Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF.
MurF is a key enzyme in the biosynthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. This enzyme has not been extensively exploited as a drug target, possibly due to the difficulty in obtaining one of the substrates, UDP-MurNAc-L-Ala-gamma-D-Glu-meso-diaminopimelate, which is usually purified from bacteria. We have identified putative inhibitors of Escherichia coli MurF by a binding assay, thus bypassing the need for substrate. Inhibition of enzymatic activity was demonstrated in a high-performance liquid chromatography-based secondary assay with UDP-MurNAc-L-Ala-gamma-D-Glu-diaminopimelate substrate prepared in a novel way by using muropeptide ligase enzyme to add UDP-MurNAc to synthetic L-Ala-gamma-D-Glu-diaminopimelate; the substrate specificity of muropeptide ligase for peptides containing L-Lys in place of diaminopimelate was also investigated. Using the muropeptide ligase-generated MurF substrate, a thiazolylaminopyrimidine series of MurF enzyme inhibitors with 50% inhibitory concentration values as low as 2.5 microM was identified.
Baum
Ellen Z
EZ
Johnson & Johnson Pharmaceutical Research & Development, LLC, 1000 Route 202, Raritan, New Jersey 08869, USA. ebaum@prdus.jnj.com
Crespo-Carbone
Steven M
SM
Abbanat
Darren
D
Foleno
Barbara
B
Maden
Amy
A
Goldschmidt
Raul
R
Bush
Karen
K
D002473:Cell Wall / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010457:Peptidoglycan / Q000096:biosynthesis*
D004791:Enzyme Inhibitors; D010457:Peptidoglycan; D010453:Peptide Synthases; C060628:UDP-N-acetylmuramoylalanyl-D-glutamyllysine-D-alanyl-D-alanine ligase; C049145:UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase