Identification and characterization of new inhibitors of the Escherichia coli MurA enzyme

The bacterial enzyme MurA catalyzes the transfer of enolpyruvate from phosp hoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first committed step of bacterial cell wall biosynthesis. From high -throughput screening of a chemical library, three novel inhibitors of the Escherichia coli MurA enzyme were identified: the cyclic disulfide RWJ-3981 , the purine analog RWJ-140998, and the pyrazolopyrimidine RWJ-110192. When MurA was preincubated with inhibitor, followed by addition of UNAG and PEP , the 50% inhibitory concentrations (IC(50)s) were 0.2 to 0.9 muM, compared to 8.8 muM for the known MurA inhibitor, fosfomycin. The three compounds e xhibited MICs of 4 to 32 mug/ml against Staphylococcus aureus; however, the inhibition of DNA, RNA, and protein synthesis in addition to peptidoglycan synthesis by all three inhibitors indicated that antibacterial activity wa s not due specifically to MurA inhibition. The presence of UNAG during the MurA and inhibitor preincubation lowered the IC50 at least fivefold, sugges ting that, like fosfomycin, the three compounds may interact with the enzym e in a specific fashion that is enhanced by UNAG. Ultrafiltration and mass spectrometry experiments suggested that the compounds were tightly, but not covalently, associated with MurA. Molecular modeling studies demonstrated that the compounds could fit into the site occupied by fosfomycin; exposure of MurA to each compound reduced the labeling of MurA by tritiated fosfomy cin. Taken together, the evidence indicates that these inhibitors may bind noncovalently to the MurA enzyme, at or near the site where fosfomycin bind s.