A cell-based assay that targets methionine aminopeptidase in a physiologically relevant environment.
Methionine aminopeptidase (MetAP) is a promising target for the development of novel antibiotics. However, many potent inhibitors of the purified enzyme failed to show significant antibacterial activity. It is uncertain which divalent metal MetAP uses as its native cofactor in bacterial cells. Herein, we describe a cell-based assay that monitors the hydrolysis of a fluorogenic substrate by overexpressed MetAP in permeabilized Escherichia coli cells and its validation with a set of MetAP inhibitors. This cell-based assay is applicable to those cellular targets with poorly defined native cofactor, increasing the chances of identifying inhibitors that can inhibit the cellular target.
Chai
Sergio C
SC
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Ye
Qi-Zhuang
QZ
D000626:Aminopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D063208:Methionyl Aminopeptidases; D015854:Up-Regulation
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases