臺灣博碩士論文加值系統

抗生素抗藥性的議題已躍為全世界的醫療危機，在缺乏有效治療的情況下，研發新型抗生素以對付抗藥性細菌的感染，無庸置疑是人類亟需努力的方向。在針對細菌轉醣酶(bacterial transglycosylase)為目標發展新型抗生素的研究中，我們建立一個以親和性為基礎的篩選平台，利用固定在微珠上的盤尼西林結合蛋白(penicillin-binding protein)，從天然萃取物中分離純化出細菌轉醣酶抑制分子。首先，我們結合質譜技術並憑藉兩種已知的細菌轉醣酶抑制分子，默諾黴素(moenomycin A)與水楊酸苯胺類似物(salicylanilide analogues)，證實此平台的應用與可行性。我們發現只有具備抑制細菌轉醣酶功能的水楊酸苯胺類似物才能夠被平台篩選出來，顯示此平台具有從混合物中區分出有效分子的能力。由於實驗平台無需進行複雜且冗長的小分子標記與細胞壁基質(Lipid II)合成，更利於快速且高效率地篩選細菌轉醣酶抑制分子。接著，我們藉由此平台從帶有抑菌效果的天然萃取物中得到四個細菌轉醣酶抑制分子(TAN1532B、bequinostatin E、benastatin B、albofungin)。而後續研究中發現，這些天然物分子不僅能夠抑制數種細菌轉醣酶的活性，更具有廣泛且良好的抑菌效果。其中TAN1532B與benastatin B能夠有效抑制絕大部分革蘭氏陽性細菌的生長，尤其是可以成功對付抗藥性葡萄球菌與抗藥性腸球菌。而albofungin除了對於革蘭氏陽性細菌有顯著效果外，更能對抗大部分的革蘭氏陰性菌，其中包括鮑氏不動桿菌(Acinetobacter baumannii)、大腸桿菌(Escherichia coli)、綠膿桿菌(Pseudomonas aeruginosa)與克雷伯氏肺炎菌(Klebsiella pneumonia)。總結上述研究，我們成功地利用以親和性為基礎的篩選平台，從天然萃取物中分離出四種新的細菌轉醣酶抑制分子，不僅建立起我們研究細菌轉醣酶抑制分子的信心，未來更可依照這些天然物分子的結構為基石，做為設計與研發新型抗生素的藍圖。

The issue of antibiotic resistance has provoked a mounting crisis across the globe. With a lack of new therapeutic agents, there is an undoubted need for human to identify new antibiotics to combat resistant bacterial infections. As part of our efforts to develop new antibiotics to target the uncharted surface bacterial transglycosylase (TGase), we report an affinity-based ligand screening platform equipped with functional penicillin-binding proteins (PBPs) immobilized beads to selectively isolate TGase binders from complex natural products. In combination with mass spectrometry, the feasibility and applicability of this platform was demonstrated with moenomycin A (Moe A) and salicylanilide analogues (1−10) serving as reference ligands. Only the group of salicylanilide analogues bearing TGase inhibitory activity was selected, indicating that the potency of an analyte mixture could be clearly discriminated. This platform is advantageous since neither labeling of compounds nor tedious synthesis of lipid II analogues is required, promoting a more rapid and efficient screening approach for new TGase inhibitors. Using this platform, we isolated four potent antibacterials confirmed to be benastatin derivatives (11−13) and albofungin (14) from crude nature product extracts. The enzymatic studies showed that 11−14 were able to inhibit in vitro polymerization of substrate lipid II analogues catalyzed by several different bacterial TGases. Our findings revealed that 11 and 13 were potent inhibitors against a broad-spectrum of Gram-positive bacteria including drug-resistant strains, especially enterococci and staphylococci. 14 was active against both Gram-positive and Gram-negative bacteria including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumonia. Collectively, the successful identification of 11−14 to inhibit bacterial growth and TGase activity not only builds the confidence to search for new TGase inhibitors, but lays the foundation for the development of new scaffolds for TGase-targeted small molecules.

CONTENTS I
LIST OF TABLES III
LIST OF FIGURES V
LIST OF APPENDIXES VII
LIST OF ABBREVIATIONS IX
中文摘要 X
ABSTRACT XI
INTRODUCTION 1
Growing Emergence of Antibiotic Resistance 1
Bacterial Cell Wall Transglycosylase as an Attractive Drug Target 1
Current Methods for Drug Discovery from Natural Products 2
OBJECTIVE 3
RESULTS AND DISCUSSION 4
Immobilization of Functional Penicillin-Binding Proteins on Nickel Chelation Beads 4
Activity Determination of Immobilized PBPs 4
Stability Characterization of Immobilized PBPs 5
Optimization of Operative Conditions for Affinity-Based Ligand Screening 5
Demonstration with Moenomycin A 6
Demonstration with Salicylanilide Analogues 7
Binding Capacity of TGases-Immobilized Beads 7
Enrichment of TGase Inhibitors by TGases-Immobilized Beads 8
Identification of Benastatin Derivatives (11−13) and Albofungin (14) as TGase Inhibitors 9
Assessment of Inhibitory Activity toward Purified PBPs 10
Assessment of Antibacterial Activity against Various Bacteria 11
CONCLUSION 13
MATERIAL AND METHODS 15
Materials 15
General Procedures 15
Plasmid Construction 16
Cloning, Expression, and Immobilization of Full-Length or Truncated Penicillin-Binding Proteins 16
Quantification of Immobilized Penicillin-Binding Proteins 17
Determination of Minimum Inhibitory Concentration 17
Bacterial Transglycosylase Activity Assay 18
Detection of Ligands by Mass Spectrometry 19
Quantification of Ligand Recovery by Antibacterial Activity Determination 19
Analysis of Intracellular Accumulation of Cell Wall Precursor 20
Affinity-Based Ligand Screening 20
Isolation and Purification of Benastatin Derivatives (11−13) and Albofungin (14) 21
Time-Dependent Killing Assay 22
REFERENCE 23
TABLES 26
FIGURES 51
APPENDIXES 76

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