臺灣博碩士論文加值系統

血球凝集素及神經氨酸酶為流感病毒株表面上兩種主要的醣蛋白，此類的醣蛋白皆為抑制病毒侵犯宿主的治療目標。自2009年，許多報導指出H1N1流感病毒的大流行，另外抗藥性的問題也越來越被重視。因此，本研究的目的在於設計出針對病毒株兩種主要的醣蛋白具有雙重抑制效果的新穎藥物。在實驗中，以基於結構設計藥物與配體設計藥物的方式來分析目標蛋白與配體之間的作用關係，並以分子動力的方式了解受體與配體的作用。新型的H1N1表面醣蛋白之三維結構利用同源模擬的技術建構，所建立的三維結構用於篩選TCM資料庫。分子對接的結果中，根據H1及N1的Dock Score總和做排序並與克流感及瑞樂莎做比較，挑選出前10名候選的化合物。這10個化合物的對接結果提出五個主要的特性，在構效關係的模組中皆符合力場、電性、氫鍵受體與供體的性質，而這些特性也符合H1及N1的胺基酸。最後在20ns的模擬過程上，發現到2-aminopyridinium基團在鍵結方面演著重要的特性。基於結構設計藥物與配體設計藥物的方式，除了能提供對H1及N1在藥物設計上的資訊外，所挑選出化合物也於日後的藥物發展上可被建議為抑制流感病毒的藥物。

Influenza viruses contain two major surface glycoproteins, hemagglutinin and neuraminidase, which are therapeutic targets for inhibiting influenza viruses from infecting host cell. Pandemic of H1N1/09 virus has been reported, and drug resistance was regarded as an important issue since 2009. Thus, the purpose of this research is to design novel potent dual inhibitors for the two surface glycoproteins on H1N1 virus. In this study, structure-based and ligand-based drug designs were performed to analyze interactions between target proteins and ligands, and molecular dynamics (MD) simulations were carried out to analyze the interaction of receptor-ligand complexes. Potent derivatives from structure-based design were ranked by sum of DockScore of two target proteins (H1 and N1) and were compared with Tamiflu (Oseltamivir) and Relenza (Zanamivir) to select the top 10 candidates. Among the scaffold of top 10 candidates, five key features were recognized for binding to H1 and N1. In quantitative structure-activity relationship models, these features were able to fit with their steric, electrostatic, hydrogen bond acceptor and donor fields. These fields were close to key residues of H1 and N1 binding site. Finally, 2-aminopyridinium group was noticed to play an important role in binding ability during 20ns MD simulations. From structure-based and ligand-based designs, we hope that we provided useful information for designing anti-viral compounds targeting H1 and N1, and we recommend the top 10 candidates from our experiments for further drug development testing.

總目錄
摘要...............................................................................................................i
Abstract........................................................................................................ii
總目錄..........................................................................................................iii
表目錄...........................................................................................................v
圖目錄..........................................................................................................vi
一、研究之緣起與目的.................................................................................1
二、研究之方法與理論.................................................................................4
2-1病毒之序列比對與結構模擬..........................................................4
2-2驗證模擬的結構..............................................................................6
2-3中草藥資料庫的建立......................................................................7
2-4目標蛋白的分子對接......................................................................7 2-5以 H1及N1的模擬蛋白生成衍生物.............................................8
2-6衍生物的五法則(Lipinski''s Rule of Five)篩選...............................8
2-7建立構效關係(QSAR)的預測模組.................................................9
2-8分子動力模擬的設定....................................................................10
三、結果與討論..........................................................................................12
3-1同源模擬的結果............................................................................12
3-2篩選的結果與衍生物結構的分析................................................13
3-3衍生物的特性與結合能分析........................................................14
3-4衍生物的特性與目標蛋白對接姿勢分析....................................15
3-5 CoMFA 與CoMSIA預測模組對五種主要特性的分析............18
3-6 分子動力模擬對分子作用力的分析...........................................20
四、結論.......................................................................................................24
五、參考文獻...............................................................................................25六、發表論文...............................................................................................74
6-1 SCI論文.........................................................................................74
6-2 EI論文............................................................................................75
6-3研討會論文....................................................................................77

表目錄

表1. 用於建立CoMFA模組與CoMSIA模組的訓練組結構.................30
表2. 對照組的分子對接結果....................................................................31
表3. 從中草藥資料庫初步篩選的81個化合物對接結果......................32
表4. 基於H1結構生成的衍生物之分子對接結果.................................35
表5. 基於N1結構生成的衍生物之分子對接結果..................................39
表6. 前十名衍生物的分子對接結果........................................................47
表7. 篩選後前十名衍生物與其初始化合物的化學結構........................48
表8. CoMFA模組與CoMSIA模組的PLS分析結果.............................49
表9. CoMFA模組與CoMSIA模組對訓練組化合物的預測能力..........50
表10. Xylopine_2 在H1與N1模擬結構中，從20ns分子動力模擬下所取得的氫鍵資料...............................................................................51
表11. Rosmaricine_14在H1與N1模擬結構中，從20ns分子動力模擬下所取得的氫鍵資料.......................................................................52

圖目錄

圖1. 序列比對結果....................................................................................53
圖2. H1與N1的模擬結構..........................................................................54
圖3. Ramachandran plot的分析結果.........................................................55
圖4. Profile-3D的分析結果.......................................................................56
圖5. 前10名的衍生物在H1模擬結構所預測出來的作用情形.............57
圖6. 前10名的衍生物在N1模擬結構所預測出來的作用情形.............58
圖7. 五個重要特性....................................................................................59
圖8. 以Atom-Fit的方式對24個訓練組化合物其骨架疊合的結果......60
圖9. CoMFA在Rosmaricine_14與H1蛋白的堆疊結果.........................61
圖10. CoMSIA在Rosmaricine_14與H1蛋白的堆疊結果......................62
圖11. CoMFA在Rosmaricine_14與N1蛋白的堆疊結果......................63
圖12. CoMSIA在Rosmaricine_14與N1蛋白的堆疊結果......................64
圖13. Xylopine_2在(a) H1與(b)N1模擬結構於20ns分子動力模擬下的RMSD結果.................................................................................................65
圖14. Rosmaricine_14在(a) H1與(b) N1模擬結構於20ns分子動力模擬下的RMSD結果.........................................................................................66

圖15. 在20ns分子動力模擬下，(a) Xylopine_2 與(b) Rosmaricine_14 的三個關鍵特性與其原子的位置.............................................................67
圖16. H1-Xylopine_2複合體在20ns模擬過程中產生氫鍵的分子間距離.................................................................................................................68
圖17 : N1- Xylopine_2複合體在20ns模擬過程中產生氫鍵的分子間距離.................................................................................................................69
圖18: N1在20ns模擬過程中與2-aminopyridinium基團產生氫鍵的分子間距離.....................................................................................................70
圖19: H1- Rosmaricine_14複合體在20ns模擬過程中產生氫鍵的分子間距離.............................................................................................................71
圖20: 2-aminopyridinium與Hydroxyl 基團與在20ns模擬過程中對H1產生氫鍵的分子間距離.............................................................................72
圖21: N1- Rosmaricine_14複合體在20ns模擬過程中產生氫鍵的分子間距離.............................................................................................................73

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