It was recently shown that PfCLKs can phosphorylate recombinant PfASF-1 in vitro

ment and SAC function, arguing that Bub1 largely operates as a scaffolding protein. However, even though Bub1 inhibition per se exerts only minor effects on mitotic fidelity, BAY-320 and BAY-524 treatment sensitizes cells to clinically relevant low doses of Paclitaxel, resulting in remarkable impairment of chromosome segregation and cell proliferation. Results BAY-320 and BAY-524 specifically inhibit Bub1 kinase The chemical synthesis of small molecule inhibitors against Bub1 has recently been described. In this study, we used the two substituted benzylpyrazole compounds, 2–5-methoxy-N-pyrimidin-4-amine and 2–5methoxy-N-pyrimidin-4-amine, abbreviated as BAY-320 and BAY-524, respectively. In vitro inhibition of Bub1 by BAY-320 and BAY-524 was demonstrated by monitoring both Bub1 autophosphorylation and phosphorylation of histone H2A on T120 . In presence of 2 mM ATP, both compounds inhibited the recombinant catalytic domain of human Bub1 with an IC50 of 680 280 nM and 450 60 nM, respectively. When tested against a panel of 222 protein kinases, BAY-320 showed only modest cross reactivity with other kinases, even when used at a concentration of 10 mM. Furthermore, quantitative measurements of BAY-320 interactions with 403 human kinases, using an active site-directed competition-binding assay, showed exquisite binding selectivity for Bub1. To test whether BAY-320 and BAY-524 also inhibit Bub1 in intact cells, increasing doses of inhibitors were applied to mitotically synchronized hTERT-RPE1 and HeLa cells, and phospho-histone H2A-T120 staining at kinetochores was monitored by immunofluorescence and in-cell western assays. These studies revealed that near-maximal inhibition of Bub1 could be achieved by using BAY-320 at 310 mM and BAY-524 at 710 mM and these concentrations were therefore used in all future experiments on intact cells. To corroborate the above immunofluorescence data, histones were purified from control and inhibitor-treated cells. Examination of histone H2A phosphorylation by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826300 Western blotting revealed that treatment of cells with either BAY-320 or BAY-524 drastically reduced T120 phosphorylation. Thus, BAY-320 and BAY-524 act as potent and selective inhibitors of Bub1 kinase in both biochemical and cellular assays and thus constitute attractive tools to study Bub1 catalytic function during mitosis. Impact of Bub1 kinase inhibition and Bub1 depletion on mitotic progression Next, we set out to directly compare the impact of Bub1 kinase inhibition with the previously reported consequences of Bub1 depletion or genetic Bub1 knock-out. In a first series of experiments, we used time-lapse imaging to compare progression through mitosis in asynchronously growing HeLa and RPE1 cells in response to either Bub1 inhibition or siRNA-mediated Bub1 depletion. In line with previous results, depletion of Bub1 from HeLa cells MedChemExpress A-83-01 significantly prolonged duration of mitosis, due to delayed chromosome alignment and delays in prometa- and metaphase. In stark contrast, treatment with either BAY-320 or BAY-524 provoked at most minor effects on mitotic progression, marked by a short delay of anaphase onset. Furthermore, in contrast to aneuploid HeLa cells, diploid RPE1 cells were not significantly affected by either Bub1 inhibition or depletion. Efficiency of siRNAmediated depletion was monitored by Western blotting. Flow- Baron et al. eLife 2016;5:e12187. DOI: 10.7554/eLife.12187 4 of 26 Research article Cell biology cytometric analyses confirmed that