Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells.
An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.
Pharma Research Basel, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland. christian.apfel@roche.com
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D000581:Amidohydrolases; D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000187:drug effects; D002457:Cell Extracts / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology; D008715:Methionine / Q000378:metabolism; D008826:Microbial Sensitivity Tests / Q000379:methods*; D014176:Protein Biosynthesis / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002457:Cell Extracts; D004791:Enzyme Inhibitors; D008715:Methionine; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase