Amalina Ilma, Bennett Ailsa, Whalley Helen, Perera David, McGrail Joanne C, Tighe Anthony, Procter David J, Taylor Stephen S
Department of Chemistry, School of Natural Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Cancer Research Centre, 555 Wilmslow Road, Manchester M20 4GJ, UK.
R Soc Open Sci. 2021 Dec 15;8(12):210854. doi: 10.1098/rsos.210854. eCollection 2021 Dec.
Bub1 is a serine/threonine kinase proposed to function centrally in mitotic chromosome alignment and the spindle assembly checkpoint (SAC); however, its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors are invaluable tools for investigating kinase function, we evaluated two potential Bub1 inhibitors: 2OH-BNPPI and BAY-320. After confirming that both inhibit Bub1 , we developed a cell-based assay for Bub1 inhibition. We overexpressed a fusion of Histone 2B and Bub1 kinase region, tethering it in proximity to H2A to generate a strong ectopic H2ApT120 signal along chromosome arms. Ectopic signal was effectively inhibited by BAY-320, but not 2OH-BNPP1 at concentrations tested. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localization. Preliminary experiments using BAY-320 suggest a minor role for Bub1 kinase activity in chromosome alignment and the SAC; however, BAY-320 may exhibit off-target effects at the concentration required. Thus, 2OH-BNPP1 may not be an effective Bub1 inhibitor , and while BAY-320 can inhibit Bub1 in cells, off-target effects highlight the need for improved Bub1 inhibitors.
Bub1是一种丝氨酸/苏氨酸激酶，被认为在有丝分裂染色体排列和纺锤体组装检查点（SAC）中发挥核心作用；然而，其作用仍存在争议。尽管有充分的文献记载，Bub1在组蛋白2A的T120位点（H2ApT120）进行磷酸化会将Sgo1/2募集到动粒，但关于其激酶活性对染色体排列和SAC的必要性仍存在争议。由于小分子抑制剂是研究激酶功能的宝贵工具，我们评估了两种潜在的Bub1抑制剂：2OH-BNPPI和BAY-320。在确认两者均能抑制Bub1后，我们开发了一种基于细胞的Bub1抑制检测方法。我们过表达了组蛋白2B和Bub1激酶区域的融合蛋白，将其 tethering 在H2A附近，以沿染色体臂产生强烈的异位H2ApT120信号。在测试浓度下，异位信号被BAY-320有效抑制，但未被2OH-BNPP1抑制。此外，只有BAY-320能够抑制内源性Bub1介导的Sgo1定位。使用BAY-320的初步实验表明，Bub1激酶活性在染色体排列和SAC中起次要作用；然而，BAY-320在所需浓度下可能会表现出脱靶效应。因此，2OH-BNPP1可能不是一种有效的Bub1抑制剂，虽然BAY-320可以在细胞中抑制Bub1，但脱靶效应凸显了开发改进的Bub1抑制剂的必要性。 （注：原文中“tethering”一词可能有误，不太明确其准确含义，按字面翻译为“拴系”，可能影响对整体内容的理解）
