Studies On Discovery Of Protein Kinase Inhibitors And Pharmacology Of Active Compound

Reversible phosphorylation is a fundamental molecular mechanism that is critical for regulating cell division. Deregulated phosphorylation of the components important to cell cycle control frequently leads to abnormal cell division and/or transformation. To date, several families of protein kinases such as cyclindependent kinases (Cdks), polo-like kinases (PLKs), and Aurora family of kinases have been characterized, which are important to cell cycle regulation. While PLKs comprise a family of four related serine/threonine kinases with different subcellular localization and functions in mammals, PLK1 is the best-studied and arguably most important regulators of cell proliferation within the PLK family. The human aurora family of serine-threonine kinases comprises three members, which act in concert with many other proteins to control chromosome assembly and segregation during mitosis. The two kinase families are all positively correlated with tumorigenesis and are considered as attractive mitotic targets for anticancer drugs.In order to screen kinase inhibitors more effectively, the whole RNA was taken from Hela cells and was used as template to RT-PCR. Then the DNA fragments encoding Aurora A and PLK1 kinases were amplified by PCR. Aurora C kinase was amplified by using human testicle cDNA as template. The recombinant plasmids pMD-18T-A,pMD-18T-PLK1 and pMD-18T-C were obtained by ligating the DNA fragments of AURKA,AURKC and PLK1 to pMD-18T plasmid respectively and used for sequence determination. Also DNA fragments of AURKA,AURKC and PLK1 were respectively cloned into the expression pET-30-a-c(+) vector to form recombinant expression plasmids which were then transformed into the host of Escherichia coli BL21. The expressed products of E. coli induced by IPTG were then puried by Ni²⁺ chelating affinity chromatography and relatively highly puried Aurora A,Aurora C and PLK1 kinase were obtained for further study.In this research, we also used budding yeast as a model system to identify additional PLK1 inhibitors. Here, we report the identification of DH166, aβ-carboline derivative, which inhibits the growth of cdc5-2 temperature-sensitive mutant more profoundly than wild-type yeast cells. Because CDC5 is the human Plk1 homologue in budding yeast, different approaches are used to investigate the effect of DH166 on PLK1 kinase activity in vitro. Interestingly, DH166 inhibits the kinase activity of purified PLK1 at low micromolar concentrations in an ATP-competitive manner. DH166 competes for the kinase domain, which is consistent with the docking results based on the crystal structure of PLK1. In addition, DH166 blocks cell proliferation, increases cyclin B1 accumulation, and induces apoptosis, presumably due to the down-regulation of PLK1. Althoughβ-carboline derivatives have been demonstrated to show antitumor activities through multiple mechanisms, our data for the first time indicate that their cytotoxicity to tumor cells might be attributable to the inhibition of PLK1 as well.Because of its easiness to cultivate and quick proliferation, the single-celled eukaryote budding yeast is an excellent model system to screen the PLK1 inhibitors. Utilizing this system, we for the first time found theβ-carboline derivative's inhibition activity against PLK1. Therefore, this work lays the foundation for further employing budding yeast cells to screen compounds that target a particular protein or a specific cellular process that could be used as anticancer drugs.