θ聚合酶（Polθ）在DNA复制和修复中发挥作用，其抑制在BRCA1和BRCA2缺陷的肿瘤细胞中具有合成致命性。Novobiocin（NVB）是第一类抑制剂，可抑制Polθ ATP酶活性，并且目前正在临床试验中作为一种抗癌药物。在本研究中，我们研究了NVB介导的Polθ抑制的分子机制。通过氢氘交换质谱（HX-MS），生物物理、生物化学、计算和细胞实验，我们发现NVB是ATP水解的非竞争性抑制剂。NVB糖基缺失导致其效力降低和HX-MS相互作用减少，支持特定的NVB结合方向。综合结果揭示了NVB结合于一个变构位点以阻断DNA结合，无论在体外还是体内均如此。对癌症基因组图谱（TCGA）肿瘤和匹配对照的比较表明，肿瘤中POLQ的上调源于其在复制应激响应和细胞增殖增加中的作用：现在可以通过NVB阻断ssDNA对ATP酶活性的刺激来测试这一点，这在15种肿瘤类型中是必需的，这种刺激对于Polθ在复制叉和DNA损伤部位的功能非常重要。本研究提供的结构和功能见解为开发以增强NVB对Polθ抑制作用的新型衍生物提供了途径，这些衍生物通过以熵约束小分子靶向ssDNA结合。© 作者团队2023. 由牛津大学出版社代表《核酸研究》杂志发表。

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.