Inhibitors of the Bub1 spindle assembly checkpoint kinase: synthesis of BAY-320 and comparison with 2OH-BNPP1

Ilma Amalina, Ailsa Bennett, Helen Whalley, David Perera, Joanne C McGrail, Anthony Tighe, David J Procter, Stephen S Taylor

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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 in vitro, 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 in cellulo, and while BAY-320 can inhibit Bub1 in cells, off-target effects highlight the need for improved Bub1 inhibitors.

Original languageEnglish
Pages (from-to)210854
JournalRoyal Society Open Science
Issue number12
Publication statusPublished - 15 Dec 2021

Research Beacons, Institutes and Platforms

  • Manchester Cancer Research Centre


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