Antibacterial and antibiofilm activities of novel bacterial topoisomerase inhibitors against Staphylococcus aureus and other clinically relevant pathogens.

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.