High-throughput Screening Of Aurora B Kinase Inhibitors And Pharmacology Research On Small Molecule Compound S2

Mitosis,the process by which a complete copy of the duplicated genome is precisely segregated into two daughter cells,is an extraordinarily complex biological process.The three members of Aurora kinase family (Aurora A/B/C)plays multipleroles in mitosis.Expression and activity of the Aurora kinases are tightly regulated during the cell cycle. Activity of all three proteins peaks during the G2 and mitotic phases of the cell cycle,while expression is low or undetectable in resting cells. Based on the known function of the Aurora kinases,inhibition of their activity should disrupt the cell cycle and block proliferation.Here we report the development of high-throughput screening platform and function study on one of the hit compounds.In our study,we designed a microplate format which was suitable for screening compound libraries and screened approximately 70,000 compounds. Seventy six compounds showed an IC₅₀less than 1μM and eight of these compounds showed an IC₅₀less than 100 nM.To confirme the interaction between hit compounds and Aurora kinases, we performed assays with Biacore 3000.The results indicated that hit compounds bind to Aurora kinases with an affinity(KD)of 1×10-8 mol/L to 1×10-6 mol/L.Then we tested the selectivity of hit compounds targeting Aurora kinases over a panel of 20 kinases.The compounds were relatively inactive on most of the other kinases.To evaluat the antiproliferative effects of hit compounds in cell models,we treated cell lines with compound and analyzed with MTS assay. Potent antiproliferative activity was showed in a wide range of cell types. In cell cycle analysis,effects of hit compounds on the cell-cycle profile by flow cytometry was examined.In the colony formation assay,the colony number of various cells lines showed dose dependent decrease compared to the control,which indicated hit compound could inhibit cell proliferation.After that,we investigated the reversibility of growth effects with MTS assay.The growth curve indicated that cells underwent growth inhibition in the presence of compounds and reversed to normal proliferation after it was released.Detection of dose-dependent phosphorylation suppression induced by hit compounds by western blotting and immunofluorescence confirmed the mechanism of cell proliferation inhibition.To examine the ability of hit compound to suppress the tumor growth in vivo,we established human cancer cell xenografts in nude mice. Treatment with hit compounds at a dose of 5mg/ i.p.resulted in significant tumor growth inhibition.Immunohistochemistry revealed that phosphorylated Histone H3(Ser10)was suppressed by 50%to 70%during the dosing period.In acute toxicity tests,no abnormality reactions were found and the LD₅₀is 183 mg/kg.Glycerophosphodiester phosphodiesterase(GDPD)catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol.GDPD5 has been reported in Mus musculus and Gallus gallus,but not in Homo sapiens.Here we report the cloning and characterization of a novel human GDPD domain-containing gene,GDPD5,isolated from human testis cDNA library,and mapped to 11q13.4-13.5 by searching the UCSC genomic database.The GDPD5 cDNA sequence of 3442 base pairs contains an open reading frame encoding 605 amino acids.The GDPD5 gene consists of 17 exons and encodes a putative protein with six transmembrane regions and a GDPD motif.Subcellular localization of GDPD5 demonstrated that the protein was localized in the cytoplasm when overexpressed in COS-7.cells. RT-PCR analysis showed that GDPD5 was widely expressed in human tissues and the expression levels in kidney and prostate were relatively low.