Background: The novel bacterial topoisomerase inhibitors (NBTIs) developed in our laboratory show potent on-target enzyme inhibition but suffer from low activity against Gram-negative bacteria. Methods: With the aim of improving the antibacterial activity of our compounds against Gram-negative bacteria, we tested them in combination with different efflux pump inhibitors (EPIs), a strategy that showed promise in several other classes of antimicrobials. We also investigated the combined effect of NBTIs with ATP-competitive inhibitors of bacterial type II topoisomerases (ACIs), as well as the antibiofilm properties of our compounds and the combination with EPIs against early and mature Acietobacter baumannii biofilm. Results: Our results demonstrate that combinations of NBTIs with EPI Phenylalanine-arginyl-β-naphthylamide significantly reduce the corresponding NBTIs’ minimal inhibitory concentration values and show potentiation of A. baumannii biofilm inhibition as compared to NBTIs alone. Although combinations of NBITs and ACIs did not show synergistic effects, the FIC index value calculations revealed additive effects for all the combinations of a selected NBTI in combination with three ACIs in all the assayed Gram-negative bacteria from the ESKAPE group. Conclusions: These results show for the first time that combinations of NBTIs with either EPIs or a different class of the topoisomerase inhibitors may be a beneficial strategy to combat difficult-to-treat bacterial infections.

Antibiotic-resistant Staphylococcus aureus poses a major global health challenge as current therapies lose effectiveness. Because most S. aureus infections are biofilm-associated, there is a critical need for new agents capable of targeting biofilm-embedded cells, and not just their planktonic form. Novel bacterial type II topoisomerase inhibitors (NBTIs) are known for their excellent antibacterial properties, against planktonic bacteria, and could also possess anti-biofilm activity. To investigate this, we designed and synthesized a new series of NBTIs featuring alternative linker scaffolds and a modified DNA-intercalating moiety to improve antibacterial and antibiofilm activity. Comprehensive MIC profiling against clinically relevant Gram-negative and Gram-positive bacteria revealed clear structure-activity trends, with compounds 20 and 40 emerging as the most promising candidates. Compound 20 showed exceptional potency against S. aureus and multiple MRSA strains (MICs 0.004-0.008 μg/mL), while compound 40 displayed the most balanced broad-spectrum activity. Both compounds exhibited dual inhibition of DNA gyrase and topoisomerase IV, maintained high selectivity over human topoisomerase IIα. In vivo evaluation in zebrafish embryos demonstrated robust protection against lethal S. aureus infection within a well-tolerated concentration range. Kinetic studies demonstrated dose-dependent bactericidal activity and measurable post-antibiotic effects. Additionally, both compounds inhibited biofilm formation at 1/2 MIC and reduced metabolic activity within mature biofilms at 2 × MIC, outperforming gepotidacin, an FDA-approved NBTI, in several assays. Together, these findings identify new NBTI chemotypes with enhanced potency and promising antibiofilm activity, providing a strong foundation for future antibacterial development.