Ignatius J. Turchi

Identification and Characterization of New Inhibitors of the Escherichia coli MurA Enzyme

ABSTRACT The bacterial enzyme MurA catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (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 (IC50s) were 0.2 to 0.9 μM, compared to 8.8 μM for the known MurA inhibitor, fosfomycin. The three compounds exhibited MICs of 4 to 32 μg/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 was not due specifically to MurA inhibition. The presence of UNAG during the MurA and inhibitor preincubation lowered the IC50 at least fivefold, suggesting that, like fosfomycin, the three compounds may interact with the enzyme 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 fosfomycin. Taken together, the evidence indicates that these inhibitors may bind noncovalently to the MurA enzyme, at or near the site where fosfomycin binds.

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 μg/ml against permeable E. coli) including cell lysis and a 5-log10-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.

On the mechanism of succinyl transfer from aryl enolsuccinates to enolates of arylketones: addition–elimination vs. alkoxide-assisted retro-ene reaction

The mechanism of succinyl transfer from aryl enolsuccinates to the enolates of aryl ketones has been studied by deuterium exchange experiments and semi-empirical calculations. The calculations indicate that direct elimination is favoured over the ene mechanism. The results of a deuterium labelling study were also inconsistent with the ene mechanism.

Ground states of molecules. Part 35. MINDO/3 study of the Cornforth rearrangement

MINDO/3 calculations are reported for the Cornforth rearrangement of 5-methoxyoxazole-4-carboxamide and of the 2-vinyl derivative to the corresponding methyl 5-amino-oxazole-4-carboxylates. The reaction involves an unusual intermediate for which no satisfactory classical structure can be written. The results account well for the experimental evidence concerning such reactions.

Ground states of molecules. Part XXXII. A MINDO/3 study of mesoionic oxazoles and imidazoles

MINDO/3 Calculations are reported for a number of mesoionic species derived from oxazole and imidazole and for isomeric ‘classical’ species. The mesoionic compounds are predicted to be highly polar and much less stable than their classical counterparts. The calculated geometries of the latter correspond to the expected classical structures. Those of the mesoionic compounds on the other hand mostly differ from the structures expected in terms of current treatments of resonance theory.