Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway.
The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes. Inhibition of ATP consumed during the MurC reaction, using an adaptation of a kinase assay format, identified a number of potential inhibitory chemotypes. After nonspecific inhibition testing and chemical attractiveness were assessed, C-1 emerged as a compound for further characterization. The inhibition of MurC by this compound was confirmed in both a kinetic-coupled enzyme assay and a direct nuclear magnetic resonance product detection assay. C-1 was found to be a low micromolar inhibitor of the E. coli MurC reaction, with preferential inhibition by one of two enantiomeric forms. Experiments indicated that it was a competitive inhibitor of ATP binding to the MurC enzyme. Further work with MurC enzymes from several bacterial sources revealed that while the compound was equally effective at inhibiting MurC from genera (Proteus mirabilis and Klebsiella pneumoniae) closely related to E. coli, MurC enzymes from more distant Gram-negative species such as Haemophilus influenzae, Acinetobacter baylyi, and Pseudomonas aeruginosa were not inhibited.
Zawadzke
Laura E
LE
HTS Center of Emphasis, Pfizer Global Research & Development, Groton, CT, USA. laura.e.zawadzke@pfizer.com
Norcia
Michael
M
Desbonnet
Charlene R
CR
Wang
Hong
H
Freeman-Cook
Kevin
K
Dougherty
Thomas J
TJ
D000900:Anti-Bacterial Agents; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D058619:Maltose-Binding Proteins; D008826:Microbial Sensitivity Tests; D010453:Peptide Synthases / Q000037:antagonists & inhibitors*; D010457:Peptidoglycan / Q000096:biosynthesis*; D011513:Proteus mirabilis / Q000187:drug effects / Q000201:enzymology; D018048:Receptors, Purinergic P2 / Q000187:drug effects; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D058619:Maltose-Binding Proteins; D010457:Peptidoglycan; D018048:Receptors, Purinergic P2; D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase