The discovery of multi-targeting ligands of bacterial enzymes is an important strategy to combat rapidly spreading antimicrobial resistance. Bacterial DNA gyrase and topoisomerase IV are validated targets for the development of antibiotics. They can be inhibited at their catalytic sites or at their ATP binding sites. Here we present the design of new hybrids between the catalytic inhibitor ciprofloxacin and ATP-competitive inhibitors that show low nanomolar inhibition of DNA gyrase and antibacterial activity against Gram-negative pathogens. The most potent hybrid 3a has MICs of 0.5 µg/mL against Klebsiella pneumoniae, 4 µg/mL against Enterobacter cloacae, and 2 µg/mL against Escherichia coli. In addition, inhibition of mutant E. coli strains shows that these hybrid inhibitors interact with both subunits of DNA gyrase (GyrA, GyrB), and that binding to both of these sites contributes to their antibacterial activity.

DNA gyrase is an important target for the development of novel antibiotics. Although ATP-competitive DNA gyrase (GyrB) inhibitors are a well-studied class of antibacterial agents, there is currently no representative used in therapy, largely due to unwanted off-target activities. Selectivity of GyrB inhibitors against closely related human ATP-binding enzymes should be evaluated early in development to avoid off-target binding to homologous binding domains. To address this challenge, we developed selective 3D-pharmacophore models for GyrB, human topoisomerase IIα (TopoII), and the Hsp90 N-terminal domain (NTD) to be used in in silico activity profiling paradigms to identify molecules selective for GyrB over TopoII and Hsp90, as starting points for hit expansion and lead optimization. The models were used to profile highly active GyrB, TopoII, and Hsp90 inhibitors. Selected compounds were tested in in vitro assays. GyrB inhibitors 1 and 2 were inactive against TopoII and Hsp90, while 3 and 4, potent Hsp90 inhibitors, displayed no inhibition of GyrB and TopoII, and TopoII inhibitors 5 and 6 were inactive at GyrB and Hsp90. The results provide a proof of concept for the use of target activity profiling methods to identify selective starting points for hit and lead identification.

New N-phenylpyrrolamide inhibitors of DNA gyrase were developed with excellent on-target activity and improved antibacterial activity.