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 (PD F) inhibitors under conditions as close as possible to the physiological si tuation. The assay principle is the detection of N-terminal [S-35]methionin e labeling of a protein that contains no internal methionine. If PDF is act ive, the deformylation of the methionine renders the peptide a substrate fo r methionine aminopeptidase, resulting in the removal of the N-terminal met hionine label. In the presence of a PDF inhibitor, the deformylation is blo cked so that the N-formylated peptide is not processed and the label is det ected, Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation sys tem as well as in intact bacterial cells.