Thiosemicarbazide Derivatives Decrease the ATPase Activity of Topoisomerase IV, Inhibit Mycobacterial Growth, and Affect Replication in .

Compounds targeting bacterial topoisomerases are of interest for the development of antibacterial agents. Our previous studies culminated in the synthesis and characterization of small-molecular weight thiosemicarbazides as the initial prototypes of a novel class of gyrase and topoisomerase IV inhibitors. To expand these findings with further details on the mode of action of the most potent compounds, enzymatic studies combined with a molecular docking approach were carried out, the results of which are presented herein. The biochemical assay for 1-(indol-2-oyl)-4-(4-nitrophenyl) thiosemicarbazide () and 4-benzoyl-1-(indol-2-oyl) thiosemicarbazide (), showing strong inhibitory activity against topoisomerase IV, confirmed that these compounds reduce the ability of the ParE subunit to hydrolyze ATP rather than act by stabilizing the cleavage complex. Compound showed better antibacterial activity than compound against clinical strains of and representatives of the genus. In vivo studies using time-lapse microfluidic microscopy, which allowed for the monitoring of fluorescently labelled replisomes, revealed that compound caused an extension of the replication process duration in , as well as the growth arrest of bacterial cells. Despite some similarities to the mechanism of action of novobiocin, these compounds show additional, unique properties, and can thus be considered a novel group of inhibitors of the ATPase activity of bacterial type IIA topoisomerases.