HCV相關(guān)靶標(biāo)與抑制劑相互作用的分子模擬研究
發(fā)布時(shí)間：2019-06-20 23:53
【摘要】：發(fā)現(xiàn)高效專一地抑制在丙型肝炎病毒(hepatitis C virus,HCV)生命周期中起關(guān)鍵作用的蛋白的抑制劑是抗HCV藥物研發(fā)的重要環(huán)節(jié)。非結(jié)構(gòu)蛋白3/4A(nonsturcture protein 3/4A,NS3/4A)、非結(jié)構(gòu)蛋白3解旋酶(nonsturcture protein3 helicase,NS3解旋酶)和非結(jié)構(gòu)蛋白5B(nonsturcture protein 5B,NS5B)是抗HCV藥物的重要靶標(biāo),它們?cè)贖CV復(fù)制和翻譯過(guò)程中擔(dān)負(fù)著重要的角色,如對(duì)核酸的解旋和易位等。目前,一系列能夠有效地抑制這些蛋白的藥物已經(jīng)被發(fā)現(xiàn),如已批準(zhǔn)上市的針對(duì)NS3/4A的藥物波普瑞韋、特拉瑞韋和司美匹韋等。但是HCV耐藥病毒株的出現(xiàn)使得對(duì)現(xiàn)有藥物的耐藥越來(lái)越嚴(yán)重,耐藥性的產(chǎn)生嚴(yán)重影響了藥物的療效。因此尋找新穎的能夠有效地抗HCV耐藥病毒株的抑制劑迫在眉睫。隨著計(jì)算機(jī)技術(shù)的不斷進(jìn)步,分子模擬方法作為尋找新型抑制劑的重要工具已被廣泛應(yīng)用于靶標(biāo)與藥物的作用機(jī)制、耐藥機(jī)制等研究中。如分子動(dòng)力學(xué)模擬、拉伸分子動(dòng)力學(xué)模擬、自適應(yīng)偏置力模擬和Metadynamics模擬等分子模擬方法都已經(jīng)廣泛應(yīng)用于HCV相關(guān)靶標(biāo)結(jié)構(gòu)和功能的研究當(dāng)中。本論文將從結(jié)構(gòu)和能量的角度詳細(xì)地闡釋相關(guān)抑制劑與HCV靶標(biāo)NS5B、NS3解旋酶和NS3/4A的相互作用機(jī)制和抑制劑的解離機(jī)制。這些實(shí)驗(yàn)結(jié)果將為設(shè)計(jì)全新的抗HCV藥物提供一定的理論基礎(chǔ)。本論文首先簡(jiǎn)述了HCV生命周期的各個(gè)階段、主要的HCV藥物作用靶標(biāo)的結(jié)構(gòu)和功能以及目前針對(duì)這些靶標(biāo)上市或在研的藥物。并總結(jié)了分子模擬方法在HCV相關(guān)靶標(biāo)與抑制劑之間的相互作用,靶標(biāo)對(duì)抑制劑的耐藥機(jī)制等方面的運(yùn)用情況。然后簡(jiǎn)單介紹了本論文用到的幾種分子模擬方法:自適應(yīng)偏置力模擬、拉伸分子動(dòng)力學(xué)模擬和Metadynamics模擬等�；谶@些模擬方法,本論文的研究?jī)?nèi)容包括4個(gè)部分。論文第一部分闡釋了NS5B突變對(duì)抑制劑BMS-791325的耐藥機(jī)制。我們運(yùn)用分子動(dòng)力學(xué)模擬、結(jié)合自由能計(jì)算、氨基酸殘基能量分解和自適應(yīng)偏置力模擬等方法探討了BMS-791325與NS5B蛋白的野生型(WT)、突變型A421V、L392I和P495L的作用機(jī)制。模擬結(jié)果表明NS5B與BMS-791325結(jié)合的關(guān)鍵作用能為疏水相互作用能,NS5B中氨基酸殘基L392、A393、A396、T399、H428、V494、P495和W500的能量貢獻(xiàn)值超過(guò)1 kcal/mol。BMS-791325從NS5B解離的第一步為氨基酸殘基R503與BMS-791325之間的親水作用能降低,第二步為拇指區(qū)Ⅰ變構(gòu)位點(diǎn)與BMS-791325之間疏水作用的消失促使BMS-791325最終逃離ns5b。氨基酸殘基突變(a421v、l392i和p495l)導(dǎo)致bms-791325與ns5b結(jié)合親和力和bms-791325從ns5b解離的平均力勢(shì)降低。發(fā)生p495l之后,495位氨基酸殘基骨架環(huán)結(jié)構(gòu)消失,使其周圍的蛋白柔性增大,蛋白不能很好地錨定抑制劑。在a421v和l392i突變型ns5b體系中,突變后氨基酸殘基與抑制劑之間疏水相互作用能的降低是其產(chǎn)生耐藥的主要原因。論文第二部分研究了藥物索菲布韋的三磷酸活性代謝產(chǎn)物gs-461203及底物utp與ns5b靶標(biāo)的結(jié)合與解離機(jī)制。我們從晶體結(jié)構(gòu)出發(fā)分別構(gòu)建了三元復(fù)合物ns5b-rna-gs-461203和ns5b-rna-utp。gs-461203和utp分別與ns5b-rna的分子動(dòng)力學(xué)模擬結(jié)果表明:極性和非極性相互作用能對(duì)gs-461203與ns5b和utp與ns5b的結(jié)合是有利的;與utp相比,gs-461203的2’-氟-2’-碳甲基核糖能夠與氨基酸殘基s282和i160形成很強(qiáng)的作用,使gs-461203能夠競(jìng)爭(zhēng)性地結(jié)合到ns5b-rna結(jié)合位點(diǎn)中。運(yùn)用隨機(jī)加速分子動(dòng)力學(xué)模擬的方法預(yù)測(cè)得到utp和gs-461203從ns5b-rna結(jié)合位點(diǎn)的解離路徑是從ns5b手掌區(qū)的背面解離。通過(guò)拉伸分子動(dòng)力學(xué)模擬gs-461203和utp的解離過(guò)程發(fā)現(xiàn),它們的解離過(guò)程大致分為3步:小分子的平移,小分子堿基和核糖的翻轉(zhuǎn)和小分子與靶標(biāo)完全分開(kāi)。s282t對(duì)utp與ns5b-rna的結(jié)合影響較小,但是對(duì)gs-461203與ns5b-rna結(jié)合影響較大。論文第三部分研究了ns3解旋酶與其3個(gè)吲哚環(huán)類抑制劑之間的相互作用。我們運(yùn)用metadynamics模擬方法研究了抑制劑在ns3解旋酶活性口袋中的作用機(jī)制及解離過(guò)程,并構(gòu)建了抑制劑解離過(guò)程的自由能表面變化圖。抑制劑的解離過(guò)程大致分為吲哚環(huán)1位連接的疏水基團(tuán)構(gòu)象發(fā)生變化之后離開(kāi)疏水空腔、吲哚環(huán)3位的乙羧基與ns3解旋酶之間的氫鍵斷裂和抑制劑調(diào)節(jié)到利于從ns3解旋酶結(jié)構(gòu)域Ⅰ和Ⅲ之間的裂縫往外逃離的構(gòu)象而完全解離3步。該類抑制劑的吲哚環(huán)與活性口袋之間有很好的幾何匹配,3位的乙羧基能夠與氨基酸殘基g255和t269形成很強(qiáng)的氫鍵作用,使其構(gòu)成了吲哚環(huán)類抑制劑的基本骨架。吲哚環(huán)1位引入疏水基團(tuán)能夠插入到活性口袋的疏水空腔而阻礙抑制劑的解離;吲哚環(huán)6位引入體積龐大的基團(tuán)能夠增加抑制劑逃離所要克服的空間位阻。論文第四部分研究了ns3/4a突變對(duì)bms-650032的耐藥機(jī)制。分子動(dòng)力學(xué)模擬和結(jié)合自由能計(jì)算的結(jié)果顯示非極性相互作用能在bms-650032與ns3/4a結(jié)合過(guò)程中起到關(guān)鍵作用。根據(jù)殘基能量分解結(jié)果定義了bms-650032與ns3/4a相互作用的11個(gè)關(guān)鍵的氨基酸殘基。metadynamics模擬bms-650032逃離ns3/4a的活性口袋表明bms-650032的p2’和p4部分先離開(kāi)疏水平面,接著p1和p1’部分逃離活性口袋,最后bms-650032整體從活性口袋中解離出來(lái)。a156t、r155k和d168a突變導(dǎo)致bms-650032與ns3/4a的結(jié)合親和力下降以及BMS-650032更加容易從活性口袋中解離。其中A156T突變,擾亂了NS3/4A的疏水口袋,使BMS-650032與NS3/4A的結(jié)合能力降低。而R155K和D168A突變通過(guò)破壞R123-D168-R155-D81之間的鹽橋來(lái)削弱NS3/4A與BMS-650032之間的結(jié)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R978.7
，
本文編號(hào)：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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R978.7
，
本文編號(hào)：2503619
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