林俊宏Lin, Chun-Hung臺灣大學:化學研究所莊育瑞Chuang, Yu-RueiYu-RueiChuang2010-06-302018-07-102010-06-302018-07-102009U0001-2007200914110900http://ntur.lib.ntu.edu.tw//handle/246246/187542岩藻糖轉移酶(fucosyltransferase)催化醣化反應(glycosylation)，將岩藻糖轉移至醣受體(acceptor substrate)。先前的研究指出，這些岩藻糖化的產物參與多種病原菌的感染和疾病的發生。因此，開發強效且具有選擇性的抑制劑，將有助於瞭解岩藻糖轉移酶與它的生理功能之間的關聯性，以及相關藥物開發的可能性。去的研究發現，這個酵素反應的副產物GDP對於岩藻糖轉移酶有很好的親和性，並會造成產物抑制作用 (product inhibition)。本實驗室設計並合成了30多種GDP衍生物，希望能探討抑制劑的結合強度與選擇性，包括幽門螺旋桿菌的alpha-1,3-FucT (HpFucT)，人類的FucT2 (hFucT2)，FucT6 (hFucT6)及FucT9 (hFucT9)。我們利用放射性標定法進行篩選，並測定其IC50和Ki值。比較碳鏈長度對岩藻糖轉移酶抑制性的影響，結果發現triazole與GDP相距2個碳的長度對於該酵素有最佳抑制性。此外相較於其他芳香環官能基，pyrrolidine能夠對於HpFucT和hFucT9產生抑制性；triazole則能夠有效地抑制hFucT2和hFucT6的活性。再者，額外連接疏水性官能基團，對岩藻糖轉移酶均有較好的抑制效果。透過雙倒數圖形判斷，所有GDP衍生物均為競爭型抑制劑，與GDP-fucose競爭相同的結合位。其中，化合物1對hFucT6有最好的抑制性，其Ki為19.9 microM。化合物18則能選擇性抑制hFucT2活性，其Ki為26.7 microM。另外，其他17種sulfone化合物中，化合物S16對於前述四種岩藻糖轉移酶均有很好的抑制性。化合物S16對HpFucT和hFucT9進行可逆型抑制作用，由雙倒數圖形判斷，為非競爭型抑制機制 (noncompetitive inhibition)。從產物生成曲線圖判斷，化合物S16對hFucT6和hFucT2的抑制性，不但會隨著抑制物濃度的增加而提升 (dose-dependent inhibition)，也因為延長預混合時間，使岩藻糖轉移酶完全失去活性 (time-dependent inhibition)。而化合物S16具有高反應性的 alpha,beta-不飽和雙鍵，確實會與dithiothreitol (DTT) 的硫醇基 (thiol group) 進行加成反應，形成共價鍵結。Fucosyltransferases (FucTs) catalyze the transfer of L-fucose from GDP-fucose to various glycoconjugate acceptors. Fucosylated glycoconjugates are known to play important roles in numerous physiological and pathological processes, but there are limited studies for FucT inhibition. The development of potent and selective inhibitors for FucTs is thus helpful to understand the relationship between FucTs and their physiological activities and eventually pursue their therapeutic use for drug discovery.GDP, the side product of FucT reactions, is known to exert product inhibition, indicating that GDP has high affinity with FucTs. Our lab synthesized more than 30 various GDP-based derivatives that were investigated in this thesis. By using radio-isotope labeled GDP-fucose, we aimed to study the inhibition of several FucTs, including alpha-1,3-FucT from Helicobacter pylori, and three human FucTs (hFucT2, hFucT6, and hFucT9). Their IC50 and Ki values were obtained. The results indicated that the optimized chain length between GDP and triazole is two carbons, and that triazole and pyrrolidine are indeed better than other aromatic functional groups. Furthermore, additional incorporation of a suitable hydrophobic group was found to enhance the inhibition potency. The analysis of double reciprocal plots indicated that all the GDP-derived inhibitors are competitive inhibitors to the donor substrate (GDP-fucose). Among them, Compound 1 is the best inhibitor of hFucT6 (Ki = 19.9 microM), while 18 is the best for hFucT2 (Ki = 26.7 microM).Moreover, among seventeen sulfones examined for FucT inhibition, S16 was found to be the best for all the four FucTs. Interestingly S16 shows reversible inhibition against HpFucT and hFucT9, while it exhibits irreversible inhibition against hFucT2 and hFucT6. The difference may be due to Michael addition of the vinyl sulfone in the presence of cysteine thiol(s).目錄(I)次(IV)次(V)錄(VIII)寫表(IX)文摘要(XI)文摘要(XII)壹章 緒論(1).岩藻糖轉移酶之簡介及重要性(1). 路易士抗原之簡介及重要性(1) 1.1 人類岩藻糖轉移酶 (hFucT)(3) 1.2 胃幽門螺旋桿菌之岩藻糖轉移酶 (HpFucT)(5). 岩藻糖轉移酶之催化反應機制(8).岩藻糖轉移酶抑制劑之開發(11). 醣受體 (acceptor) 衍生物之開發(11). 醣予體 (donor) 衍生物之開發(12). 雙受質 (bisubstrate) 衍生物之開發(12). 其他岩藻糖轉移酶抑制劑(13).Sulfone化合物之背景介紹(14).研究動機(15)貳章 材料與方法(17).實驗材料(17).實驗相關之儀器設備(17).實驗方法(18). 岩藻糖轉移酶之胺基酸序列比對(18). 岩藻糖轉移酶之純化與鑑定(18).1 胃幽門螺旋桿菌岩藻糖轉移酶之表現與純化(18).2 人類岩藻糖轉移酶之表現與純化(19).3 岩藻糖轉移酶之鑑定(19). 岩藻糖轉移酶之活性分析(20). 抑制劑活性分析(20).1 岩藻糖轉移酶之衍生物篩選(20).2 代表性抑制劑之IC50測定(21).3 最佳抑制劑之抑制常數之測定(21). 化合物S16和hFucT6 (或hFucT2)的預混合時間對其抑制性之影響(22). 測試化合物S16 + DTT對hFucT6之抑制性(22). 鑑定化合物S16和DTT之加成反應產物(22)參章 實驗結果(23). 岩藻糖轉移酶之胺基酸序列比對結果(23). 幽門螺旋桿菌岩藻糖轉移酶之表現與純化結果(23). 人類岩藻糖轉移酶之表現與純化結果(24). 岩藻糖轉移酶之活性分析結果(24). GDP衍生物抑制能力之結果(25) 5.1 GDP抑制劑篩選結果(25) 5.2 最佳抑制劑之抑制常數測定結果及作用機制(25). Sulfone化合物抑制能力之結果(26) 6.1 Sulfone抑制劑篩選結果(26) 6.2 代表性sulfone抑制劑之作用機制(26) 6.2.1 化合物S13和S16對HpFucT及hFucT9之抑制作用(27) 6.2.2 化合物S13和S16對hFucT2及hFucT6之抑制作用(27)肆章 結論(29). GDP衍生物對岩藻糖轉移酶之抑制性探討(29).1 比較碳鏈長度對岩藻糖轉移酶抑制性之影響(30).2 比較核心結構對岩藻糖轉移酶抑制性之影響(30) 1.3 GDP衍生物對岩藻糖轉移酶之選擇性抑制作用(31) 1.4 GDP衍生物對岩藻糖轉移酶之抑制作用機制探討(32). Sulfone衍生物對岩藻糖轉移酶之抑制性探討(32) 2.1 化合物S16對HpFucT之抑制作用機制(33) 2.2 化合物S16對hFucT6之抑制作用機制(34) 2.3 Sulfone衍生物之選擇性抑制作用(34)伍章 未來展望(35)六章 參考文獻(36)錄(69)2786285 bytesapplication/pdfen-US岩藻糖轉移酶抑制競爭型非競爭型不競爭型fucosyltransferaseinhibitionGDPsulfonecompetitivenoncompetitiveuncompetitivethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/187542/1/ntu-98-R96223201-1.pdf