- 學位論文
GDP及Sulfone的衍生化合物對岩藻糖轉移酶抑制性之研究
Inhibition of Fucosyltransferases by GDP- and Sulfone-based Compounds
摘要
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。 過去的研究發現，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。
並列摘要
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).
並列關鍵字
fucosyltransferase ； inhibition ； GDP ； sulfone ； competitive ； noncompetitive ； uncompetitive參考文獻
延伸閱讀
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