合成以螢光共振能量轉移為基礎之Lipid II 類似物以發展細菌肽聚醣轉醣酶的活性分析

Abstract

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.