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  • 學位論文

細菌轉醣酶之研究:受質與抑制劑的設計、合成與活性評估

Bacterial Transglycosylase Studies : Design, Synthesis and Bio-evaluation of Substrates and Inhibitors

指導教授 : 翁啟惠
共同指導教授 : 鄭偉杰
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摘要


由於日益嚴重的細菌抗藥性問題,設計與合成出有效的抗生素是刻不容緩的。細菌的細胞壁轉醣酶 (TGase) 因為其不存在於哺乳類生物、容易進入與未發現任何的抗藥性等特性,被認為是一個有潛力的藥物靶標。然而,此轉醣酶的受質 (Lipid II) 因為自然存在量低與複雜的化學結構,使得製備上並不容易。經過我們的努力,我們建立了酵素合成法與化學合成法來製備天然的Lipid II與其衍生物,同時也發展出新的化學合成路徑。不同於之前的合成方法,這個新的合成策略不只可用來製備Lipid II,也可廣泛應用於Lipid IV 的製備,甚至是Lipid VI等。有了合成Lipid II的能力之後,我們開始一系列的研究與探討,從結構活性分析、篩選平台建立、酵素活性分析,進而到抑制劑的設計和評估。 首先,我們著重於受質的結構活性探討,並利用化學合成法合成出受質與一系列在胜肽結構上的衍生物。經過結合力與活性的測試之後,我們發現在六個胺基酸中(D-Lac-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala),最前端的二個胺基酸 (D-Lac-L-Ala) 在受質的結合能力與活性上扮演著重要的角色,此外螢光團修飾在第四個胺基酸的側鏈(ε-NH2 at L-Lys)不會影響受質活性。 有鑒於此,天然的受質 (Lipid II) 經過化學法衍生出在胺基酸側鏈修飾的螢光探針。螢光探針不僅可減少受質與蛋白質在分析上的使用量,並且可以提高偵測的靈敏度。此螢光修飾的受質已用於TGase檢測平台的建立來進行酵素活性的分析與量測;同時也用於抑制劑的篩選與評估。 在抑制劑的設計上,我們應用了組合式的合成方法,合成出以iminocyclitol為主架構的化合物資料庫,並成功地篩選出第一個以iminocyclitol為主的抑制劑 (半抑制濃度為 52 μM)。同時也運用了之前在受質的結構活性上的資訊,設計與合成出以受質為架構的抑制物 (半抑制濃度為 5 μM)。

並列摘要


Due to the increasing drug resistance, design and synthesis of potent antibiotics is urgent and critical to scientists. Bacterial TGase is considered as a potential target because of the lack of a eukaryotic counterpart, easy access and no resistance to current drugs. In order to develop new antibiotics against TGase, the detailed study in protein-substrate interaction and the establishment of TGase assay are required. However, preparation of the key component, TGase substrate-Lipid II, is difficult due to the low natural abundance and complex structure. Through our efforts, we established enzymatic and chemical synthetic approaches to prepare natural Lipid II and derived analogues. Also, we developed a new chemical synthetic route and successfully applied to Lipid II and Lipid IV preparation. With Lipid II and its derivatives in hand, a systematic investigation from structural study, assay development, protein characterization, inhibitor design and evaluation was initiated. First, we focused on the structural activity study of substrate, Lipid II, and prepared several Lipid II analogues to examine the role of the peptide moiety with regard to their contribution to substrate activity. As a result, we have discovered that the first two amino acids (D-Lac-L-Ala) within the peptide chain of Lipid II is essential for substrate binding-affinity and activity in our SPR analysis and kinetic studies. Besides, we found the fluorophore modified at the ε-NH2 of lysine side chain does not affect the substrate affinity. Based on this result, we synthesized the fluorescent Lipid II with a NBD-tag at the lysine side chain as our TGase probe. The fluorescent probe not only reduces the usage amount of substrate and protein, but enhances the sensitivity of assay system. By using this NBD-Lipid II, the TGase activity assay has been established to characterize protein behaviors and to evaluate inhibitory activity against the TGase enzyme. From the view of inhibitor design, we developed a combinatorial approach to prepare iminocyclitol-based libraries and screened out the first iminicyclitol-based inhibitor with IC50 value of 52 μM, which potentially has a similar binding feature with Moenomycin A in TGase after computer modeling study. We also designed and synthesized some substrate-based inhibitors guided from the structural activity study of Lipid II. The most potent substrate-based inhibitor, GalNAc-Lipid II, has a IC50 value of 5 μM and was delivered to crystallization with TGase for binding-site and binding-mode study

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