臺灣博碩士論文加值系統

H5N1流感病毒不僅會在禽鳥類間大流行，甚至也會感染到人類造成嚴重的威脅，因此研究藥物對抗流感病毒是非常重要的。為了研發新藥，我們必須了解病毒的生長過程，尤其在於病毒進入及離開宿主細胞所利用的血球凝集素與神經氨酸酶。在此我們藉由分子模擬方法來比較不同藥物對於N1神經氨酸酶的結合作用，以及藥物對於不同神經氨酸酶的結合性分析。在藥物部份，包含Peramivir、Oseltamivir、Zanamivir以及Sialic acid；在蛋白質的部份，則是有N1神經氨酸酶的野生型(wild type)以及兩種突變型(mutant type)：N1(H274Y)、N1(N294S)。
經由分子模擬後，我們分析其能量與結構上的變化。在能量分析方面，計算結合能(binding energy)以及結合自由能(binding free energy)。其能量排序均為Peramivir<Oseltamivir<Zanamivir<Sialic acid，與實驗所得IC50順序一致。在結構方面，則是作氫鍵(hydrogen bond)、庫倫靜電作用(electrostatic interaction)以及疏水作用(hydrophobic interaction)的分析。氫鍵的分析中，四種藥物都有與野生型N1的酪胺酸 347產生氫鍵，顯示這個胺基酸對於氫鍵的作用力的重要性，而突變型N1的酪胺酸 347並沒有與藥物形成氫鍵，可見此突變對於氫鍵的影響。在庫倫靜電方面，我們分析藥物附近的蛋白質活性區帶電情況，結果顯示藥物與蛋白質活性區在電荷上具有很好的互補性。而在疏水作用的分析上，Peramivir、Oseltamivir、Zanamivir都比Sialic acid有更好的作用力，並皆與精氨酸224有交互作用，顯示此胺基酸對於疏水性作用力的重要性。另外突變型N1神經氨酸酶與藥物形成疏水作用的組數比野生型N1神經氨酸酶還少，顯示此突變會減少兩者之間的疏水作用。這些分析不僅提供更詳細的資訊，同時也對能量的計算結果加以驗證。

H5N1 avian influenza have caused a severe and overwhelming epidemic among birds and humans. To develop new drugs against the pathogen, an easily-mutated virus, much more efforts have been putting on fully understanding the viral entry and leave the cell, especially on neuraminidases and hemagglutinins. Here we used molecular dynamics simulations to test different ligands, including sialic acid, Peramivir, Oseltamivir and Zanamivir binding to N1 neuraminidases and to compare Oseltamivir binding to wild type and mutant N1 neuraminidases.
Binding energy and binding free energy of each ligand were calculated, and the binding energy order is Peramivir<Oseltamivir<Zanamivir<Sialic acid, which agrees with the ranking of experimental IC50 values. In addition, hydrogen bond, electrostatic interaction and hydrophobic interaction were analyzed and compared. Four ligands formed a stable hydrogen bond with the Tyr347 of the wildtype N1 neuraminidase, showing its importance on stabilizing the ligands. On the contrary, hydrogen bonds between ligands and the Tyr347 didn't appear in the mutant N1 neuraminidase, so the ligands' binding modes were different in mutants and in the wildtype. When it comes to the electrostatic interaction, the partial charges of the protein in the vicinity of the ligands were analyzed, and the results showed a good complementarity of charges between ligands and proteins. For the analysis of the hydrophobic interaction, three drugs have more pairs than the sialic acid, which shows that they have better hydrophobic interaction with the protein than the sialic acid. To be more specific, Arg 224 was conserved and greatly contributed to the strong hydrophobic interaction. Besides, less pairs existed in the mutant N1 neuraminidases than the wildtype, which means mutants suppressed the hydrophobic interaction. Above these analyses not only provide more detailed information but are consistent with the energy analysis.

致謝 i
Abstract iii
目錄 iv
圖目錄 vi
表目錄 vii
第一章 導論 1
1.1 流感病毒的演化過程 1
1.2 流感病毒抑制物 5
1.3 相關研究 6
1.4 目的 8
第二章 材料與方法 10
2.1 序列及結構比對 10
2.2 分子動力學模擬 10
2.2.1 材料 11
2.2.2分子動力學模擬理論 11
2.2.3 系統設定 13
2.3 結合能計算 14
2.4 結合自由能計算 15
2.5 結構分析 17
2.5.1 氫鍵分析 17
2.5.2 庫倫靜電作用力分析 17
2.5.3 疏水作用力分析 18
第三章 結果與討論 19
3.1 序列及結構比對 19
3.2 分子動力學模擬 25
3.3 結合能 29
3.4 結合自由能 30
3.5結構分析 39
3.5.1 氫鍵分析 39
3.5.2 庫倫靜電作用力分析 49
3.5.3 疏水作用力分析 53
3.6 野生型與突變型N1神經氨酸酶結構比較 61
第四章 結論 66
參考資料 71

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