High-throughput screening of peptide deformylase inhibitors.
The emergence of bacterial pathogens resistant to current antibiotics has caused an urgent demand for new treatments. Peptide deformylase (PDF) has become an exciting target for designing novel antibiotics. To facilitate the screening of PDF inhibitors, three robust, coupled assays have been developed. The first method couples the PDF reaction with that of formate dehydrogenase. Formate dehydrogenase oxidizes formate into CO2 with a concomitant reduction of NAD+ to NADH, which can be monitored spectrophotometrically. The second method involves Aeromonas aminopeptidase (AAP) as the coupling enzyme and an artificial substrate, f-Met-Leu-p-nitroanilide. The sequential action of PDF and AAP releases p-nitroanilide as a highly chromogenic product. In the third method, f-Met-Lys-7-amino-4-methylcoumarin is used as the substrate. Deformylation by PDF gives an excellent substrate for dipeptidyl peptidase I, which releases the dipeptide Met-Lys and fluorogenic 7-amino-4-methylcoumarin. The combination of these assay methods should meet the needs of most laboratories.
Nguyen
Kiet T
KT
Wadsworth Center, New York State Department of Health, CMS, Albany, NY.
Pei
Dehua
D
D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D000626:Aminopeptidases / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000378:metabolism; D020789:Cathepsin C / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000378:metabolism; D006864:Hydrogenase / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; D000626:Aminopeptidases; C038222:bacterial leucyl aminopeptidase; D020789:Cathepsin C; D000581:Amidohydrolases; C084507:peptide deformylase