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研究生:林裕凱
研究生(外文):Yu-Kie Lin
論文名稱:小分子藥物普拉辛活化EphA2受體產生的生物物理特徵探討
論文名稱(外文):Biophysics Characterization of EphA2 Receptor Upon Activation by Prazosin
指導教授:周綠蘋周綠蘋引用關係
指導教授(外文):LU-PING CHOW
口試委員:黃楓婷林榮信孫英傑
口試委員(外文):FENG-TING HUANGJUNG-HSIN LINYING-CHIEH SUN
口試日期:2018-06-05
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:75
中文關鍵詞:EphA2普拉辛分子動力學模擬喹唑啉
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根據實驗室過去的研究發現,在具有蕾莎瓦藥物抗性的肝癌細胞株中有大量EphA2受體(Ephrin type-A receptor 2 or EphA2 receptor)的表現。近期文獻顯示,在有配體結合誘導的情況下,EphA2受體絲胺酸897位點的磷酸化下降並抑制Akt的磷酸化,使得癌症細胞的增生及轉移受阻,成為有效抑制癌症生長的途徑。然而,尚未有人發表強力有效的促效劑可以應用在癌症治療上。而且近期關於EphA2受體分子作用層面的研究也相當有限,使得找尋EphA2受體的促效劑受阻。因此,我們將在本篇研究中,透過實驗室過去研究發現的,具有活化EphA2受體潛力的促效劑普拉辛(prazosin),作為研究的材料。我們嘗試更專注於EphA2 LBD(ligand binding domain)的作用機轉以及與普拉辛之間的作用力,並成功純化了EphA2 LBD重組蛋白,也進行了表面電漿共振的分析。更進一步利用分子動力學模擬分析EphA2受體與普拉辛結合後產生的結構變化及動性變化,並將之與EphA2受體及其天然配體Ephrin A5結合的結果作比較,藉此了解兩組模擬模型分子作用的異同處。

在我們的研究中觀察到EphA2受體中,由組胺酸54(GLN 54)到纈胺酸69(VAL 69)組成的β折板,在EphA2受體與配體結合後產生的角度偏折可能與EphA2受體活化有關。在普拉辛的分子動力學模擬中,普拉辛結合下的EphA2受體組胺酸54(GLN 54)到纈胺酸69(VAL 69)組成的β折板,呈現出與Ephrin A5相同的結構動性特徵。這個二級結構的動性變化使得未來在藥物設計上有一個可能的篩選指標,成為EphA2受體促效劑開發上的一大助力。
The EphA2 receptor is overexpressed in sorafenib-resistance hepatic cancer cells. When the ligand is absent, EphA2 has been reported to be a pro-oncogenic factor due to Akt activation. However, the effect can be reversed when ligand binds to EphA2. Currently, none of therapeutic agonist of the EphA2 receptor has been reported. Because current understanding of molecular mechanism of the EphA2 receptor activation is limited, designing an effective agonist on the EphA2 receptor is a difficult task. In this work, we successfully purified the EphA2 LBD recombinant protein and used surface plasma resonance analysis to understand the relationship between prazosin and EphA2 LBD recombinant protein. Subsequently, we have built EphA2-ligand docking models: EphA2-prazosin, in which prazosin is a potential EphA2 agonist that was previous identified in our lab. We have also conducted molecular dynamics simulations (MDS) to investigate the difference of conformational and dynamics changes upon EphA2 receptor activation.

Based on the analysis of MDS trajectories, we found that there is a significant dynamics change of the EphA2 β-sheet between Gln54 and Val69, which may be related to EphA2 receptor activation. Prazosin-EphA2 MDS showed that the dynamics changes of the EphA2 β-sheet were correlated with the activation of the EphA2 receptor-Ephrin A5. Therefore, it is anticipated that this finding could serve as a structural and dynamical fingerprint for discovering novel EphA2 agonists.
口試委員會審定書 II
謝誌 III
中文摘要 IV
英文摘要 V
目次 VI
圖表目錄 IX
導論 1
一、 肝癌 1
1. 肝癌臨床特徵 1
2. 肝癌的分期 2
3. 肝癌的治療策略 3
4. 蕾莎瓦的作用機制及抗藥性 4
二、 EphA2 5
三、 藥物分子 8
1. 喹唑啉(quinazolines) 8
2. 喹唑啉衍生物 8
3. 普拉辛(prazosin) 9
研究目的 10
實驗方法 : 11
一、 EphA2 LBD蛋白質分析 11
1. EphA2重組蛋白質純化 11
2. 表面電漿共振分析 12
二、 分子動力學模擬 12
1. 建置模擬模組 12
2. 建置嵌合模型 14
3. 力場參數 15
4. 系統能量最佳化 16
5. 系統加熱 16
6. 分子動力學模擬運算 17
實驗結果 : 18
一、 EphA2 LBD重組蛋白質純化 18
二、 表面電漿共振分析 19
三、 普拉辛與EphA2受體之嵌合模型 19
四、 分子動力學模擬結果 21
1. 普拉辛與EphA2 LBD之A組模型 21
2. 普拉辛與EphA2 LBD之B組模型 22
3. EphA2 LBD之配體結合位分析 24
討論 : 27
1. 普拉辛 27
2. EphA2 LBD之配體結合位分析 30
參考文獻 : 32
圖表 : 38
1. 實驗方法 38
i. 系統建置 38
ii. 系統加熱 39
2. 實驗結果 40
i. EphA2 LBD重組蛋白質純化 40
ii. 表面電漿共振分析結果 42
iii. 普拉辛嵌合模型 43
iv. 普拉辛A組分子動力學模擬 46
v. 普拉辛B組分子動力學模擬 52
vi. EphA2 LBD 之配體結合位分析 58
3. 討論 64
附錄 68
1. 背景介紹 68
2. 系統能量最佳化參數 70
3. 系統加熱參數 71
4. 分子動力學模擬運算 72
5. 蛋白質純化 73
6. 表面電漿共振分析 74
7. 討論 75
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