Abstract
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.
Highlights
Ilma Amalina1, Ailsa Bennett2, Helen Whalley2, David Perera2, Joanne C
To better define the role of Bub1 kinase activity in mitosis, a potent and selective Bub1 inhibitor would be of great value
Exposure to 10 μM BAY-320 for 3 h prevented the distribution of H2ApT120 along chromosome arms in tetracycline-induced cells; H2ApT120 remained visible on the arms in the presence of 10 μM 2OH-BNPP1
Summary
To better define the role of Bub kinase activity in mitosis, a potent and selective Bub inhibitor would be of great value. We sought to compare the characteristics of two previously described inhibitors, the bulky ATP analogue 2OH-BNPP1 [16] and the substituted benzylpyrazole compound BAY-320 [23,39]. We first sought to synthesize BAY-320 in our laboratory by adapting the methodology of Hitchcock et al [39]. Subsequent reaction of Weinreb amide 2 with ethylmagnesium bromide generated cyclopropyl ethyl ketone 3, which was used to deliver 1,3-dicarbonyl 4. The pyrazole core in 5 was formed using a Knorr reaction. Subsequent alkylation of the pyrazole core with benzyl bromide 6 gave ester 7 and functional group interconversion delivered amidine 8. After construction of the pyrimidine ring in 10, using reagent 9, cyclopropylbenzylpyrazole 11 (BAY-320) was obtained by Buchwald–Hartwig amination
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