Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP’s Distribution on Microtubules

Mitotic regulators exhibiting gain of function in tumor cells are considered useful cancer therapeutic targets for the development of small-molecule inhibitors. The human Aurora kinases are a family of such targets. In this study, from a panel of 105 potential small-molecule inhibitors, two compounds Tripolin A and Tripolin B, inhibited Aurora A kinase activity in vitro. In human cells however, only Tripolin A acted as an Aurora A inhibitor. We combined in vitro, in vivo single cell and in silico studies to demonstrate the biological action of Tripolin A, a non-ATP competitive inhibitor. Tripolin A reduced the localization of pAurora A on spindle microtubules (MTs), affected centrosome integrity, spindle formation and length, as well as MT dynamics in interphase, consistent with Aurora A inhibition by RNAi or other specific inhibitors, such as MLN8054 or MLN8237. Interestingly, Tripolin A affected the gradient distribution towards the chromosomes, but not the MT binding of HURP (Hepatoma Up-Regulated Protein), a MT-associated protein (MAP) and substrate of the Aurora A kinase. Therefore Tripolin A reveals a new way of regulating mitotic MT stabilizers through Aurora A phosphorylation. Tripolin A is predicted to bind Aurora A similarly but not identical to MLN8054, therefore it could be used to dissect pathways orchestrated by Aurora kinases as well as a scaffold for further inhibitor development. © 2013 Kesisova et al

Chemical structure of Tripolin A and Tripolin B.

<p>Chemical structure of Tripolin A and Tripolin B.</p

Selectivity of Tripolins against a panel of kinases.

<p>IC₅₀ values of Tripolin A and Tripolin B against Aurora A, Aurora B and a panel of other selected kinases.</p

Tripolin A alters pole-to-pole distance and MT stability in mitotic cells and influences interphase MT array.

<p>(A) Maximum projections from z-stacks of a representative control cell and representative cells treated with Tripolin A. In the merged images α-tubulin is pseudocolored red; pericentrin is green, DNA is blue. Yellow arrows indicate interpolar distance. (B) Interpolar distances were measured based on pericentrin staining in HeLa cells (n≥100 cells for each group, from at least three independent experiments). ***: p<0.0001; (Student's t-test, two-tailed). Error bars indicate SD. (C) Longitudinal line scans of tubulin intensity from metaphase spindles of control and Tripolin A treated HeLa cells (n = 5 for each group). Intensities were normalized to the maximum value of the control curve, and spindle size was interpolated. Curves indicate mean values. (D) Representative immunofluorescence images of HeLa cells in interphase treated with DMSO, 100 nM MLN8237 for 1 h or 20 µM Tripolin A for 1 h and 24 h. In the merged images α-tubulin is pseudocolored red, DNA blue. (Scale bar 10 µm). (E) Graph showing the percentages of interphase cells with altered MT array, classified in the indicated arbitrary categories in control cells (DMSO) and cells treated with MLN8237 or Tripolin A (n = 150 cells for each group, from three independent experiments).</p

Tripolin A treatment results in spindle and centrosomal defects.

<p>(A) Representative immunofluorescence images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 24 h, 100 nM MLN8237 for 24 h or Aurora A siRNAs. In the merged images α-tubulin is pseudocolored red, DNA blue. (Scale bars, 5 µm). (B) Graph showing the percentage of normal, multipolar, misaligned, disorganized and monopolar figures in control mitotic cells (DMSO or control siRNAs) and mitotic cells treated with Tripolin A, MLN8237 or Aurora A siRNA (n = 300 cells for each group, from three independent experiments). (C) Western Blot analysis for Aurora A levels in Aurora A siRNA treated cells. α-tubulin was used as a loading control. (D) Images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 5 h and 24 h or Aurora A siRNA. In the merged images Aurora A is pseudocolored red, pericentrin green, DNA blue. (Scale bar 5 µm). (E) Graph showing the percentage of mitotic cells with fragmented centrosomes (up), or acentrosomal poles (down) in control mitotic cells (DMSO or control siRNA) and mitotic cells treated with Tripolin A, or Aurora A siRNA (n = 150 cells for each group, from three independent experiments).</p

Tripolins inhibit Aurora kinase activity <i>in vitro</i>.

<p><i>(</i>A) Chemical structure of Tripolin A and Tripolin B. (B) Graph showing IC₅₀ values (in µM) of Tripolin A (red) and Tripolin B (green) in the presence of different ATP concentrations, using an <i>in vitro</i> kinase assay. (C) Differential Scanning Fluorimetry results for Aurora A in the presence and absence of the inhibitors. Blue curve determines the melting temperature of Aurora A alone (45°C), red in the presence of Tripolin A (47°C) and green in the presence of Tripolin B (53°C).</p

Tripolin A selectively inhibits Aurora A over Aurora B in cultured tumor cells.

<p>(A) Representative immunofluorescence images of HeLa cells in metaphase treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 5 h and 24 h. In the merged images Aurora A is pseudocolored red, pAurora T288 green, DNA blue. (Scale bars, 5 µm). (B) Fluorescence intensity (% percentage) of pAurora A T288 on centrosomes and total Aurora A on spindles were quantified in control metaphase cells or cells treated with Tripolin A or Tripolin B (n≥20 cells for each group, from at least two independent experiments). **: 0.0010.05; (Mann-Whitney test, two-tailed). Error bars represent SEM. (C) Western Blot analysis for Aurora A, Aurora B and pHistone H3 Ser10 in Tripolin A and Tripolin B-treated mitotic cells. α-tubulin was used as a loading control. (D) Representative immunofluorescence images of bipolar metaphase HeLa cells treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 24 h. In the merged images pHistone H3 Ser10 is pseudocolored red, Aurora B green, DNA blue. (Scale bars, 5 µm).</p