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-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.

FIG. 1.

Structures and molecular masses of the products of Mpl and MurF reactions using various substrates. The products of Mpl reactions using peptide substrates l-Ala-γ-d-Glu-A₂pm (A), l-Ala-γ-d-Glu-l-Lys (C), l-Ala-γ-d-Glu-l-Lys-d-Ala (D), and l-Ala-γ-d-Glu-l-Lys-d-Ala-d-Ala (E) are depicted. The products of MurF reactions using substrates UDP-MurNAc-l-Ala-γ-d-Glu-A₂pm (A) and UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys (C) are shown in B and E, respectively.

FIG. 2.

HPLC chromatograms of Mpl and MurF reactions. The HPLC profiles at 260 nM are displayed for the following reactions: l-Ala-γ-d-Glu-A₂pm without enzyme (A) or incubated with Mpl (B) or with Mpl and MurF (F), l-Ala-γ-d-Glu-l-Lys incubated with Mpl (C) or with Mpl and MurF (G), l-Ala-γ-d-Glu-l-Lys-d-Ala incubated with Mpl (D), and l-Ala-γ-d-Glu-l-Lys-d-Ala-d-Ala incubated with Mpl (E). Panel A is representative of the elution profile of all peptides in the absence of enzymes. Arrows indicate product peaks. For the MurF reactions (F and G), the peak corresponding to residual Mpl product is denoted by an asterisk. A standard curve of pure UDP-MurNAc (H) was analyzed by HPLC in parallel with samples A to G; the absorbance at 260 nm of the UDP-MurNAc peak detected at 4.2 min is shown.