Nitroimidazoles such as metronidazole are used as anti-infective drugs against anaerobic bacteria. Upon in vivo reduction of the nitro group, reactive radicals damage DNA and proteins in the absence of oxygen. Unexpectedly, a recent study of nitroimidazoles linked to an indolin-2-one substituent revealed potent activities against aerobic bacteria. This suggests a different, yet undiscovered mode of action (MoA). To decipher this MoA, we first performed whole proteome analysis of compound-treated cells, revealing an upregulation of bacteriophage-associated proteins, indicative of DNA damage. Since DNA binding of the compound was not observed, we applied activity-based protein profiling (ABPP) for direct target discovery. Labeling studies revealed topoisomerase IV, an essential enzyme for DNA replication, as the most enriched hit in pathogenic Staphylococcus aureus cells. Subsequent topoisomerase assays confirmed the inhibition of DNA decatenation in the presence of indolin-2-one nitroimidazole with an activity comparable to ciprofloxacin, a known inhibitor of this enzyme. Furthermore, we determined significantly increased redox potentials of indolin-2-one nitroimidazoles compared to classic 5-nitroimidazoles such as metronidazole, which facilitates in vivo reduction. Overall, this study unravels that indolin-2-one-functionalized nitroimidazoles feature an unexpected dual MoA: first, the direct inhibition of the topoisomerase IV and second the classic nitroimidazole MoA of reductive bioactivation leading to damaging reactive species. Importantly, this dual MoA impairs resistance development. Given the clinical application of this compound class, the new mechanism could be a starting point to mitigate resistance.

中文翻译：

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Indolin-2-one nitroimidazole 抗生素表现出意想不到的双重作用模式

硝基咪唑类如甲硝唑用作抗厌氧菌的抗感染药物。在体内硝基的还原，活性自由基在缺氧的情况下破坏DNA和蛋白质。出乎意料的是，最近对与二氢吲哚-2-一取代基连接的硝基咪唑的研究揭示了对需氧细菌的有效活性。这表明存在一种不同但尚未被发现的作用方式 (MoA)。为了破译这个 MoA，我们首先对化合物处理的细胞进行了全蛋白质组分析，揭示了噬菌体相关蛋白的上调，表明 DNA 损伤。由于未观察到化合物的 DNA 结合，我们应用基于活性的蛋白质分析 (ABPP) 进行直接靶标发现。标记研究表明，拓扑异构酶 IV 是 DNA 复制的必需酶，是致病性金黄色葡萄球菌中最富集的酶细胞。随后的拓扑异构酶测定证实了在 indolin-2-one 硝基咪唑存在下抑制 DNA 分解，其活性与环丙沙星（一种已知的该酶抑制剂）相当。此外，我们确定与经典的 5-硝基咪唑（如甲硝唑）相比，indolin-2-one 硝基咪唑的氧化还原电位显着增加，这有助于体内减少。总的来说，这项研究揭示了 indolin-2-one-functionalized nitroimidazoles 具有意想不到的双重 MoA：首先是拓扑异构酶 IV 的直接抑制，其次是经典的硝基咪唑 MoA 还原生物活化导致破坏性反应物种。重要的是，这种双重 MoA 会削弱耐药性的发展。鉴于此类化合物的临床应用，新机制可能成为减轻耐药性的起点。