N4-Substituted Piperazinyl Norfloxacin Derivatives with Broad-Spectrum Activity and Multiple Mechanisms on Gyrase, Topoisomerase IV, and Bacterial Cell Wall Synthesis

Abstract

Fluoroquinolones are an important class of antibiotics with broad-spectrum antibacterial and antitubercular activity. Here, we describe the design and synthesis of a series of 38 N4-substituted piperazinyl norfloxacin derivatives. Their activity and mechanism of action were characterized using in silico, in vitro, and in vivo approaches. Several compounds displayed interesting activities against both Gramnegative and Gram-positive bacteria, and few displayed antimycobacterial activity, whereby some were as potent as norfloxacin and ciprofloxacin. Molecular docking experiments suggested that the new derivatives inhibit both DNA gyrase and DNA topoisomerase IV in a similar manner as norfloxacin. Selecting the most promising candidates for experimental mode of action analysis, we confirmed DNA gyrase and topoisomerase IV as targets of all tested compounds using enzymatic in vitro assays. Phenotypic analysis of both Escherichia coli and Bacillus subtilis confirmed a typical gyrase inhibition phenotype for all of the tested compounds. Assessment of possible additional targets revealed three compounds with unique effects on the B. subtilis cell wall synthesis machinery, suggesting that they may have an additional target in this pathway. Comparison with known cell wall synthesis inhibitors showed that the new compounds elicit a distinct and, so far, unique phenotype, suggesting that they act differently from known cell wall synthesis inhibitors. Interestingly, our phenotypic analysis revealed that both norfloxacin and ciprofloxacin displayed additional cellular effects as well, which may be indicative of the so far unknown additional mechanisms of fluoroquinolones.

Dissecting antibiotic effects on the cell envelope using bacterial cytological profiling: a phenotypic analysis starter kit

“…Stalled MreB movement was also observed with tunicamycin and D-cycloserine (Fig. S14) as well as with all previously tested cell wall synthesis inhibitors ( 61 – 63 ). Membrane-active compounds may detach MreB from the cell membrane (see gramicidin and CCCP; Fig.…”

“…However, MreB moves in a spiraling movement along the long axis of the cell, driving forward cell wall synthesis and ensuring rod shape ( 57 – 59 ). This movement is dependent on lipid-linked cell wall precursors, and all cell wall synthesis inhibitors tested so far have stalled MreB movement, while even aggressive membrane-active compounds did not completely abolish MreB mobility ( 60 – 63 ). Thus, MreB mobility can be used as a specific and robust reporter for cell wall synthesis activity.…”

“…Membrane-active compounds may detach MreB from the cell membrane (see gramicidin and CCCP; Fig. S14) or induce distinct, static clusters, yet as long as non-clustered MreB remains at the cell membrane, it retains its mobility ( 61 – 63 ). None of the other test compounds affected MreB mobility either (Fig.…”

“…The promoter may also react to indirect inhibition of the lipid II cycle, e.g., by clustering of RIFs or dissociation of MurG ( 24 , 34 ). Therefore, it can be a valuable complementation of the MreB mobility assay, which reacts to all types of direct cell wall synthesis inhibition but not to membrane-mediated, indirect effects on this pathway ( 61 – 63 ). Together, these two assays allow reliable identification of cell wall synthesis inhibitors as well as narrowing down of the target to the membrane-bound lipid II cycle as opposed to intracellular and extracellular steps of cell wall synthesis.…”

Reduced Graphene Oxide–SnSe Nanocomposite Photocatalyst with High Apparent Quantum Yield for the Photodegradation of Norfloxacin

“…As a member of the fluoroquinolone antibiotic class, norfloxacin has been used and abused in recent decades due to its wide-range antibacterial activity. , Antibiotic-resistant microbial infections may arise due to the widespread and increasing concentration of norfloxacin in aquatic environments, rendering current antibiotic treatments ineffective. However, because of its weak metabolism, molecules of norfloxacin gather extensively in the water bodies, endangering the balance of the ecosystem and having major impacts on public health.…”