Establishment Models To Screen For New Antitumor Drugs Targeting Aurora A Kinase Inhibitors

Kinases including Serine/Threoine kinases and protein tyrosine kinases,can catalyze the phosphorylation of certain signaling proteins.Protein phosphorylation plays an important role in cellular signal transduction processes.Kinases act an important factor in tumorigenesis,such as proliferation,invasive,metastatic and angiogenesis.With the deep research in tumor signal transduction,some important protein kinases have become the targets of screening antitumor inhibitors.The kinases inhibitors have already become the research focus.Some of them come into the market and most of them currently being studied in clinical trials.Aurora A,located within mitotic spindle and centrosome,belongs to serine/threonine subclass Aurora kinases and it regulates the functions of centrosomes, spindles mitotic entry and nuclear envelope broken in mitosis events.The research demonstrates that Aurora A is strongly expressed in a broad range of solid tumors. Aurora A over-expression contributes to genetic instability and tumorigenesis,and it has some relationship with the prognosis of some tumors.Recently,Aurora kinase inhibitors as antitumor drugs have been developed and a number of these novel agents are currently being studied in phaseⅠandⅡclinical trials exhibiting an promising clinical efficacy.This paper aims to establish kinase inhibitors screening model which can be utilized to screen for specific Aurora A inhibitors.On one hand,the full length Aurora A was expressed as a soluble active kinase using the Bac-to-Bac Baculoviruse Expression System of Invitrogen Company.After that we purified the recombinant protein utilizing the Ni-NTA column.Then,the recombinant protein was identified via SDS-PAGE and Western blot.The result is that we got the recombinant proteins full length Aurora A successfully,nominated as Aurora A.Then,we established a high-throughput,stable and sensitive screening model based on the fluorescence polarization.On the other hand,utilizing retroviral vector pMSCV to package virus,then infected the host cell—HEK293.Then,we got the over-expression cell line named HEK293-Aurora A and identified HEK293-Aurora A cell line by using RT-PCR and Western blot.Finally,we successfully structured the Aurora A over-expression cell line and the over-expression cell line can be used to further evaluate the inhibitors screened from the model based on the fluorescence polarization.