Studies On Discovery Of Novel Aurora-B Inhibitors Based On Rational Screening System And Pharmacology Of Active Compounds

Mitosis-the most basic life process by which a complete copy of the duplicated genome is precisely segregated by the microtubule spindle apparatus into two daughter cells-has developed multiple fidelity-monitoring checkpoint systems to ensure its correct temporal and spatial coordination. The Auroras-the serine/threonine kinases, which have been conserved throughout eukaryotic evolution, have recently emerged as key mitotic regulators for genome stability. They are required for multiple aspects of mitosis, including centrosome maturation, formation of a bipolar mitotic spindle, chromosome alignment and segregation, cytokinesis and et al,by regulating the phosphorylation of their substrates such as BubRl,Mad2,Cenp-E and histoneH3. At the same time their activity and localization and function are regulated by their "partners" and upstream regulators. Many studies in recent years showed that Auroras are frequently overexpressed in human tumors and clearly implicated in tumorigenesis, indicating that their encoding genes maybe play a role of oncogene.In this study, the whole RNA was taken from Hela cells and was used as template to RT-PCR. Then the DNA fragment encoding Aurora-B kinase was amplified by PCR. The recombinant plasmid pMD-18T-B1 was obtained by ligating the AURKB DNA fragment to pMD-18T plasmid and used for sequence determination. Also the AURKB DNA fragment was cloned into the expression vector- pET-20b(+) to form recombinant expression plasmid which was then transformed into the host of Escherichia coli DH5α. The expressed products of E.coli induced by IPTG were then puried by Ni²⁺ chelating affinity chromatography and highly puried Aurora-B kinase was obtained for further study.Based on the crystal structure of Aurora-B kinase, we screened the Microbial Natural Products Database by virtual screening. Budding yeast strains-Y300 and ipl1-321 temperature sensitive mutant, were used as initial model organism to test the inhibitory effect of 22 compounds obtained on them. Jadomycin B shows preferred inhibition on ipl1-321 mutnat cells and the kinase activity of recombinant Aurora-B kinase is inhibited by Jadomycin B in vitro. We also demonstrated that Aurora-B-dependent phosphorylation of histone H3 at serine 10 decreased in the presence of Aurora-B. The docking model shows that JB enters the catalytic cleft of Aurora-B and forms multiple interactions with residues surrounding the active site. On the basis of these observations, we conclude that Jadomycin B is a new Aurora-B inhibitor. The SAR(structure-activity-relationship) studies demonstrate that the side chains of the oxazolone ring make a significant impact on the biological activity of Jadomycin which offers an ideal scaffold to generate novel derivatives with a hydrophobic side chain on their oxazolone rings, from which we may find Aurora-B inhibitors with improved activity. Also it is demonstrated that yeast cell models can be used for HTS because of their economy and convenience.