The discovery and optimization of a new class of bacterial topoisomerase (DNA gyrase and topoisomerase IV) inhibitors binding in the ATP domain are described. A fragment molecule, 1-ethyl-3-(2-pyridyl)urea, provided sufficiently potent enzyme inhibition (32 μM) to prompt further analogue work. Acids and acid isosteres were incorporated at the 5-pyridyl position of this fragment, bridging to a key asparagine residue, improving enzyme inhibition, and leading to measurable antibacterial activity. A CF3-thiazole substituent at the 4-pyridyl position improved inhibitory potency due to a favorable lipophilic interaction. Promising antibacterial activity was seen versus the Gram-positive pathogens Staphylococcus aureus and Streptococcus pneumoniae and the Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis . Precursor metabolite incorporation and mutant analysis studies support the mode-of-action, blockage of DNA synthesis by dual target topoisomerase inhibition. Compound 35 was efficacious in a mouse S. aureus disease model, where a 4.5-log reduction in colony forming units versus control was demonstrated.

Background: Inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and TopoIV by the antibacterial spiropyrimidinetrione AZD0914 was investigated. Results: AZD0914 stabilized the gyrase-DNA complex with double strand DNA cleavage, retaining potency in a fluoroquinolone-resistant mutant, with little inhibition of human type II topoisomerases. Conclusion: AZD0914 displays mechanistic differences from fluoroquinolones. Significance: AZD0914 has the potential to combat drug-resistant gonorrhea.

Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D 1H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo-β-lactamase subclass 1 (NDM-1), an emerging antibiotic-resistance threat. Cell-based NMR studies demonstrated that two known NDM-1 inhibitors, L-captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem in vivo. NDM-1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM-1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole-cell settings, including mammalian cells.

The intrinsic resistance of P. aeruginosa PAO1 to the peptide deformylase inhibitor (PDF-I) LBM415 was mediated by the MexAB-OprM and MexXY-OprM efflux pumps, the latter of which was strongly induced by LBM415. Single-step exposure of PAO1 deleted for mexAB-oprM (therefore lacking both MexAB-OprM and MexXY-OprM functions) to PDF-Is selected for nfxB mutants, which express the MexCD-OprJ efflux pump, indicating that these compounds are also substrates for this pump. Selection of resistant mutants by use of levels of LBM415 greater than that accommodated by efflux yielded two additional groups of mutations, in the methionyl-tRNA fmet formyltransferase (fmt) and folD genes. Both mechanisms are known to impose an in vitro growth deficit (also observed here), presumably due to impairment of protein synthesis. We surmised that this inherent impairment of protein synthesis would upregulate expression of mexXY in a fashion similar to upregulation by LBM415 or by ribosome inhibitory compounds. Transcriptional profiling and/or mexX::lux promoter fusion analysis revealed that fmt and folD mutants were strongly upregulated for mexXY and another gene known to be required for upregulation of the pump, PA5471. Complementation of the fmt mutation in trans reversed this constitutive expression. This supports the notion that MexXY has a natural physiological function responding to impairment of ribosome function or protein synthesis and that fmt mutation (Fmt bypass) and folD mutation generate the intracellular mexXY-inducing signal.

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