亞胺醣與岩藻醣水解酶結合行為的熱力學分析

Abstract

亞胺醣 (iminosugar) 為著名的水解酶抑制物，由於其分子結構中環內的氮在正常生理環境中容易質子化，因此，亞胺醣與醣類水解酶的反應過渡態- 鎓陽離子(oxocarbenium ion) 相似，同樣具有正電荷的特徵，成為良好的醣類水解酶抑制物。目前，由於亞胺醣具有成為藥物開發的價值而受到學術界高度重視，舉例而言，第一型亞胺醣藥物 GlysetTM (也被稱作Miglitol TM)可有效地抑制小腸內的雙醣水解酶，可成為第一代經由FDA認證的第二型糖尿病的治療藥物。 先前，本實驗室利用此概念成功地開發出一系列對於岩藻醣水解酶具有良好抑制性的衍生物，此系列抑制物由以fuconojirimycin (FNJ, fuco-type iminosugar)為核心結構進行不同的官能基修飾，根據修飾官能基的不同，抑制常數Ki 分布範圍為micro- to pico-層級。本論文針對上述FNJ系列的抑制物(ZY1 ZY2 ZY3)，對於Thermotoga maritima 岩藻醣水解酶進行結合行為的分析。實驗主軸為利用等溫滴定卡計測量相關的熱力學參數，包跨解離常數、焓變化、熵變化、誘導質子數以及熱容變化量。依據實驗結果發現在不同pH 值下，FNJ抑制物與岩藻醣水解酶結合過程中的驅動力呈現enthalpy-driven以及 entropy-cost 的過程，這類型的熱力學的改變推測與質子轉移現象及疏水性作用力有關。 針對抑制常數為pico的抑制物，由於等溫滴定為卡計的偵測極限為micro- to nano-molar.，因此，我們設計了競爭型滴定的方式進行實驗，先以低親和性分子結合酵素後，再加入高親合的抑制物進行競爭，此種實驗方式成功的解決高親和性方的無法精準量測結合常數的問題。

Iminosugars are well known as glycosidase inhibitors because their ring nitrogen is protonated at physiological pH to mimic the positive-charge feature of the oxocarbonium-ion-like transition state. The related research has drawn increasing attention due to the current pharmaceutical interest. For instance, GlysetTM (also named as Miglitol TM), developed to target the intestinal disaccharidase, is the first iminosugar approved by FDA for the treatment of type II diabetes. We previously developed a series of potent alpha--L-fucosidase inhibitors that are fuconojirimycin (FNJ, fuco-type iminosugar) derivatives that have Ki values spanning the micro- to pico- molar. This thesis aims at examining the binding interactions of the aforementioned FNJs (compounds ZY1 ZY2 ZY3) with Thermotoga maritima alpha-fucosidase (TmF). We mainly relied on the studies of isothermal titration calorimetry (ITC) to measure the thermodynamic parameters, including KD, DH, DS and Dn. According to the preliminary result, the binding of these FNJs with TmF is an enthalpy-driven and entropy-cost process. The observed thermodynamic changes are likely correlated with the combined effect of proton transfer and hydrophobic interaction. Furthermore, we have tried several ways to precisely measure the KD values of pM inhibitors because generally ITC is only suitable for measuring the binding affinity in the range of micro- to nano-molar. The replacement method seems to provide a satisfying solution, i.e. the enzyme of interest is first titrated with a weak inhibitor that is then replaced by titration with a more potent inhibitor.