A MurF inhibitor that disrupts cell wall biosynthesis in Escherichia coli

Abstract

MurF is an essential enzyme of bacterial cell wall biosynthesis. Few MurF inhibitors have been reported, and none have displayed measurable antibacterial activity. Through the use of a MurF binding assay, a series of 8-hydroxyquinolines that bound to the Escherichia coli enzyme and inhibited its activity was identified. To derive additional chemotypes lacking 8-hydroxyquinoline, a known chelating moiety, a pharmacophore model was constructed from the series and used to select compounds for testing in the MurF binding and enzymatic inhibition assays. Whereas the original diverse library yielded 0.01% positive compounds in the binding assay, of which 6% inhibited MurF enzymatic activity, the pharmacophore-selected set yielded 14% positive compounds, of which 37% inhibited the enzyme, suggesting that the model enriched for compounds with affinity to MurF. A 4-phenylpiperidine (4-PP) derivative identified by this process displayed antibacterial activity (MIC of 8 microg/ml against permeable E. coli) including cell lysis and a 5-log(10)-unit decrease in CFU. Importantly, treatment of E. coli with 4-PP resulted in a 15-fold increase in the amount of the MurF UDP-MurNAc-tripeptide substrate, and a 50% reduction in the amount of the MurF UDP-MurNAc-pentapeptide product, consistent with inhibition of the MurF enzyme within bacterial cells. Thus, 4-PP is the first reported inhibitor of the MurF enzyme that may contribute to antibacterial activity by interfering with cell wall biosynthesis.

FIG. 1.

(A) Pharmacophore model of the 8-hydroxyquinoline series derived from the Common Features algorithm of the Catalyst program. Me, methyl. (B) Catalyst depiction of the pharmacophore model including directionality of the aromatic rings and H-bond donors. (C) Structure of the MurF inhibitor, 4-PP.

FIG. 2.

ThermoFluor traces of normalized fluorescence intensity as a function of temperature for MurF. The vertical dashed lines indicate the T_ms of MurF without ATP (triangles) and with 1 mM ATP (squares). The change in T_m is shown.

FIG. 3.

(A) Growth curves of E. coli treated with MurF inhibitor 4-PP or cycloserine. Strain OC2530 was grown to mid-log phase as described in Materials and Methods, and compounds were added to a final concentration of 2× MIC. Aliquots of cultures were transferred to the Bioscreen C plate, and culture growth was monitored by A₅₈₀. A representative well of 10 wells for each culture (control, squares; cycloserine, triangles; 4-PP, open circles) is shown. (B) Quantitation of CFU after treatment with MurF inhibitor 4-PP or cycloserine. Aliquots of cultures shown in panel A were removed from the Bioscreen C plate at the indicated times for determination of CFU. The averages ± standard deviations (error bars) for two independent experiments are shown.

FIG. 4.

Altered muropeptide profile in 4-PP-treated E. coli. Cells were treated with 4-PP or cycloserine (2× MIC for 30 min) as described in Materials and Methods, and muropeptides were extracted and quantitated. White bars, UDP-MurNAc-tripeptide; gray bars, UDP-MurNAc-pentapeptide. The results of one representative experiment of two independent experiments are shown. mAu, milli absorbance units.