Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA (‘poisoning’) in an analogous fashion to fluoroquinolones, but without the requirement for the water–metal–ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase–DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases.

中文翻译：

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一类新型抗菌药物——咪唑并吡嗪酮类药物，揭示了 DNA 旋转酶中毒中涉及的结构转变和耐药机制

咪唑并吡嗪酮 (IPY) 是一类新型化合物，以细菌拓扑异构酶为靶点，作为其抗菌活性的基础。我们通过结构/机制研究表征了这些化合物的机制，表明它们以与氟喹诺酮类药物类似的方式结合并稳定 DNA 旋转酶和 DNA 之间的裂解复合物（“中毒”），但不需要水-金属-离子桥。与未切割的旋转酶-DNA 复合物相比，IPY 切割复合物的生化实验和结构研究揭示了与 DNA 门处 DNA 切割耦合的构象转变。这些涉及 GyrA 界面处的运动以及 TOPRIM 结构域向可裂解磷酸盐倾斜，从而捕获催化金属离子。我们的实验表明，这些结构转变通常与治疗化合物对旋转酶的中毒有关，并且类似于该酶在其三磷酸腺苷偶联的链传代循环期间所经历的结构转变。除了影响与化合物直接相互作用的残基的抗性突变外，我们还鉴定了一种突变体（D82N），该突变体抑制未中毒的酶形成裂解复合物。D82N 突变体似乎通过稳定 DNA 旋转酶与未切割 DNA 的二元构象来发挥作用，而不与化合物直接相互作用。这提供了对针对细菌 II 型拓扑异构酶的抗生素耐药机制的一般见解。