Fuconojirimycin C1取代衍生物對嗜熱菌岩藻糖水解酶之抑制研究

Abstract

中文摘要 岩藻糖水解酶 (alpha-L-fucosidases) 為催化自糖鏈末端水解出左旋岩藻糖之酵素，其活性之喪失會造成許多重要疾病相關，包括癌症、纖維性囊腫化 (cystic fibrosis) 和岩藻糖代謝儲積症 (fucosidosis)。因此，開發具有強效及選擇性的抑制劑將有助於瞭解岩藻糖水解酶之功能和相關藥物的研究。 本實驗室先前建立一套具有fuconojirimycin結構抑制劑之快速合成篩選方法，並快速找到Thermotoga maritima及人類組織 (human tissue) 岩藻糖水解酶之強效抑制劑。值得注意的是，這些抑制劑對前者造成slow, tight-binding inhibition，然而對後者卻無此現象。這些分子因此對Thermotoga maritima岩藻糖水解酶有較佳之抑制效果，其中最強的抑制劑對兩種酵素的結合強度差異高達5400倍。 藉由序列比對及電腦模擬分析，初步推測這種截然不同的抑制情形應和Thermotoga maritima岩藻糖水解酶對於抑制劑本身aglycon的結合區有關，而人類組織岩藻糖水解酶則缺乏此一結合區。為由分子層次來研究此一現象，本論文選取6個胺基酸 (W58、F59、Y64、L191、M225、Y267) 進行定點突變分析，在突變成丙胺酸之後，slow-binding inhibition在此6個突變株均有不同程度的減弱。由蛋白質螢光實驗發現，突變型岩藻糖水解酶Y64A及Y267A在抑制劑作用下沒有明顯的結構轉變(conformational change)，此點與人類岩藻糖水解酶相同。其結果不但與動力學實驗相符合，亦證實了這些疏水性胺基酸對於抑制劑algycon之結合扮演重要角色。

Abstract alpha-L-Fucosidases are exo-glycosidases to cleave alpha-linked L-fucose residues from the glycoconjugates that usually participate in a variety of important biological processes. Decreased α-L-fucosidase activity is related to a number of pathological conditions such as inflammation, cancer, cystic fibrosis and fucosidosis. Therefore, discovery of potent and selective inhibitors certainly sheds light on the study of their functions and development of therapeutic agents. Previously, our lab established rapid synthesis of fuconojirimycin-based inhibitors in microtiter plates for high-throughput screening, which rapidly identified potent and selective inhibitors against alpha-L-fucosidases from Thermotoga maritima and human tissue. Interestingly, slow, tight-binding inhibition was found in the study of the former enzyme, distinct from the reversible inhibition of the latter. These molecules thus have higher affinity with the Thermotoga maritima alpha-L-fucosidases than that with the human enzyme, with the best inhibitor exhibiting inhibitory difference up to 5,400-fold . According to the homology modeling and molecular docking, the drastic difference in the inhibition was probably attributed to the hydrophobic aglycon binding site that was absent in the human fucosidase. To decipher the interesting distinction at molecular level, six residues (W58, F59, Y64, L191, M225 and Y267) likely involved in the aglycon binding were changed to alanine by site-directed mutagenesis. Slow-binding inhibition was diminished to different degrees in activity assays of these mutants. Futhermore, intrinsic fluorescence analysis indicated that Y64A and Y267A mutants had no significant change upon inhibitor titration and thus behaved like their human counterpart. The result is not only consistent with the corresponding kinetic data, but also corroborates the role of proposed hydrophobic residues in the aglycon binding site.