AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme.

中文翻译：

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表征人 AP 核酸内切酶 1 的抑制剂。

AP 核酸内切酶 1 (APE1) 处理 DNA 损伤，包括脱嘌呤/脱嘧啶位点和 3'-阻断基团，介导碱基切除修复和单链断裂修复。许多努力都集中在开发 APE1 的特异性抑制剂上，这可能在基础研究中有重要的应用，并可能导致临床抗癌药物。我们使用结构、生物物理学和生化方法来表征几种已报道的抑制剂，包括 7-硝基吲哚-2-羧酸 (CRT0044876)，鉴于其体积小、报道的效力和广泛用于研究 APE1。有趣的是，NMR 化学位移扰动 (CSP) 实验表明 CRT0044876 和三种类似的吲哚-2-羧酸结合远离 APE1 活性位点的口袋。晶体结构证实了这些发现并定义了 5-nitroindole-2-carboxylic acid 的结构。然而，动态光散射实验显示吲哚化合物形成胶体聚集体，可以结合（螯合）APE1，导致非特异性抑制。核酸内切酶测定表明，这些化合物在破坏聚集的条件（洗涤剂）下缺乏显着的 APE1 抑制作用。因此，在远程口袋处结合吲哚-2-羧酸不会抑制 APE1 修复活动。杨梅素还会形成聚集体，并且在聚集体破坏条件下缺乏 APE1 抑制作用。其他两种已报道的化合物（MLS000552981、MLS000419194）在体外以低微摩尔 IC50 抑制 APE1，并且在此浓度范围内似乎不会聚集。然而，NMR CSP 实验表明这些化合物不会特异性结合 apo 或 Mg2+ 结合的 APE1，表明存在非特异性抑制模式，可能是 DNA 结合。我们的结果突出了对推定的 APE1 抑制剂进行严格审查的方法，并且应该有助于未来发现特异性抑制这种重要修复酶的化合物的努力。