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Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
High-throughput screening assay for identification of small molecule inhibitors of Aurora2/STK15 kinase
Chongbo Sun1, Yvette Newbatt, Leon Douglas
1Cancer Research UK, Centre for Cancer Therapeutics, Sutton, Surrey SM2 5NG, UK.
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, gamma-(33)P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate(R) 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.
Insights
Researchers developed a new assay to measure STK15 kinase activity, crucial for cancer drug discovery. This high-throughput screening method aids in identifying inhibitors targeting STK15 (Aurora2) for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- STK15 (Aurora2) is a centrosome-associated kinase involved in cell cycle regulation.
- Elevated STK15 levels and activity are linked to tumorigenesis and amplified in various human cancers.
- STK15 is a significant therapeutic target for developing small molecule inhibitors to block cancer cell proliferation.
Purpose of the Study:
- To develop a novel, fast, and simple microplate assay for STK15 activity.
- To establish a high-throughput screening (HTS) method for identifying STK15 inhibitors.
- To investigate the regulatory mechanisms of STK15 activity.
Main Methods:
- Development of a microplate assay using gamma-(33)P-ATP and STK15 incubated on myelin basic protein (MBP)-coated FlashPlate(R).
- Assay validation for reproducibility, signal-to-noise ratio, and Z' factor.
- Adaptation of the assay for robotic systems in drug discovery.
- Investigation of STK15 regulation by phosphorylation and its N-terminal domain.
Main Results:
- A robust and reproducible microplate assay for STK15 activity was established with a high signal-to-noise ratio (11) and a Z' factor of 0.69.
- The assay is suitable for high-throughput screening and adaptable to robotic systems for drug discovery.
- STK15 activity is regulated by phosphorylation and its N-amino terminal domain, with increased activity observed upon phosphatase inhibition or N-terminal domain deletion.
Conclusions:
- The developed microplate assay provides a valuable tool for high-throughput screening of STK15 inhibitors.
- This assay facilitates drug discovery efforts targeting STK15 for cancer therapy.
- Further understanding of STK15 regulation can inform the development of more effective cancer treatments.
