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

Chongbo Sun

Cancer Research UK, Centre for Cancer Therapeutics, Sutton, Surrey SM2 5NG, UK.

Yvette Newbatt

Cancer Research UK, Centre for Cancer Therapeutics, Sutton, Surrey SM2 5NG, UK.

Leon Douglas

The Breakthrough Toby Robins Breast Cancer Research Centre, Fulham Road, London SW3 6JB, UK.

Paul Workman

Cancer Research UK, Centre for Cancer Therapeutics, Sutton, Surrey SM2 5NG, UK.

Wynne Aherne

Cancer Research UK, Centre for Cancer Therapeutics, Sutton, Surrey SM2 5NG, UK.

Spiros Linardopoulos

Cancer Research UK, Centre for Cancer Therapeutics, Sutton, Surrey SM2 5NG, UK, The Breakthrough Toby Robins Breast Cancer Research Centre, Fulham Road, London SW3 6JB, UK.

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, {gamma}-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.

Key Words: STK15/Aurora2 • HTS • FlashPlate® • small molecule • inhibitor

Journal of Biomolecular Screening, Vol. 9, No. 5, 391-397 (2004)
DOI: 10.1177/1087057104264071
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F. Chan, C. Sun, M. Perumal, Q.-D. Nguyen, V. Bavetsias, E. McDonald, V. Martins, N. E. Wilsher, F. I. Raynaud, M. Valenti, et al.
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[Abstract] [Full Text] [PDF]