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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.


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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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