ABSTRACT
The emergence of antibiotic resistance has prompted scientists to search for new antibiotics. Transglycosylase (TGase) is an attractive target for new antibiotic discovery due to its location on the outer membrane of bacteria and its essential role in peptidoglycan synthesis. Though there have been a few molecules identified as TGase inhibitors in the past thirty years, none of them have been developed into antibiotics for humans. The slow pace of development is perhaps due to the lack of continuous, quantitative, and high-throughput assay available for the enzyme. Herein, we report a new continuous fluorescent assay based on Förster resonance energy transfer, using lipid II analogues with a dimethylamino-azobenzenesulfonyl quencher in the lipid chain and a coumarin fluorophore in the peptide chain. During the process of transglycosylation, the quencher-appended polyprenol is released and the fluorescence of coumarin can be detected. Using this system, the substrate specificity and affinity of lipid II analogues bearing various numbers and configurations of isoprene units were investigated. Moreover, the inhibition constants of moenomycin and two previously identified small molecules were also determined. In addition, a high-throughput screening using the new assay was conducted to identify potent TGase inhibitors from a 120,000 compound library. This new continuous fluorescent assay not only provides an efficient and convenient way to study TGase activities, but also enables the high-throughput screening of potential TGase inhibitors for antibiotic discovery.
Subject(s)
Bacteria/enzymology , Fluorescence Resonance Energy Transfer/methods , Peptidoglycan Glycosyltransferase/metabolism , Coumarins/chemistry , Coumarins/metabolism , Enzyme Assays/methods , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Peptidoglycan Glycosyltransferase/antagonists & inhibitors , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Uridine Diphosphate N-Acetylmuramic Acid/chemistry , Uridine Diphosphate N-Acetylmuramic Acid/metabolismABSTRACT
Breaking down barriers: A rapid, inexpensive preparation of the structurally complex mycobacterial N-glycolyl Lipidâ I, Lipidâ II, and their analogues from a range of different synthetic N-glycolyl and N-glycinyl Park's nucleotides is described (see scheme). The biotransformations were catalyzed by a readily available biocatalyst obtained from a bacterial cell-free membrane fraction. The unnatural N-glycinyl Lipidâ II was found to be a substrate of Mycobacterium tuberculosis (Mtb) transglycosylase, PonA, and N-glycolyl Lipidâ I was a weak inhibitor against PonA.
