its three inhibitors. The mechanism and the dissociation process of the inhibitor in the active pocket of the ns3 helicase were studied by using the metadnics simulation method, and the free energy surface changes of the inhibitor dissociation process were constructed. the dissociation process of the inhibitor is roughly divided into a hydrophobic cavity, The hydrogen bond cleavage and the inhibitor of the 3-position ethoxyline and the ns3 helicase are fully dissociated for 3 steps from the conformation that facilitates the escape of the crack from the ns3 helicase domain I and III. The inhibitor has a good geometric matching with the active pocket, and the 3-position ethoxy group can form a strong hydrogen bond with the amino acid residues g255 and t269, so as to form the basic framework of the alicyclic inhibitor. The introduction of a hydrophobic group into the hydrophobic cavity of the active pocket can prevent the dissociation of the inhibitor; the introduction of a bulky group into the alicyclic ring 6 can increase the steric hindrance to which the inhibitor is to be overcome. In the fourth part of the paper, the resistance mechanism of ns3/ 4a mutation to bms-650032 was studied. The results of the molecular dynamics simulation and the combined free-energy calculation show that the non-polar interaction can play a key role in the process of bms-650032 and ns3/ 4a. The residue energy decomposition results define 11 key amino acid residues for the interaction of bms-650032 with ns3/ 4a. The metadnics simulation of bms-650032 from the active pocket of ns3/ 4a indicates that the p2 'and p4 parts of the bms-650032 leave the hydrophobic level first, and then the p1 and p1' portions escape the active pocket, and the last bms-650032 is entirely dissociated from the active pocket. The mutation of a156t, r155k, and d168a results in a decrease in binding affinity between bms-650032 and ns3/ 4a and the easier dissociation of BMS-650032 from the active pocket. The A156T mutation, which disrupted the hydrophobic pocket of NS3/ 4A, reduced the binding capacity of BMS-650032 to NS3/ 4A. The R155K and D168A mutations weaken the binding capacity between NS3/ 4A and BMS-650032 by destroying the salt bridge between R123-D168-R155-D81.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R978.7
，
本文编号：2503619
[Abstract]:It is found that the inhibitor of protein that plays a key role in the life cycle of hepatitis C virus (HCV) is an important link to the development of anti-HCV drugs. Non-structural protein 3/ 4A (NS3/ 4A), nonstructural protein 3 (nonstructural protein 3 help, NS3 helicase) and nonstructural protein 5B (NS5B) are important targets for anti-HCV drugs. They play an important role in the process of HCV replication and translation, such as the derotation and translocation of nucleic acids. Currently, a series of drugs that are able to effectively inhibit these proteins have been found, such as those that have been approved to be listed for NS3/ 4A, Pooprius, Trarebet, and the Division. But the emergence of the HCV-resistant virus strain makes the drug resistance of the existing medicine more and more serious, and the production of the drug resistance seriously affects the curative effect of the medicine. Therefore, it is urgent to find novel inhibitors capable of effectively resisting the HCV-resistant strains. With the development of computer technology, the molecular simulation method, as an important tool for finding new inhibitors, has been widely used in the research of target and drug action mechanism, drug resistance mechanism and so on. Molecular simulation methods such as molecular dynamics simulation, tensile molecular dynamics simulation, self-adaptive bias force simulation and Metadynamics simulation have been widely used in the research of HCV-related target structures and functions. The mechanism of the interaction between the inhibitor and the HCV target NS5B, the NS3 helicase and the NS3/ 4A and the dissociation mechanism of the inhibitor will be explained in detail from the structure and energy. These results will provide a theoretical basis for designing new anti-HCV drugs. This paper first describes the various stages of the HCV life cycle, the structure and function of the main HCV drug action targets, as well as the currently marketed or otherwise developed drugs for these targets. The application of the molecular simulation method to the interaction between the target and the inhibitor of HCV and the mechanism of drug resistance of the target to the inhibitor are also summarized. In this paper, several molecular simulation methods, such as self-adaptive bias force simulation, tensile molecular dynamics simulation and Metadnics simulation, are briefly introduced. Based on these simulation methods, the research content of this thesis includes four parts. The first part of the paper illustrates the resistance mechanism of the NS5B mutation to the inhibitor BMS-791325. The effects of wild-type (WT), mutant A421V, L392I and P495L of BMS-791325 and NS5B were discussed by molecular dynamics simulation, free energy calculation, amino acid residue energy decomposition and self-adaptive bias force simulation. The simulation results show that the key role of the combination of NS5B and BMS-791325 is that the energy contribution of the amino acid residues L392, A393, A396, T399, H428, V494, P495 and W500 in the NS5B exceeds 1 kcal/ mol. The first step of the dissociation of the BMS-791325 from the NS5B is that the hydrophilic action between the amino acid residue R503 and the BMS-791325 can be reduced, The second step is that the disappearance of the hydrophobic interaction between the allosteric site of the thumb region and the BMS-791325 causes the BMS-791325 to finally escape the ns5b. The amino acid residue mutations (a421v, l392i, and p495l) resulted in a decrease in the average force potential for the binding affinity of bms-791325 and ns5b and the dissociation of bms-791325 from ns5b. After the occurrence of p495l, the structure of the 495-position amino acid residue skeleton ring disappeared, so that the protein in the periphery of the 495-position amino acid residue was increased flexibly, and the protein could not be well anchored to the inhibitor. In that mutant n5b system of a421v and l392i, the decrease in the hydrophobic interaction between the amino acid residue and the inhibitor after the mutation is the main cause of the drug resistance. The second part of the paper studies the binding and dissociation mechanism of the triphosphate active metabolite gs-461203 and the substrate utp and the ns5b target of the drug. The results of the molecular dynamics simulation of ns5b-rna-gs-461203 and ns5-rnab-utp. g-461203 and utp, respectively, with ns5b-rna show that the polarity and non-polar interaction can be advantageous for the combination of gs-461203 and ns5b and utp and ns5b. The 2 '-fluoro-2'-carboxyribose of the gs-461203 is capable of forming a strong effect with the amino acid residues s282 and i160, allowing the gs-461203 to be competitively bound to the ns5b-rna binding site. The dissociation path of utp and gs-461203 from the ns5b-rna binding site was predicted from the back of the ns5b palmar region using a method of random accelerated molecular dynamics simulation. The dissociation process of gs-461203 and utp, which is simulated by the stretching of the molecular dynamics, is found to be roughly divided into three steps: the translation of small molecules, the inversion of the small molecular bases and the ribose, and the complete separation of the small molecules from the target. S282t has a smaller binding effect on utp and ns5b-rna, but the combination of gs-461203 and ns5b-rna is significant. The third part of the paper deals with the interaction between the ns3 helicase and its three inhibitors. The mechanism and the dissociation process of the inhibitor in the active pocket of the ns3 helicase were studied by using the metadnics simulation method, and the free energy surface changes of the inhibitor dissociation process were constructed. the dissociation process of the inhibitor is roughly divided into a hydrophobic cavity, The hydrogen bond cleavage and the inhibitor of the 3-position ethoxyline and the ns3 helicase are fully dissociated for 3 steps from the conformation that facilitates the escape of the crack from the ns3 helicase domain I and III. The inhibitor has a good geometric matching with the active pocket, and the 3-position ethoxy group can form a strong hydrogen bond with the amino acid residues g255 and t269, so as to form the basic framework of the alicyclic inhibitor. The introduction of a hydrophobic group into the hydrophobic cavity of the active pocket can prevent the dissociation of the inhibitor; the introduction of a bulky group into the alicyclic ring 6 can increase the steric hindrance to which the inhibitor is to be overcome. In the fourth part of the paper, the resistance mechanism of ns3/ 4a mutation to bms-650032 was studied. The results of the molecular dynamics simulation and the combined free-energy calculation show that the non-polar interaction can play a key role in the process of bms-650032 and ns3/ 4a. The residue energy decomposition results define 11 key amino acid residues for the interaction of bms-650032 with ns3/ 4a. The metadnics simulation of bms-650032 from the active pocket of ns3/ 4a indicates that the p2 'and p4 parts of the bms-650032 leave the hydrophobic level first, and then the p1 and p1' portions escape the active pocket, and the last bms-650032 is entirely dissociated from the active pocket. The mutation of a156t, r155k, and d168a results in a decrease in binding affinity between bms-650032 and ns3/ 4a and the easier dissociation of BMS-650032 from the active pocket. The A156T mutation, which disrupted the hydrophobic pocket of NS3/ 4A, reduced the binding capacity of BMS-650032 to NS3/ 4A. The R155K and D168A mutations weaken the binding capacity between NS3/ 4A and BMS-650032 by destroying the salt bridge between R123-D168-R155-D81.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R978.7
，
本文编号：2503619
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