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

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 Gram-negative 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.