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Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function

Abstract

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and for the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, the development of a potent, selective small-molecule inhibitor of Mps1 should facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 cocrystal structures of new, selective small-molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells having extra centrosomes (an abnormality found in some cancers), Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

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Figure 1: Mps1-IN-1 and Mps1-IN-2 inhibit Mps1 kinase activity and bind Mps1 in the ATP binding site.
Figure 2: Mps1-IN-1 and Mps1-IN-2 induce bypass of a checkpoint-mediated mitotic arrest.
Figure 3: Mps1-IN-1 treatment causes disruption in recruitment of Mad2 to kinetochores.
Figure 4: Mps1-IN-1 treatment decreases intracellular Aurora B kinase activity.
Figure 5: Mps1-IN-1 compound treatment does not affect centrosome duplication.
Figure 6: Mps1-IN-1 drives cancer cells with extra centrosomes into a catastrophic anaphase.
Figure 7: Mps1 is required for cell viability.

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Acknowledgements

We thank R. King (Harvard Medical School), B. Vogelstein (Howard Hughes Medical Institute and Johns Hopkins Medical Institutions) and E. Nigg (Max Planck Institute of Biochemistry, Munich) for reagents and R. King, T. Mitchison, A. Abrieu, U. Eggert, C. Walsh, F. Sigoillot and E. Chung for helpful discussions. We also thank Ambit Biosciences and Invitrogen Corporation for technical support in the initial compound screening and enzymatic activity assays, respectively, as well as the Nikon Imaging Facility (Harvard Medical School) and the Dana-Farber Flow Cytometry Lab for technical help and instrument use. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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Authors and Affiliations

Authors

Contributions

N.S.G. and T.S. conceived and directed the chemistry effort. H.G.C. and N.K. performed the chemical synthesis and small-molecule structure determination. N.K., N.J., M.S.M., M.K., Q.L.D., S.R., D.P., J.V.S., G.J.P.L.K. and N.S.G. designed the biological experimental research. N.K., N.J., M.S.M. and M.K. performed experimental research and analysis. S.K. conceived and directed the X-ray crystallography research. P.F. and M.S. performed the X-ray crystallography research and analysis. N.K., P.F., M.K. and N.S.G. cowrote the paper. All authors read and edited the manuscript.

Corresponding author

Correspondence to Nathanael S Gray.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Results, Supplementary Figures 1–12 and Supplementary Tables 1–4 (PDF 6941 kb)

Supplementary Movie 1

This representative time-lapse movie, related to Fig. 2d and Supplementary Fig. S5a, shows U2OS H2B-GFP cells treated with DMSO vehicle. Mitotic progression and DNA segregation were followed by Histone H2B movement in fluorescent channel (serves as control for Movie S2). Based on time from nuclear envelope breakdown (NEBD, t=0 min.) to anaphase initiation these cells spent 14 and 20 minutes in mitosis (90% cells completed mitosis in 40 minutes). Frame rate equals five frames per second. Time is given in minutes.seconds. (AVI 5129 kb)

Supplementary Movie 2

This representative time-lapse movie, related to Fig. 2d and Supplementary Fig. S5a, shows a U2OS H2B-GFP cell treated with Mps1-IN-1 (10 μM). Mitotic progression and DNA segregation were followed by Histone H2B movement in fluorescent channel. Based on time from nuclear envelope breakdown (NEBD, t=0 min.) to anaphase initiation this cell spent 8 minutes in mitosis (90% cells completed mitosis in 18 minutes). Frame rate equals five frames per second. Time is given in minutes.seconds. (AVI 5044 kb)

Supplementary Movie 3

This representative time-lapse movie, related to Fig. 3a and 3b, shows a Ptk2 cell stably expressing HsMad2-EYFP treated with vehicle control (serves as control for Movie S4). Clear fluorescent signal was detectable above background at the kinetochores as the cell entered mitosis. Scale bar is equal to 10 micron. Frame rate equals seven frames per second. Time is given in minutes.seconds. (AVI 3779 kb)

Supplementary Movie 4

This representative time-lapse movie, related to Fig. 3a and 3b, shows a Ptk2 cell stably expressing HsMad2-EYFP treated with Mps1-IN-1 (10 μM). No fluorescent signal was detectable above background at the kinetochores as the cell entered mitosis. Scale bar is equal to 10 micron. Frame rate equals seven frames per second. Time is given in minutes.seconds. (AVI 4431 kb)

Supplementary Movie 5

This representative time-lapse movie, related to Fig. 3a and 3b, shows a Ptk2 cell stably expressing HsMad2-EYFP treated with nocodazole (serves as control for Movie S6). Clear fluorescent signal was detectable above background at the kinetochores as the cell entered mitosis. Scale bar is equal to 10 micron. Frame rate equals seven frames per second. Time is given in minutes.seconds. (AVI 4202 kb)

Supplementary Movie 6

This representative time-lapse movie, related to Fig. 3a and 3b, shows a Ptk2 cell stably expressing HsMad2-EYFP treated with Mps1-IN-1 (10 μM) and nocodazole. Relative to untreated cells and nocodazole control limited fluorescent signal was detectable above background at the kinetochores as the cell entered mitosis. Scale bar is equal to 10 micron. Frame rate equals seven frames per second. Time is given in minutes.seconds. (AVI 3180 kb)

Supplementary Movie 7

This representative time-lapse movie, related to Fig. 6e and 6f, shows an U2OS H2B-GFP cell treated with DMSO vehicle after induced PLK4 overexpression (serves as a control for Movie S8). Mitotic progression and DNA segregation were followed by Histone H2B movement in fluorescent channel. Based on time from nuclear envelope breakdown (NEBD, t=20 min.) to anaphase initiation this cell spent 100 minutes in mitosis. Frame rate equals five frames per second. Time is given in minutes.seconds. (AVI 3007 kb)

Supplementary Movie 8

This representative time-lapse movie, related to Fig. 6e and 6f, shows an U2OS H2B-GFP cells treated with Mps1-IN-1 (10 μM) after induced PLK4 overexpression. Mitotic progression and DNA segregation were followed by Histone H2B movement in fluorescent channel. Based on time from nuclear envelope breakdown (NEBD, t=30 min.) to anaphase initiation this cell spent 20 minutes in mitosis. Frame rate equals five frames per second. Time is given in minutes.seconds. (AVI 3045 kb)

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Kwiatkowski, N., Jelluma, N., Filippakopoulos, P. et al. Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function. Nat Chem Biol 6, 359–368 (2010). https://doi.org/10.1038/nchembio.345

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