Study On Anti - Tumor Effect And Mechanism Of Three Small Molecule Lead Compounds Targeting Aurora Kinase Activity

The Aurora family of serine/threonine protein kinases is known to be critical for the regulation of mitosis. Two of them, Aurora A and Aurora B, are widely expressed in human tumours, with expression peaking at G2and mitotic phases. They have distinct but important roles in mitosis. Aurora B is a chromosomal passenger protein and localises to centromeres in early mitosis and then the spindle midzone in anaphase. Aurora B and its binding partners INCENP and survivin are required for chromosome alignment, mitotic checkpoint function and completion of cytokinesis. Since these discoveries about aurora kinases, inhibition of the kinases should inhibit histone H3phosphorylation, disrupt the cell cycle, lead to aneuploidy or cell death. They are thought to be promising targets for anticancer drug development.In the previous study of our lab, about70,000compounds were screened for the ability to inhibit the kinase activity of recombinant human Aurora B against the substrate MBP. One of the compounds, S2, was selected for further research.Using Kinase-Glo Luminescence assay, we found S2has a equal effect with VX680and more effective than its cis-isomer, S2-Z, to inhibit the activity of Aurora B kinase.Then we treated a panel of20cancer cell lines with S2,5Fu, VX680and ZM447439, analyzed using cck-8kit and calculated their IC50. S2could potently inhibited the proliferation of HepG2with IC500.82p M (micromole), Hep3B, SW620.Inhibition of Aurora B activity is reported to generate polyploidy cells as a result of repeated rounds of DNA duplication in the absence of cytokinesis. Using FACS, we found the majority of S2-treated cells were delayed in G2/M phase of the cell-cycle, failed to divide, became polyploidy and then went to apoptosis.The result of immunofluorescence technic and western blot revealed that phospho-histone H3(Ser10) signal and phosphor-Aurora B (Thr232) signal in S2-treated cells were both greatly reduced compared with controls. Using RNA interference, we found that when the level of Aurora B protein was substantially reduced, the proliferation of the cells was not so sensitive to S2as control cells. S2is able to disrupt the cell cycle and inhibit proliferation by targeting aurora kinase in vitro.As the result of our previous research, S2was not only able to inhibit the activity of Aurora B, but also inhibit VEGFR2&3, and PDGFR. The VEGFR and PDGFR tyrosine kinase families play important role in tumor angiogenesis (and inhibition of them is another particularly attractive therapeutic approach). We established human A375xenograft in nude mice and treated them with S2. Immunohistochemistry revealed that MVD was suppressed by S2. S2has potential to be a multi-target drug.As the targeted therapies have been substantially practiced clinically, New Drug Combination Strategy was considered to be more effective. So the antitumor activity of S2combined with other clinical drugs was also evaluated. We selected15drugs with different mechanisms. The combination effect was detected in9tumor cell lines. After analysis of all the8cell lines, I select rapamycin to do further research.Using FACS (flow cytometry analysis), we observed in the combination group, there is a percentage increase of sub-Gl cells compared with the single agent treatment group. Then we examined the combination effect of S2and rapamycin in vivo, sorafenib plus rapamycin group was used as the positive control. Both of combinations of S2-rapamycin and sorafenib-rapamycin produced statistically significant inhibition compared with single drugs. The survival rate of the S2+rapamycin group is better than the sorafenib+rapamycin group.As a potent and selective inhibitor of Aurora kinases and VEGFR kinases, S2is a successful a multi-targetted small-molecule leader compound with antitumor and antiangiogenesis activity. It also has potential to be used a part of drug combination.We then also evaluated the antitumor effect of two of the hit compounds S7, S39. Using Kinase-Glo Luminescence assay, we found S7and S39has an equal effect with VX680to inhibit the kinase activity of aurora b.Then we treated a panel of10cancer cell lines with S7, S39analyzed using cck-8kit and calculated their IC50. Both could potently inhibit the proliferation of different cancer cell lines. Though colony formation assay and growth curves, we found S7and S39could inhibit the proliferation of cancer cell lines dose-and time-dependently.Using FACS, we found the majority of S7-treated and S39-treated cells were delayed in G2/M phase of the cell-cycle, and then went to apoptosis. The result of immunofluorescence technic and Western blot revealed that phospho-histone H3(Ser10) signal and phosphor-Aurora B(Thr232) signal in S2-treated cells was greatly reduced compared with controls. Using RNA interference, we found that when the level of Aurora B protein was substantially reduced, the proliferation of the cells was not so sensitive to S7and S39as control cells. S2is able to disrupt the cell cycle and inhibit proliferation by targeting aurora B kinase in vitro.Using a a human SMMC7721xenograft in nude mice, we evaluate the ability of S39to suppress the tumor growth in vivo. Treatment of S39resulted in significant tumor growth inhibition with low toxicity.The antitumor activity of the two compounds combined with other clinical drugs was also evaluated. We selected8drugs with different mechanisms. The combination effect was detected in SMMC7721cell lines. We found the combination of S7and rapamycin has a synergistic effect to inhibit the cell proliferation.