High-Throughput Screening Assay for Identification of Small Molecule Inhibitors of Aurora2/STK15 Kinase

Abstract

STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, γ-³³P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate® to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z′ factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity.

Keywords

STK15/Aurora2 HTS FlashPlate®small molecule inhibitor

References

1. Francisco L , Wang W , Chan CS : Type 1 protein phosphatase acts in opposition to Ipl1 protein kinase in regulating yeast chromosome segregation. Mol Cell Biol 1994;14:4731-4740.

2. Glover DM , Leibowitz MH , McLean DA , Parry H : Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 1995;81:95-105.

3. Giet R , Prigent C : Aurora/IpI1p related kinases a new oncongenic family of mitotic serine threonine kinases. J Cell Sci 1999;112:3591-3601.

4. Bernard M , Sanseau P , Henry C , Couturier A , Prigent C : Cloning of STK13, a third human protein kinase related to Drosophila Aurora and budding yeast Ipl1 that maps on chromosome 19q13. 3-ter. Genomics 1998;53:406-409.

5. Bischoff JR , Anderson L , Zu Y , Mossie K , Ng L , Souza B , et al : A homologue of Drosophila aurora kinase is oncongenic and amplified in human colorectal cancers. EMBO J 1998;17:3052-3065.

6. Zhou H , Kuang J , Zhong L , Kuo W-L , Gray JW , Sahin A , et al : Tumour amplified STK15/BTAK induce centrosome amplification, aneuploidy and transformation. Nature Genet 1998;20:189-193.

7. Kallioniemi A , Kallioniemi O-P , Piper J , Tanner M , Stoke T , Chen L , et al : Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc Natl Acad Sci U S A 1994;91:2156-2160.

8. Littlepage LE , Wu H , Andresson T , Deanehan JK , Amundadottir LT , Ruderman JV : Identification of phosphorylated residues that affect activity of the mitotic kinase Aurora-A. Proc Natl Acad Sci U S A 2002;99:15440-15445.

9. Walter A , Seghezzi W , Korver W , Sheung J , Lees E : The mitotic serine threonine kinase Aurora2/STK15 is regulated by phosphorylation and degradation. Oncogene 2000;19:4906-4916.

10.

10. Hauf S , Cole RW , LaTerra S , Zimmer C , Schnapp G , Walter R , et al : The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J Cell Biol 2003;161:281-294.

11.

11. Ditchfield C , Johnson VL , Tighe A , Ellston R , Haworth C , Johnson T , et al : Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 2003;161:267-280.

12.

12. Warner SL , Bearss DJ , Han H , Von Hoff DD : Targeting Aurora-2 kinase in cancer. Mol Cancer Ther 2003;6:589-595.

13.

13. Bischoff JR , Plowman GD : the Aurora/Ipl1p kinase family: regulators of chromosome segregation and cytokinesis. Trends Cell Biol 1999;9:454-459.

14.

14. Zhang JH , Chung T , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of HTS assays. J Biomol Screen 1999;4:67-73.

15.

15. Andresson T , Ruderman JV : The kinase Eg2 is a component of the Xenopus oocyte progesterone-activated signaling pathway. EMBO J 1998;17:5627-5637.

16.

16. Crosio C , Fimia GM , Loury R , Kimura M , Okano Y , Zhou H , et al : Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol Cell Biol 2002;22:874-885.

17.

17. Hemmila I : LANCE™: homogeneous assay platform for HTS. JBiomol Screen 1999;6:303-307.

18.

18. Turlais F , Hardcastle A , Rowlands M , Newbatt Y , Bannister A , Kouzarides T , et al : High-throughput screening for identification of small molecule inhibitors of histone acetyltransferases using scintillating microplates (FlashPlate). Anal Biochem 2001;298:62-68.

19.

19. Meggio F , Donella DA , Ruzzene M , Brunati AM , Cesaro L , Guerra B , et al : Different susceptibility of protein kinases to staurosporine inhibition. Kinetic studies and molecular bases for the resistance of protein kinase CK2. Eur J Biochem 1995;234:317-322.

20.

20. Delle FS , Piubelli C , Domenici E : Development of a high-throughput scintillation proximity assay for the identification of C-domain translational initiation factor 2 inhibitors. J Biomol Screen 2002;7:541-546.