Tilmicosin derivatives as topoisomerase I/II inhibitors: Rational design, synthesis, and antibacterial evaluation

The antibacterial mechanisms of macrolides primarily focus on ribosome inhibition, while their potential interactions with topoisomerases (Topo) remain to be thoroughly explored. In this study, novel tilmicosin (TIM) derivatives were designed and screened with strong binding affinities to Topo I/II by using molecular docking. Then, four TIM derivatives (T-1, T-2, T-16, and T-17) were selected as potential candidates for subsequent synthesis. In vitro antibacterial activities were evaluated, among them, T-1 and T-16 exhibited superior antibacterial effects against most bacteria, with a particularly notable MIC of 1 μg/mL against E. coli ATCC 8739. The inhibitory activities of T-1 (IC₅₀ = 17 μM) and T-16 (IC₅₀ = 15 μM) against Topo II were significantly higher than that of TIM. The inhibitory effects of T-1/T-16 may stem from optimized binding to the residue Asp or Met via hydrogen bonding. Gel electrophoresis analysis demonstrated that T-1 and T-16 effectively induce unwinding of supercoiled pUC19 plasmid, further confirming their interference with bacterial Topo II activity, thereby leading to disruption of DNA metabolism. The antibacterial mechanisms were also investigated by molecular dynamics (MD), which forms a closed-loop verification with the above-described inhibitory effect against Topo II and unwinding ability towards pUC19 plasmid. HPLC combined with electrochemical analysis indicated that T-1 and T-16 possessed better stability in acidic aqueous solutions and a longer metabolic half-life in mice compared to TIM. To further evaluate the in vivo anti-infective efficacy, different mice infection models were established. The results suggested that T-1 and T-16 not only exhibit excellent in vivo antibacterial activity and biocompatibility but also effectively treat infections.