Indolin-2-one Nitroimidazole Antibiotics Exhibit an Unexpected Dual Mode of Action

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.

甲硝唑（metronidazole）等硝基咪唑类（nitroimidazoles）可用作抗厌氧菌感染药物。在体内发生硝基还原后，其产生的活性自由基可在无氧环境下损伤细胞DNA与蛋白质。此前学界普遍认为这类化合物仅对厌氧菌有效，但近期一项针对连有吲哚啉-2-酮取代基的硝基咪唑类化合物的研究却意外发现，该类化合物对需氧菌同样具有强效抗菌活性，这提示其存在尚未被阐明的全新作用模式（mode of action, MoA）。为解析该作用模式，研究团队首先对经化合物处理的细胞开展全蛋白质组分析，结果显示噬菌体相关蛋白表达上调，提示细胞存在DNA损伤。由于未观察到该化合物与DNA结合，研究团队转而采用基于活性的蛋白质谱分析（activity-based protein profiling, ABPP）进行直接靶点发现。标记实验结果显示，在致病性金黄色葡萄球菌（Staphylococcus aureus）细胞中，拓扑异构酶IV（topoisomerase IV）——一种参与DNA复制的必需酶——为富集程度最高的命中靶点。后续拓扑异构酶活性测定证实，该吲哚啉-2-酮取代的硝基咪唑类化合物可抑制DNA解连环过程，其活性与已知的该酶抑制剂环丙沙星（ciprofloxacin）相当。此外，研究团队测定发现，相较于甲硝唑这类经典5-硝基咪唑类化合物，吲哚啉-2-酮取代的硝基咪唑类的氧化还原电位显著升高，这更利于其在体内发生还原反应。综上，本研究揭示吲哚啉-2-酮功能化修饰的硝基咪唑类化合物具有意外的双重作用模式：其一为直接抑制拓扑异构酶IV，其二为经典硝基咪唑类的还原性生物活化途径，即产生损伤性活性物种。值得注意的是，这种双重作用模式可延缓耐药性的产生。鉴于该类化合物已应用于临床，这一新的作用机制可为缓解临床耐药性提供新的研发起点。