Design, Synthesis, Molecular Modeling, Biological Activity, and Mechanism of Action of Novel Amino Acid Derivatives of Norfloxacin

Abstract

Two series of N4-substituted piperazinyl amino acid derivatives of norfloxacin (24 new compounds) were designed and synthesized to attain structural surrogates with additional binding sites and enhanced antibacterial activity. Synthesized derivatives showed increased antibacterial and antimycobacterial activity compared to their lead structure, norfloxacin. Molecular modeling studies supported the notion that the derivatives can establish additional bonds with the target enzymes gyrase and topoisomerase IV. In vitro enzyme inhibition assays confirmed that the tested compounds were significant inhibitors of these enzymes. Inhibition of gyrase and topoisomerase IV was then confirmed in living bacterial cells using bacterial cytological profiling of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, revealing a typical topoisomerase inhibition phenotype characterized by severe nucleoid packing defects. Several derivatives exhibited additional effects on the Gram-positive cell wall synthesis machinery and/or the cytoplasmic membrane, which likely contributed to their increased antibacterial activity. While we could not identify specific cell wall or membrane targets, membrane depolarization was not observed. Our experiments further suggest that cell wall synthesis inhibition most likely occurs outside the membrane-bound lipid II cycle.

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.…”