Johns Hopkins University School of Medicine, Department of Biophysics and Biophysical Chemistry, Baltimore, MD 21205, USA.
Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Nucleic Acids Res. 2019 Sep 5;47(15):8163-8179. doi: 10.1093/nar/gkz579.
Type II topoisomerases catalyze essential DNA transactions and are proven drug targets. Drug discrimination by prokaryotic and eukaryotic topoisomerases is vital to therapeutic utility, but is poorly understood. We developed a next-generation sequencing (NGS) approach to identify drug-resistance mutations in eukaryotic topoisomerases. We show that alterations conferring resistance to poisons of human and yeast topoisomerase II derive from a rich mutational 'landscape' of amino acid substitutions broadly distributed throughout the entire enzyme. Both general and discriminatory drug-resistant behaviors are found to arise from different point mutations found at the same amino acid position and to occur far outside known drug-binding sites. Studies of selected resistant enzymes confirm the NGS data and further show that the anti-cancer quinolone vosaroxin acts solely as an intercalating poison, and that the antibacterial ciprofloxacin can poison yeast topoisomerase II. The innate drug-sensitivity of the DNA binding and cleavage region of human and yeast topoisomerases (particularly hTOP2β) is additionally revealed to be significantly regulated by the enzymes' adenosine triphosphatase regions. Collectively, these studies highlight the utility of using NGS-based methods to rapidly map drug resistance landscapes and reveal that the nucleotide turnover elements of type II topoisomerases impact drug specificity.
II 型拓扑异构酶催化重要的 DNA 转化，是已被证实的药物靶点。原核和真核拓扑异构酶对药物的区分对于治疗用途至关重要，但目前对此了解甚少。我们开发了一种下一代测序 (NGS) 方法来鉴定真核拓扑异构酶的耐药突变。我们表明，赋予人类和酵母拓扑异构酶 II 型毒素耐药性的改变源自于广泛分布在整个酶中的氨基酸取代的丰富突变景观。发现具有普遍和区分性的耐药行为源自于同一氨基酸位置的不同点突变，并且发生在已知的药物结合位点之外。对选定的耐药酶的研究证实了 NGS 数据，并进一步表明抗癌喹诺酮类药物 vosaroxin 仅作为嵌入毒素，而抗菌环丙沙星可毒害酵母拓扑异构酶 II。此外，还揭示了人类和酵母拓扑异构酶（尤其是 hTOP2β）的 DNA 结合和切割区域的固有药物敏感性受到其腺苷三磷酸酶区域的显著调节。总的来说，这些研究强调了使用基于 NGS 的方法快速绘制药物耐药性图谱的实用性，并表明 II 型拓扑异构酶的核苷酸转换元件会影响药物特异性。
