MurF Inhibitors with Antibacterial Activity: Effect on Muropeptide Levels

Abstract

MurF catalyzes the last cytoplasmic step of bacterial cell wall synthesis and is essential for bacterial survival. Our previous studies used a pharmacophore model of a MurF inhibitor to identify additional inhibitors with improved properties. We now present the characterization of two such inhibitors, the diarylquinolines DQ1 and DQ2. DQ1 inhibited Escherichia coli MurF (50% inhibitory concentration, 24 M) and had modest activity Cell wall biosynthesis and the cell wall structure have long been considered useful targets for antibacterial agents, as demonstrated by antibiotics such as the ␤-lactams and glycopeptides (14, 29). However, of the series of steps catalyzed by the enzymes MurA through MurF that produce UDP-MurNAcpentapeptide, a useful antibiotic, fosfomycin, has been generated only against the MurA target (6, 18), despite extensive screening efforts against all of the enzymes in this pathway (for reviews, see references 8, 11, 14, 20, 29, and 31).MurF catalyzes the last cytoplasmic step of bacterial cell wall biosynthesis, generating UDP-MurNAc-pentapeptide from UDP-MurNAc-tripeptide and D-Ala-D-Ala (37). Previously identified inhibitors of MurF include a nonhydrolyzable ATP analog (1), phosphinate transition state analogs (25), sulfonamides (15, 22), thiazolylaminopyrimidines (4), and 8-hydroxyquinolines (5). These compounds inhibited the purified MurF enzyme but lacked antibacterial activity, presumably due to poor penetration into cells. A pharmacophore model based on the 8-hydroxyquinoline series was used to search for compounds with antibacterial activity, and this process identified several classes of compounds, including a 4-phenylpiperidine derivative (5). This inhibitor had the distinction of being the first inhibitor of the MurF enzyme which appeared to inhibit MurF within Escherichia coli cells.Observations of conditional lethal MurF mutants of E. coli (24) and Staphylococcus aureus (33, 34) are useful for predicting the effects of a MurF inhibitor on bacteria. In E. coli, a temperature-sensitive mutant with a mutated MurF enzyme that possessed Ͻ1% of the relative activity of the wild-type enzyme exhibited (i) cell lysis at the nonpermissive temperature, (ii) the accumulation of UDP-MurNAc-tripeptide (the MurF substrate), and (iii) a decrease in the level of the UDPMurNAc-pentapeptide product (24). In S. aureus, similar effects on the abundance of muropeptide were observed (33). These and other parameters were examined for two new MurF inhibitors, the diarylquinolines DQ1 and DQ2. MATERIALS AND METHODSMurF enzymatic assay. The 50% inhibitory concentrations (IC 50 s) for E. coli MurF were determined as described previously (5).Microbiology studies. All bacterial strains were from the strain collection ofMICs were determined by CLSI broth microdilution assays (9). The checkerboard methodology was used for determination of the MICs of compound DQ2 in combination with vancomycin (10). For growth curve generation, CFU quantitation, and muropeptide analysis, 125-ml cultures of E. coli OC2530 or S. ...

The biology of Mur ligases as an antibacterial target

“…has identified three inhibitors with antibacterial activity: diaryquinolines 1 and 2 (Baum et al ., ), and phenylpiperidine (Baum et al ., ). These compounds have been shown to inhibit E. coli MurF and to consequently decrease the amount of the MurF product in vivo , thus inhibiting PG synthesis in vivo and causing bacterial cell lysis (Baum et al ., 2007; 2009). Recently, a new class of pyrazolopyrimidine inhibitors with sub‐nanomolar potency against E. coli and Pseudomonas aeruginosa MurC enzymes have been reported.…”

Challenges of Antibacterial Discovery

“…The best pieces of evidence for MurB to -F inhibitors having any target-related activity in whole cells are the MurF inhibitors of Baum et al (31,32). The 4-PP compound (32) and the diarylquinoline DQ1 (Fig.…”

“…The 4-PP compound (32) and the diarylquinoline DQ1 (Fig. 2F) (31) were shown to moderately inhibit E. coli MurF (IC 50 , 24 to 29 M) and to cause an intracellular build-up of the pool of UDP-muramyl-L-Ala-DGlu-m-DAP, the MurF substrate, and a decrease in the UDPmuramyl-pentapeptide product, as would be expected for inhibitors of MurF. Treatment with DQ1 eventually led to cell lysis.…”

Toward a structome of Acinetobacter baumannii drug targets

“…One exception, there is a diarylquinoline compound which hinders cell wall synthesis due to MurF inhibition in gram‐positive bacteria like S . aureus …”