Utility of Muropeptide Ligase for Identification of Inhibitors of the Cell Wall Biosynthesis Enzyme MurF

Abstract

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-␥-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-␥-D-Glu-diaminopimelate substrate prepared in a novel way by using muropeptide ligase enzyme to add UDP-MurNAc to synthetic L-Ala-␥-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 M was identified.The steps involved in the synthesis of the bacterial cell wall have long been considered to be good targets for antibacterial agents, as evidenced by drugs such as ␤-lactams and vancomycin (18). The MurF enzyme catalyzes the last cytoplasmic step of bacterial cell wall synthesis, the ligation of D-Ala-D-Ala to UDP-MurNAc-tripeptide with the concomitant hydrolysis of ATP. In gram-negative bacteria, the tripeptide is L-Ala-␥-DGlu-meso-A 2 pm (where A 2 pm represents diaminopimelate); in gram-positive bacteria, L-Lys replaces meso-A 2 pm (23).MurF is an attractive antibacterial drug target for several reasons: (i) it carries out an essential step of cell wall biosynthesis as demonstrated by the study of a temperature-sensitive lethal mutation in this gene in Escherichia coli (12); (ii) it is a single-copy gene in both gram-positive and gram-negative bacteria with extensive amino acid sequence conservation, raising the possibility of broad-spectrum inhibitors; and (iii) an earlier step in this pathway, MurA, is the target of the antibacterial drug fosfomycin (9), suggesting that interference with MurF function would likewise disrupt bacterial replication. In addition, normal MurF activity has been shown to be necessary for ␤-lactam resistance in methicillin-resistant Staphylococcus aureus (20).Despite these attractive features, MurF has not been used extensively as a target in high-throughput screening, possibly due to the difficulty in obtaining sufficient quantities of its substrate, UDP-MurNAc-tripeptide. Previous efforts to assay MurF that bypassed the need for substrate included the use of a coupled reaction containing the enzymes MurA, B, C, D, E, and F (8, 24) or permeabilized cells (2). A more direct approach would be an assay to detect compounds that bind to MurF. We have recently reported the use of capillary electrophoresis to identify compounds that bind to E. coli MurF.Similarly, Gu et al. (10) utilized an unspecified affinity selection screening technol...

Biochemical Characterization and Physiological Properties ofEscherichia coliUDP-N-Acetylmuramate:l-Alanyl-γ-d-Glutamyl-meso- Diaminopimelate Ligase

“…Changing A 2 pm to L-Lys did not eliminate the activity with tri-to pentapeptides but resulted in significant decrease in the catalytic efficiency ( Table 2). The ability of the Mpl enzyme to add Lys-containing tri-, tetra-, and pentapeptides to UDP-MurNAc has been reported previously and was used to generate the corresponding UDP-MurNAcpeptides (2).…”

Challenges of Antibacterial Discovery

“…Phosphinate transition-state analogs of the MurC to -F enzymes were synthesized by various groups in the 1990s, and some of these showed potent enzyme inhibition (at low nanomolar levels for some, illustrating druggability of the targets) but no antibacterial activity, presumably due to a lack of cell entry (127,254,310,362,403). A number of inhibitors of MurB, -C, -D, and -F, found via screens for enzyme inhibition or binding, had micromolar (in some cases, low micromolar) 50% inhibitory concentrations (IC 50 s) and no (or weak in one case [370]) antibacterial activity (11,30,103,141,364,370).…”

How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan)

“…Mpl was shown to utilize tripeptide, tetrapeptide, and pentapeptide with similar efficiencies (46). Mpl also accepts peptides containing lysine in place of Dap (5). During entry into stationary phase, the expression level of Mpl increases under the control of sigma S (114).…”