Probing cell-division phenotype space and Polo-like kinase function using small molecules


Cell-permeable small molecules that inhibit their targets on fast timescales are powerful probes of cell-division mechanisms. Such inhibitors have been identified using phenotype-based screens with chemical libraries. However, the characteristics of compound libraries needed to effectively span cell-division phenotype space, to find probes that target different mechanisms, are not known. Here we show that a small collection of 100 diaminopyrimidines (DAPs) yields a range of cell-division phenotypes, including changes in spindle geometry, chromosome positioning and mitotic index. Monopolar mitotic spindles are induced by four inhibitors, including one that targets Polo-like kinases (Plks), evolutionarily conserved serine/threonine kinases. Using chemical inhibitors and high-resolution live-cell microscopy, we found that Plk activity is needed for the assembly and maintenance of bipolar mitotic spindles. Plk inhibition destabilizes kinetochore microtubules while stabilizing other spindle microtubules, leading to monopolar spindles. Further testing of compounds based on 'privileged scaffolds', such as the DAP scaffold, could lead to new cell-division probes and antimitotic agents.

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Figure 1: Cell-division phenotype space is effectively spanned by a small collection of DAPs mapping to the chemical space occupied by known bioactive compounds.
Figure 2: Monopolar mitotic spindles are induced by two DAPs, including one that inhibits Plk1 activity in vitro.
Figure 3: Comparison of cell-division phenotypes upon Plk1 knockdown and chemical inhibitor treatments.
Figure 4: DAP-81 and BTO-1 inhibit in vivo phosphorylation of a Plk1 substrate, Cdc25C, at concentrations that do not inhibit phosphorylation of an Aurora B kinase substrate, histone H3.
Figure 5: Plk activity is required for the maintenance of spindle bipolarity.
Figure 6: Plk inhibition leads to collapsing spindles with stable astral-microtubule bundles without loss of centromere tension.
Figure 7: The selective stabilization of astral microtubules and the destabilization of K-fibers can be reversed by Plk activation.


  1. 1

    Rajagopalan, H. & Lengauer, C. Aneuploidy and cancer. Nature 432, 338–341 (2004).

    CAS  Article  Google Scholar 

  2. 2

    Mitchison, T.J. & Salmon, E.D. Mitosis: a history of division. Nat. Cell Biol. 3, E17–E21 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Inoue, S. & Salmon, E.D. Force generation by microtubule assembly/disassembly in mitosis and related movements. Mol. Biol. Cell 6, 1619–1640 (1995).

    CAS  Article  Google Scholar 

  4. 4

    Lampson, M.A. & Kapoor, T.M. Unraveling cell division mechanisms with small-molecule inhibitors. Nat. Chem. Biol. 2, 19–27 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Jordan, M.A. & Wilson, L. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 4, 253–265 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Bettencourt-Dias, M. et al. Genome-wide survey of protein kinases required for cell cycle progression. Nature 432, 980–987 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Kittler, R. & Buchholz, F. Functional genomic analysis of cell division by endoribonuclease-prepared siRNAs. Cell Cycle 4, 564–567 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Fitzgerald, K. RNAi versus small molecules: different mechanisms and specificities can lead to different outcomes. Curr. Opin. Drug Discov. Devel. 8, 557–566 (2005).

    CAS  PubMed  Google Scholar 

  9. 9

    Specht, K.M. & Shokat, K.M. The emerging power of chemical genetics. Curr. Opin. Cell Biol. 14, 155–159 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Tan, D.S. Diversity-oriented synthesis: exploring the intersections between chemistry and biology. Nat. Chem. Biol. 1, 74–84 (2005).

    CAS  Article  Google Scholar 

  11. 11

    Lipinski, C. & Hopkins, A. Navigating chemical space for biology and medicine. Nature 432, 855–861 (2004).

    CAS  Article  Google Scholar 

  12. 12

    Evans, B.E. et al. Methods for drug discovery: development of potent, selective, orally effective cholecystokinin antagonists. J. Med. Chem. 31, 2235–2246 (1988).

    CAS  Article  Google Scholar 

  13. 13

    De Corte, B.L. From 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk](1,4)benzodiazepin-2(1H)-one (TIBO) to etravirine (TMC125): fifteen years of research on non-nucleoside Inhibitors of HIV-1 reverse transcriptase. J. Med. Chem. 48, 1689–1696 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Sorbera, L.A., Castaner, J. & Martin, L. Revaprazan hydrochloride: treatment of GERD, H+/K+-ATPase inhibitor, antiulcer drug. Drugs Future 29, 455–459 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Breault, G.A. et al. Cyclin-dependent kinase 4 inhibitors as a treatment for cancer. Part 2: identification and optimisation of substituted 2,4-bis anilino pyrimidines. Bioorg. Med. Chem. Lett. 13, 2961–2966 (2003).

    CAS  Article  Google Scholar 

  16. 16

    Legendre, P. & Legendre, L. Numerical ecology (Elsevier, New York, 1998).

    Google Scholar 

  17. 17

    Hotha, S. et al. HR22C16: a potent small-molecule probe for the dynamics of cell division. Angew. Chem. Int. Edn. Engl. 42, 2379–2382 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Aronov, A.M. & Murcko, M.A. Toward a pharmacophore for kinase frequent hitters. J. Med. Chem. 47, 5616–5619 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Feng, B.Y., Shelat, A., Doman, T.N., Guy, R.K. & Shoichet, B.K. High-throughput assays for promiscuous inhibitors. Nat. Chem. Biol. 1, 146–148 (2005).

    CAS  Article  Google Scholar 

  20. 20

    Burdine, L. & Kodadek, T. Target identification in chemical genetics: the (often) missing link. Chem. Biol. 11, 593–597 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Davis-Ward, R., Mook, R.A., Jr., Neeb, M.J. & Salovich, J.M. Preparation of pyrimidine derivatives as Polo-like kinases inhibitors for treatment of cancers (World Patent WO 2004074244, 2004).

  22. 22

    Barr, F.A., Sillje, H.H. & Nigg, E.A. Polo-like kinases and the orchestration of cell division. Nat. Rev. Mol. Cell Biol. 5, 429–440 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Takai, N., Hamanaka, R., Yoshimatsu, J. & Miyakawa, I. Polo-like kinases (Plks) and cancer. Oncogene 24, 287–291 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Compton, D.A. Spindle assembly in animal cells. Annu. Rev. Biochem. 69, 95–114 (2000).

    CAS  Article  Google Scholar 

  25. 25

    Sharp, D.J., Rogers, G.C. & Scholey, J.M. Microtubule motors in mitosis. Nature 407, 41–47 (2000).

    CAS  Article  Google Scholar 

  26. 26

    McInnes, C., Mezna, M. & Fischer, P.M. Progress in the discovery of polo-like kinase inhibitors. Curr. Top. Med. Chem. 5, 181–197 (2005).

    CAS  Article  Google Scholar 

  27. 27

    Gumireddy, K. et al. ON01910, a non-ATP-competitive small molecule inhibitor of Plk1, is a potent anticancer agent. Cancer Cell 7, 275–286 (2005).

    CAS  Article  Google Scholar 

  28. 28

    McInnes, C., Meades, C., Mezna, M. & Fischer, P. Benzthiazole-3 oxides useful for the treatment of proliferative disorders (World Patent WO 2004067000, 2004).

  29. 29

    van Vugt, M.A. et al. Polo-like kinase-1 is required for bipolar spindle formation but is dispensable for anaphase promoting complex/Cdc20 activation and initiation of cytokinesis. J. Biol. Chem. 279, 36841–36854 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Sumara, I. et al. Roles of polo-like kinase 1 in the assembly of functional mitotic spindles. Curr. Biol. 14, 1712–1722 (2004).

    CAS  Article  Google Scholar 

  31. 31

    Khodjakov, A., Copenagle, L., Gordon, M.B., Compton, D.A. & Kapoor, T.M. Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis. J. Cell Biol. 160, 671–683 (2003).

    CAS  Article  Google Scholar 

  32. 32

    Toyoshima-Morimoto, F., Taniguchi, E. & Nishida, E. Plk1 promotes nuclear translocation of human Cdc25C during prophase. EMBO Rep. 3, 341–348 (2002).

    CAS  Article  Google Scholar 

  33. 33

    Nigg, E.A. Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell Biol. 2, 21–32 (2001).

    CAS  Article  Google Scholar 

  34. 34

    Hauf, S. et al. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J. Cell Biol. 161, 281–294 (2003).

    CAS  Article  Google Scholar 

  35. 35

    Niiya, F., Xie, X., Lee, K.S., Inoue, H. & Miki, T. Inhibition of cyclin-dependent kinase 1 induces cytokinesis without chromosome segregation in an ECT2 and MgcRacGAP-dependent manner. J. Biol. Chem. 280, 36502–36509 (2005).

    CAS  Article  Google Scholar 

  36. 36

    Lampson, M.A., Renduchitala, K., Khodjakov, A. & Kapoor, T.M. Correcting improper chromosome-spindle attachments during cell division. Nat. Cell Biol. 6, 232–237 (2004).

    CAS  Article  Google Scholar 

  37. 37

    Dogterom, M., Kerssemakers, J.W., Romet-Lemonne, G. & Janson, M.E. Force generation by dynamic microtubules. Curr. Opin. Cell Biol. 17, 67–74 (2005).

    CAS  Article  Google Scholar 

  38. 38

    Li, X. & Nicklas, R.B. Mitotic forces control a cell-cycle checkpoint. Nature 373, 630–632 (1995).

    CAS  Article  Google Scholar 

  39. 39

    Waters, J.C., Mitchison, T.J., Rieder, C.L. & Salmon, E.D. The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work. Mol. Biol. Cell 7, 1547–1558 (1996).

    CAS  Article  Google Scholar 

  40. 40

    Gergely, F., Draviam, V.M. & Raff, J.W. The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells. Genes Dev. 17, 336–341 (2003).

    CAS  Article  Google Scholar 

  41. 41

    Joseph, J., Liu, S.T., Jablonski, S.A., Yen, T.J. & Dasso, M. The RanGAP1-RanBP2 complex is essential for microtubule-kinetochore interactions in vivo. Curr. Biol. 14, 611–617 (2004).

    CAS  Article  Google Scholar 

  42. 42

    Garrett, S., Auer, K., Compton, D.A. & Kapoor, T.M. hTPX2 is required for normal spindle morphology and centrosome integrity during vertebrate cell division. Curr. Biol. 12, 2055–2059 (2002).

    CAS  Article  Google Scholar 

  43. 43

    Gruber, J., Harborth, J., Schnabel, J., Weber, K. & Hatzfeld, M. The mitotic-spindle-associated protein astrin is essential for progression through mitosis. J. Cell Sci. 115, 4053–4059 (2002).

    CAS  Article  Google Scholar 

  44. 44

    Adams, R.R., Maiato, H., Earnshaw, W.C. & Carmena, M. Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J. Cell Biol. 153, 865–880 (2001).

    CAS  Article  Google Scholar 

  45. 45

    Holt, S.V. et al. Silencing Cenp-F weakens centromeric cohesion, prevents chromosome alignment and activates the spindle checkpoint. J. Cell Sci. 118, 4889–4900 (2005).

    CAS  Article  Google Scholar 

  46. 46

    Zhu, C. et al. Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference. Mol. Biol. Cell 16, 3187–3199 (2005).

    CAS  Article  Google Scholar 

  47. 47

    Dai, J., Sultan, S., Taylor, S.S. & Higgins, J.M. The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment. Genes Dev. 19, 472–488 (2005).

    CAS  Article  Google Scholar 

  48. 48

    McEwen, B.F., Heagle, A.B., Cassels, G.O., Buttle, K.F. & Rieder, C.L. Kinetochore fiber maturation in PtK1 cells and its implications for the mechanisms of chromosome congression and anaphase onset. J. Cell Biol. 137, 1567–1580 (1997).

    CAS  Article  Google Scholar 

  49. 49

    Knight, Z.A. & Shokat, K.M. Features of selective kinase inhibitors. Chem. Biol. 12, 621–637 (2005).

    CAS  Article  Google Scholar 

  50. 50

    Tanaka, M. et al. An unbiased cell morphology-based screen for new, biologically active small molecules. PLoS Biol. 3, e128 (2005).

    Article  Google Scholar 

  51. 51

    Qian, Y.W., Erikson, E., Li, C. & Maller, J.L. Activated polo-like kinase Plx1 is required at multiple points during mitosis in Xenopus laevis. Mol. Cell. Biol. 18, 4262–4271 (1998).

    CAS  Article  Google Scholar 

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We thank C. Karan and acknowledge the use of the Rockefeller University High Throughput Screening Resource Center. This work was supported by US National Institutes of Health grant GM71772 (to T.M.K.).

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Correspondence to Tarun M Kapoor.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Composition of the diaminopyrimidine library. (PDF 194 kb)

Supplementary Fig. 2

Overview of cell-based screening results. (PDF 257 kb)

Supplementary Fig. 3

Mitotic phenotypes induced by a subset of DAPs. (PDF 442 kb)

Supplementary Fig. 4

Monopolar mitotic spindles induced by two diaminopyrimidines do not result from changes in centrosome number or Eg5 inhibition. (PDF 908 kb)

Supplementary Fig. 5

BTO-1 inhibits Polo-like kinase 1 in vitro. (PDF 93 kb)

Supplementary Fig. 6

Additional images for comparing Plk1-knock-down and treatment with chemical inhibitors. (PDF 129 kb)

Supplementary Fig. 7

Quantitation of monopolar spindles and mitotic indices in DAP-81 and BTO-1 treated PTKαT cells. (PDF 97 kb)

Supplementary Methods (PDF 72 kb)

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Peters, U., Cherian, J., Kim, J. et al. Probing cell-division phenotype space and Polo-like kinase function using small molecules. Nat Chem Biol 2, 618–626 (2006).

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