Discovery of a novel Aurora B inhibitor GSK650394 with potent anticancer and anti- aspergillus fumigatus dual efficacies in vitro

Abstract

Invasive fungal infections including Candidiasis and Aspergillosis are associated with considerable morbidity and mortality in immunocompromised individuals, such as cancer patients. Aurora B is a key mitotic kinase required for the cell division of eukaryotes from fungus to man. Here, we identified a novel Aurora B inhibitor GSK650394 that can inhibit the recombinant Aurora B from human and Aspergillus fumigatus, with IC₅₀ values of 5.68 and 1.29 µM, respectively. In HeLa and HepG2 cells, GSK650394 diminishes the endogenous Aurora B activity and causes cell cycle arrest in G2/M phase. Further cell-based assays demonstrate that GSK650394 efficiently suppresses the proliferation of both cancer cells and Aspergillus fumigatus. Finally, the molecular docking calculation and site-directed mutagenesis analyses reveal the molecular mechanism of Aurora B inhibition by GSK650394. Our work is expected to provide new insight into the combinational therapy of cancer and Aspergillus fumigatus infection.

Keywords: Aurora B; anti-aspergillus fumigatus; anti-cancer; inhibitor.

Figure 1.

Identification of GSK650394 as a novel Aurora B inhibitor. (a) The ATPase activity of Aurora B was measured by its ability to hydrolyse ATP using the luciferase-coupled ATP assay. The luminescence of the ATP-only control was normalised to 1.0. Values are means ± SD, n = 3. SDS-PAGE of the representative eluted fraction of Aurora B/INCENP protein complex is shown in the inset. (b) The chemical structure of GSK650394. (c) Inhibition of the ATPase activity of human Aurora B by GSK650394. The values were normalised to ATP-only control set as 100% inhibition. Values are means ± SD, n = 3. (d) Western blot analysis of histone H3 Ser10 phosphorylation (pH3) by human Aurora B in the presence of indicated concentrations of GSK650394. The immunoblots are representative of two independent experiments.

Figure 2.

Effect of GSK650394 on cell cycle progression. (a) The dynamic change of histone H3 phosphorylation during the cell cycle. Cells were synchronised with thymidine and released into normal media for the indicated time, followed by immunoblotting with indicated antibodies. The immunoblots are representative of three independent experiments. (b) Effect of GSK650394 on histone H3 phosphorylation in HeLa and HepG2 cells. The thymidine arrested cells were released into media without or with indicated concentrations of drugs for 9 h. Samples were blotted with indicated antibodies. The immunoblots are representative of three independent experiments. (c, d) Effect of GSK650394 on cell cycle progression in HeLa (c) and HepG2 (d) cells. G1/S synchronised cells were released into media without or with indicated concentrations of drugs for 20 h, the resulting cells were subjected to cell cycle analysis by flow cytometry assay.

Figure 3.

GSK650394 suppresses cancer cells proliferation. (a, b) MTT cell proliferation assay for HeLa (a) and HepG2 (b) cells. Cells were treated with indicated concentrations of GSK650394 for 24–72 h. After incubation with MTT solution, the number of viable cells was determined by measuring the absorbance at 490 nm. Values are means ± SD, n = 5. ***P < 0.001 vs. vehicle. (c) Effect of GSK650394 on the nuclear morphology of HeLa cells. Cells were treated with indicated concentrations of GSK650394 for 48 h and the nuclei were stained with DAPI. Outlined regions were magnified and shown in the left bottom corner. Red arrows indicated the expanded nuclei and white arrows for cell debris.

Figure 4.

GSK650394 inhibits the growth of Aspergillus fumigatus. (a) Inhibition of GSK650394 on the ATPase activity of recombinant Af.Aurora B. The values were normalised to ATP-only control set as 100% inhibition. Values are means ± SD, n = 3. (b) Western blot analysis of histone H3 phosphorylation by the recombinant Af.Aurora B in the absence or presence of GSK650394. The immunoblots are representative of two independent experiments. (c) Effect of GSK650394 on the growth inhibition of Aspergillus fumigatus by using the plate assay. (d) Quantification of the diameters of Aspergillus fumigatus clonies in (c). Values are means ± SD, n = 3. ***P < 0.001 vs. vehicle.

Figure 5.

Molecular docking of GSK650394 to human Aurora B. (a) The best-calculated model of human Aurora B bound to GSK650394, with the crystal structure of human Aurora B/INCENP complex (PDB: 4AF3) as starting model. Aurora B was shown in a green cartoon, INCENP was in the orange cartoon, GSK650394 and its key interacting residues were shown in cyan sticks. (b) Detailed interaction between GSK650394 and Aurora B. The interaction was calculated using the program of LIGPLOT, with green dashed lines for hydrogen bonds, and spiked residues for hydrophobic contacts. GSK650394 was shown in the cyan stick. (c) Comparison of ATPase activity between wild type (WT) and mutant Aurora B proteins as measured by luciferase-coupled ATP assay. The luminescence of the ATP-only control was normalised to 1.0. (d) Effect of GSK650394 on the inhibition of wild-type (WT) and V91A Aurora B. The values were normalised to ATP-only control set as 100% inhibition. Values are means ± SD, n = 3.