臺灣博碩士論文加值系統

本文探討由八個胺基酸所組成之環狀胜肽Octreotide (SMS 201-995) D-Phe1-cyclo[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys7]-Thr8-ol，Octreotide穿過細胞膜並與體制素受體鍵結而具有類似體制素之生化活性，特別是對體制素受體SSTR-2/3/5具選擇性位於腦、下丘腦及垂體已被確定。藉由螢光實驗證實Octreotide進入SDS ( Sodium Dodecyl Sulfate )微胞溶液其色胺酸殘基位於微胞厭水性環境中，並以脈衝磁場梯度實驗可分別測量Octreotide 於水溶液及SDS微胞環境中之擴散係數，進而估計Octreotide 與SDS微胞之鍵結常數為 6.12×102 M-1，由自由能公式得△G為 -15.9 kJ/mol 顯示Octreotide 與SDS微胞之鍵結作用為自發性結合，與螢光實驗相符合。最後使用核磁共振光譜技術及分子模擬研究Octreotide之構型特徵，並根據化學位移特性、NOE及耦合常數3JNH-CαH之分析顯示，Octreotide於SDS微胞溶液環境下構型呈現β-迴路和螺旋結構(碳端310-helix)之平衡狀態，且D-W4、K5為β-迴路之中心。Octreotide之構型與芳香環支鏈的空間關係以及其對體制素受體選擇性之影響在文中進一步的討論。

This paper studies a conformational of an octapeptide D-Phe1-cyclo[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys7]-Thr8-ol (disulfide bridged), known as Octreotide (or SMS 201-995 or sandostatin) using nuclear magnetic resonance spectroscopy and molecular modeling method. Octreotide displays both somatostatin-like and opioid-like bioactivities which colud be mediated through trans-membrane receptors SSTRs. Predominant selectivity to SSTR2、SSTR3、SSTR5 located in brain, hypothalamus and pituitary has been identified for Octreotide. The interaction between Octreotide and SDS micelle is also monitored by the blue shift and attenuation of fluorescence signal of peptide upon the introduction of micelle. The association constant of Octreotide-micelle complex is estimated by a two-state approximation based on the diffusion coefficients, measured by PFG-NMR spectroscopy, for Octreotide in aqueous and micellar solutions, respectively. The binding constant is about 6.12×102 M-1. These results are evidences to show that the tryptophan residue locates in the hydrophobic environment of the micelle and the which are consistent with the observation in NMR experiments. Base on the analysis of chemical shift deviation, NOE cross-peaks and coupling constants, the backbone conformation of Octreotide, under micellar environment, was in equilibrium between β-strand and helix-like secondary structure fold, with a β-turn centered at D-Trp4-Lys5. The correlation between the relative positions of the aromatic side-chain of the peptide and its receptor affinity is further discussed.

目 錄
中文摘要...........................................Ⅰ
英文摘要...........................................Ⅱ
誌謝.............................................. Ⅲ
目錄.............................................. Ⅴ
表目錄............................................ Ⅸ
圖目錄........................................... XII
名詞簡稱及縮寫對照表........................... XVIII
第一章 緒 論.......................................1
1.1體制素...........................................1
1.2體制素受體.......................................3
1.3體制素類似物及相關藥物設計原理...................5
1.4體制素類似物-Octreotide........................ 11
1.5體制素及其類似物之構型......................... 13
1.6研究動機........................................16
第二章 材 料 與 方 法.............................40
2.1樣品鑑定....................................... 40
2.1.1 HPLC與MALDI-TOF質譜之藥品配製及實驗條件..... 40
2.1.2 HPLC與MALDI-TOF質譜所使用藥品及儀器..........40
2.2樣品與微胞作用性質測量......................... 41 2.2.1 螢光光譜.................................... 41
2.2.1.1螢光樣品配製................................41
2.2.1.2螢光實驗藥品及儀器......................... 42
2.2.1.3螢光光譜實驗條件........................... 43
2.2.2脈衝磁場梯度實驗（Pulsed Field Gradient , PFG-NMR）.43
2.2.2.1脈衝磁場梯度................................43
2.2.2.2樣品配製................................... 44
2.2.2.3磁場梯度實驗條件........................... 45
2.3構型特徵及相關參數之取得–核磁共振光譜..........45
2.3.1樣品配製..................................... 45
2.3.2核磁共振光譜實驗藥品及儀器................... 46
2.3.3化學位移標定................................. 46
2.3.4一維光譜實驗................................. 46
2.3.4.1一維光譜....................................47
2.3.4.2一維光譜實驗條件........................... 47
2.3.5二維光譜實驗..................................48
2.3.5.1二維光譜....................................48
2.3.5.2二維光譜實驗條件........................... 48
2.3.6氫-氘交換（H-D exchange）實驗................ 50
2.3.6.1氫-氘交換速率.............................. 50
2.3.6.2樣品配製................................... 50
2.3.6.3氫-氘交換實驗實驗條件...................... 50
2.3.7胜肽二級結構分析............................. 51
2.3.7.1胜肽的1H NMR光譜判定....................... 51
2.3.7.2化學位移差值（Chemical Shift Index , CSI）. 51
2.3.7.3溫度係數（Temperature coefficient）........ 52
2.3.7.4胺基質子之氫氘交換速率......................52
2.3.7.5 NOE連接（NOE connectivity）................53
2.3.7.6耦合常數3JNH-CαH（Coupling constant）...... 54
2.4分子模擬（Molecular Simulation）............... 55
2.4.1 Insight II...................................55
2.4.2 Insight II參數設定...........................58
第三章 實 驗 結 果............................... 72
3.1 Octreotide樣品鑑定.............................72
3.2樣品與微胞作用性質測量..........................72
3.2.1螢光光譜......................................72
3.2.2脈衝磁場梯度實驗..............................73
3.3核磁共振光譜....................................73
3.3.1濃度效應-纏捲實驗.............................74
3.3.2 1H NMR二維光譜共振訊號之判定.................75
3.3.3化學位移差值..................................76
3.3.4 溫度係數.....................................76
3.3.5胺基質子之氫氘交換速率........................77
3.3.6 NOE連接......................................77
3.3.7 耦合常數3JNH-CαH及雙平面角...................79
3.4分子模擬........................................79
第四章 討 論.....................................137
4.1 Octreotide與SDS微胞作用之討論.................137
4.2 Octreotide在SDS微胞下背骨構型之討論...........138
4.3 Octreotide在SDS微胞下側鏈之結構對SSTR選擇性討論...140
4.4 Octreotide與Lanreotide、Vapreotide對SSTR受體選擇性之影響...................................................141
第五章 結 論.....................................146
參考文獻..........................................147
表 目 錄
表1-1 體制素受體各子型分佈與其生理作用。...............17
表1-2 體制素及類似物對體制素受體之選擇性。.............18
表1-3 體制素受體對體制素及類似物之選擇性。.............19
表1-4 體制素及類似物對體制素受體之選擇性。............ 20
表1-5 體制素類似物之序列。.............................21
表1-6 體制素類似物背骨間之NOES。...................... 22
表2-1 10% D2O/90% H2O溶液，在不同溫度所測得之化學位移。60
表2-2 200 mM SDS微胞溶液，在不同溫度所測得之化學位移。 61
表2-3 二十種共同胺基酸於無序纏捲時的質子化學位移表。...62
表2-4 胜肽二級結構所對應之耦合常數3JNH-CαH標準值。.....63
表2-5 胜肽二級結構所對應之雙平面角標準值。.............64
表2-6 胜肽二級結構γ及β-turns所對應之雙平面角結構特徵。65
表3-1 1 mM Octreotide於100% D2O之DOSY 2D光譜-三重複之計算。....................................................82
表3-2 1 mM Octreotide於100 mM SDS微胞溶液（100%D2O）之DOSY 2D光譜-三重複之計算。..............................83
表3-3 2mM Octreotide於10% D2O / 90% H2O，溫度在278 K之質子化學位移表。.......................................... .84
表3-4 2mM Octreotide於200mM SDS-d25，溫度在298 K下之質子化學位移表。............................................ .85
表3-5 2 mM Octreotide於10%D2O / 90%H2O，溫度為298 K之背骨胺基質子化學位移測量值減去無序纏捲背骨胺基質子化學位移標準值所得到之化學位移差值。................................86
表3-6 2 mM Octreotide於200 mM SDS，溫度為298 K之背骨胺基質子化學位移測量值減去無序纏捲背骨胺基質子化學位移標準值所得到之化學位移差值。......................................87
表3-7 2mM Octreotide於200 mM SDS微胞溶液中背骨胺基質子化學位移測量值減去10% D2O / 90% H2O環境中背骨胺基質子化學位移測量值，所得到的化學位移差值，溫度為298 K。................88
表3-8 Octreotide在水溶液及SDS微胞溶液下之溫度係數表。...89
表3-9 溫度為278 K，Octreotide於10% D2O / 90% H2O之耦合常數3JNH-CαH及雙平面角。.....................................90
表3-10 溫度為278 K，Octreotide於10% D2O / 90% H2O之耦合常數3JNH-CαH，以不同文獻之常數值(A、B、C)求得雙平面角之結果。91
表3-11 溫度為298 K，Octreotide於SDS微胞溶液之耦合常數3JNH-CαH及雙平面角。...........................................92
表3-12 溫度為298 K，Octreotide於SDS微胞溶液之耦合常數3JNH-CαH，以不同文獻之常數值(A、B、C)求得雙平面角之結果。......93
表3-13 Octreotide於SDS微胞溶液之結構計算限制條件。.......94
表3-14 Octreotide於SDS微胞溶液以Insight II及MOLMOL軟體計算兩群結構之RMSD值。.......................................95
表3-15 Octreotide於SDS微胞溶液兩群分子之結構以Insight II軟體計算各殘基之RMSD值，結果顯示T6~T8-ol片段堆疊的最好。...96
表3-16 分別以Insight II及MOLMOL軟體計算比較兩群細分結構之RMSD值。............................................... .97
表3-17 Octreotide於SDS微胞溶液兩群之結構以Insight II軟體計算各殘基之RMSD值，第一群堆疊的最好為T6~T8-ol片段及第二群堆疊較佳為K5~C7片段。..................................... 98
表4-1 SRIF-14 K9→A 、Vapreotide、Lanreotide及Octreotide於D2O及SDS 微胞溶液中以PFG-NMR 實驗所測得之擴散係數及結合常數。....................................................143
表4-2 體制素受體與構型對生化活性之關聯。................144
圖 目 錄
圖1-1 第三腦室中神經內分泌系統之基本要素。..............23
圖1-2 1965年貝勒大學醫學院，純化提取適用綿羊之下丘腦。..24
圖1-3 Prosomatostatin、SRIF-28、SRIF-14 關係示意圖。....25
圖1-4 體制素受體與腺苷酸環酶之鍵結。................... 26
圖1-5 體制素受體與離子通道之鍵結。..................... 27
圖1-6 體制素與其受體鍵結後之抗分泌作用之信號傳遞路徑。..28
圖1-7 G蛋白與受體鍵結之模型。.......................... 29
圖1-8 人類的體制素受體子型之序列分別為七個跨膜片段。....30
圖1-9 人類體制素受體分為五類子型。......................31
圖1-10 體制素SRIF-14對於腸胃之作用。.....................32
圖1-11 SRIF-14之一級結構。..............................33
圖1-12 體制素類似物Octreotide、Lanreotide和Vapreotide之一級結構。...................................................34
圖1-13 模擬體制素類似物Octreotide與體制素受體SSTR-2之鍵結。.....................................................35
圖1-14 模擬體制素類似物Octreotide與體制素受體SSTR-2之鍵結。.....................................................36
圖1-15 為分子模擬圖與X-ray繞射圖。.......................37
圖1-16 體制素類似物對其體制素受體之活性部位空間結構相關位置。.....................................................38
圖1-17 比較對SSTR-2與對SSTR-2/3/5有選擇性構型之差異性。..39
圖2-1 標準二級結構MCD型態。.............................66
圖2-2 各種二級結構元素的NOEs的典型型態。................67
圖2-3 胜肽骨架上之雙平面角φ、ψ、ω、χ等示意圖。.........68
圖2-4 NMR 圖譜數據進行分子模擬之步驟。..................69
圖2-5 Insight II進行分子模擬之流程與所需之模組功能。....70
圖2-6 分子模擬過程中所設定之重要參數。..................71
圖3-1 Octreotide之HPLC圖。..............................99
圖3-2 Octreotide之MALDI-TOF質譜圖。....................100
圖3-3 20μM之Octreotide溫度在298 K下，10 mM Tris-buffer中， pH=7.1及在20 mM SDS中，p H=7.3之螢光光譜。.........101
圖3-4 1 mM Octreotide於100%D2O中，溫度為298 K下之一維光譜疊圖。..................................................102
圖3-5 1 mM Octreotide於100% D2O中，溫度為298 K下之DOSY 2D光譜，為Xwinnmr演算之結果。.............................103
圖3-6 1 mM Octreotide於100 mM SDS微胞溶液（100%D2O）中，溫度為298 K下之一維光譜疊圖。.............................104
圖3-7 1 mM Octreotide於100 mM SDS微胞溶液（100%D2O）中，溫度為298 K下之DOSY 2D光譜，為Xwinnmr演算之結果。.........105
圖3-8 Octreotide於10%D2O/90%H2O中之不同濃度(0.1 mM及2.0 mM )下之一維光譜圖，溫度為278 K。.......................106
圖3-9 Octreotide於於SDS微胞溶液中之不同濃度( 0.1 mM、1 mM及2.0 mM)下之一維光譜圖，溫度為298 K。..................107
圖3-10 2 mM Octreotide於10%D2O/90%H2O中之TOCSY圖譜，mixing time=80 ms，溫度為278K，pH=5.03。................108
圖3-11 2 mM Octreotide於10%D2O/90%H2O中之NOESY圖譜，mixing time=450 ms，溫度為278 K，pH=5.03。..............109
圖3-12 2 mM Octreotide於200 mM SDS微胞溶液中之TOCSY圖譜，mixing time= 80ms，溫度為298 K，pH=4.62。...............110
圖3-13 2 mM Octreotide於200 mM SDS微胞溶液中之NOESY圖譜，mixing time=500 ms，溫度為298 K，pH=4.62。..............111
圖3-14 2 mM Octreotide在水溶液及SDS微胞溶液環境下，溫度為298 K之背骨胺基質子化學位移測量值減去無序纏捲背骨胺基質子化學位移標準值所得到之化學位移差值。......................112
圖3-15 2 mM Octreotide於200 mM SDS微胞溶液中背骨胺基質子化學位移與10% D2O / 90% H2O環境中背骨胺基質子化學位移之差值，溫度為298 K。...........................................113
圖3-16 Octreotide於10% D2O / 90% H2O中，278 K~298 K溫度範圍之一維光譜疊圖。......................................114
圖3-17 2 mM Octreotide於200 mM SDS微胞溶液中，在不同溫度(293 K~305 K)下之變溫一維光譜疊圖。....................115
圖3-18 2 mM Octreotide於200 mM SDS微胞環境下（100% D2O），其溫度為293 K下之背骨胺基質子氫氘交換實驗一維光譜疊圖。116
圖3-19 2 mM Octreotide於10%D2O/90%H2O中，pH=5.03，溫度為278 K，mixing time=250 ms之ROESY光譜。..................117
圖3-20 2mM Octreotide10% D2O/90% H2O，溫度在278K下之NOE連接。....................................................118
圖3-21 2 mM Octreotide於SDS微胞溶液mixing time=500 ms之NOESY光譜。標示指紋區域為αH-NH範圍。....................119
圖3-22 2 mM Octreotide於SDS微胞溶液mixing time=500 ms之NOESY光譜。標示指紋區域為NH-NH範圍。....................120
圖3-23 2 mM Octreotide於SDS微胞溶液mixing time=500 ms之NOESY光譜。標示指紋區域為苯環上的氫之範圍。.............121
圖3-24 2 mM Octreotide於200 mM SDS微胞溶液之NOE連接。..122
圖3-25 2 mM Octreotide於10%D2O/90%H2O中，溫度為278 K下之DQF-COSY光譜。..........................................123
圖3-26 2 mM Octreotide於200 mM SDS微胞環境中，溫度為298 K下之DQF-COSY光譜。......................................124
圖3-27 經過分子動力模擬焠熄計算，分別以Phe3∼Thr6活性片段為中心進行Octreotide於SDS微胞溶液中結構之篩選，其篩選結果以背骨方式呈現，主要分為兩群結構之第一群。................125
圖3-28 經過分子動力模擬焠熄計算，以D-Trp4-Lys5為中心進行Octreotide於SDS微胞溶液中結構之篩選，其篩選結果以背骨方式呈現，主要分為兩群結構之第二群。..........................126
圖3-29 兩群分子結構各殘基之RMSD值以長條圖表示。........127
圖3-30 經過分子動力模擬焠熄計算，以D-Trp4-Lys5為中心進行Octreotide於SDS微胞溶液中結構之篩選，其篩選之結果得到30個重疊良好的Octreotide分子結構。............................128
圖3-31 經過分子動力模擬焠熄計算，以D-Trp4-Lys5為中心進行Octreotide於SDS微胞溶液中結構之篩選，其篩選之結果得到26個重疊良好的Octreotide分子結構。............................129
圖3-32 兩群分子結構各殘基之RMSD值以長條圖表示。........130
圖3-33 細分第一群分子結構。............................131
圖3-34 細分第二群分子結構。............................132
圖3-35 將細分兩群分子結構疊圖，背骨以緞帶方式呈現。....133
圖3-36 Octreotide在SDS微胞環境下，a、b兩群分子D-W4、K5側鏈與支鏈之空間關係圖。....................................134
圖3-37 Octreotide在SDS微胞環境下，第一群分子之藥效基團D-W4、K5側鏈與支鏈之距離關係圖。圖中呈現各殘基Cγ原子間所量測得之。..................................................135
圖3-38 Octreotide在SDS微胞環境下，第二群分子之藥效基團D-W4、K5側鏈與支鏈之距離關係圖。圖中呈現各殘基Cγ原子間所量測得之。..................................................136
圖4-1 體制素類似物側鏈與芳香環支鏈在空間中排列位置與不同受體具有選擇性之關聯。....................................145

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