臺灣博碩士論文加值系統

抗生素的抗藥性問題已經是個急迫的問題，進而促使科學家去研究以及發展新的抗生素。胜聚醣是細菌細胞壁主要的組成成分，它經由 lipid II 這個分子的轉醣以及轉胜肽作用聚合而成，形成網狀的聚合體，藉由細胞壁的堅韌與剛性，來維持細菌的形狀，以及正常的滲透壓。由於胜聚醣只存在細菌體而不存在於哺乳細胞內，因此它便成為了發展新抗生素的標的之一，也因為如此，參與細菌胜聚醣合成之酵素即被認為是可發展潛力新藥的目標。在參與胜聚醣合成的眾多酵素中，轉醣酶負責lipid II醣鏈結的聚合，為胜聚醣合成的一個重要酵素，而且轉醣酶坐落在細菌細胞壁外圍，容易與小分子接觸，是個好的抗菌劑研究方向。在過去三十年間，只有少數的化學分子已被研究確定是轉醣酶的抑制劑，因此，如果可以發展一個有效率的分析方法，再利用這個方法來研究轉醣酶活性，並進一步發展為高速篩選系統篩選出新的抑制劑，有可能加速新抗生素的發現。在本篇論文中，我們以螢光共振能量轉移的理論為基礎，合成了 lipid II分子的類似物，其在脂質部分帶有螢光的消光團，在聚胜肽部分帶有一個香豆素的螢光團﹔這樣的分子在轉醣過程中，帶有消光團的脂質部分會脫去，而香豆素的螢光可以被偵測到，以這個系統為基礎，我們可以偵測並比較含有不同脂質長度和構型的lipid II分子對轉醣酶的活性與專一性。此外，我們也利用這個系統來訂出富樂黴素對不同細菌轉醣酶的抑制常數，以及在我們之前研究中所發現到的小分子抑制劑之抑制常數。最後，我們把這個系統應用至中研院基因體中心的超高速篩選系統，在篩選了十二萬個小分子資料庫後得到數個具有抑制細菌轉醣酶潛力的小分子，這個篩選方法除了篩選出已知能抑制轉醣作用的抗生素，如萬古黴素以及teicoplanin，也發現了兩個新穎的小分子，這兩個小分子可以做為發展成為新一代抗生素的先驅結構。這個螢光分析平台提供了一個有效率和方便的方法去研究轉醣酶的活性，除此之外也能用於高速篩選以加速發展新抗生素上。

The emergence of antibiotic resistance has prompted scientists to search for new antibiotics. Peptidoglycan (PG), a major component of bacterial cell wall, is formed by transglycosylation and transpeptidation of lipid II substrate to a mesh-like polymer, which provides strength and rigidity to maintain the shape against variable internal osmotic shock. PG is an attractive target since no equivalent exists in the mammalian cells. Therefore, the enzymes participating in bacterial cell-wall biosynthesis is considered as potential drug targets. Transglycosylases (TGases) are attractive targets due to their location on the outer membrane and essential function in peptidoglycan synthesis. Since there are few molecules identified as TGase inhibitors in the past thirty years, an efficient assay for the TGase activity in a high-throughput mode is needed for discovery of new TGase inhibitors. In this thesis, I report a new continuous TGase activity assay based on Froster resonance energy transfer (FRET), using lipid II analogues with a dimethylamino-azobenzenesulfonyl (dabsyl) quencher on the lipid chain and coumarin fluorophore on the peptide chain. During the process of transglycosylation, the dabsyl-appended polyprenol is released and the fluorescence of coumarin can be detected. Using this system, the substrate specificity and affinity of lipid II analogues that contain different numbers and configurations of isoprene units were determined. Moreover, the inhibition constants (Ki) of moenomycin and two previously identified small molecules were also determined. In addition, the FRET system was also applied to identify potent TGase inhibitors by high-throughput screening. We adjusted the assay to a 1536-well plate format and screened 120,000 compounds of the small library collection at the Genomics Research Center, Academia Sinica. Several hits targeting TGase were validated and followed by confirmation based on HPLC assay and Ki determination. This new continuous fluorescent assay not only provides an efficient and convenient way to study TGases activities but also enables high-throughput screening of potential TGase inhibitors for antibiotics discovery.

Chapter 1. Introduction 1
1.1 Bacterial cell-wall biosynthesis 3
1.2 Bacterial transglycosylase as a potential drug target 5
1.3 Current assays for TGase activity determination 8
Chapter 2. Results 12
2.1 Design and retrosynthetic analysis for FRET-based Lipid II analogues (FBLAs) 12
2.2 Preparation of FBLAs 7a–7d 15
2.3 Purification and characterization of Lipid II-based FRET analogues (FBLAs) 24
2.4 FBLAs as bacterial transglycosylase substrates 26
2.5 Substrate specificity of FBLAs towards transglycosylases from different bacteria 29
2.6 Determination of kinetics parameters of FBLAs 32
2.7 Determination of inhibition constant (Ki) of TGase inhibitors 34
2.8 Ultra-high throughput screening (uHTS) for transglycosylase inhibitors 42
2.9 Synthesis of Improved efficacy of FBLA efficacy by changing fluorophore-quencher pair 45
Chapter 3. Discussion and Conclusion 49
3.1 Discussion 49
3.2 Conclusion 54
Chapter 4. Experimental section 56
4.1 Materials 56
4.2 General procedures. 56
4.3 Cloning, expression, and purification of full-length bifunctional penicillin-binding proteins from various bacterial species. 57
4.4 Transglycosylase activity analysis by high performance liquid chromatography 58
4.5 Kinetics analysis of FRET-based lipid II analogues 59
4.6 Determination of Ki values of transglycosylase inhibitors 60
4.7 High-throughput screening of transglycosylase inhibitors 60
4.8 Synthetic procedures and compound characterizations 61
References 82
Appendix 88

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