University of Leicester
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Identification by high-throughput screening of viridin analogs as biochemical and cell-based inhibitors of the cell cycle-regulated Nek2 kinase

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posted on 2012-10-24, 08:58 authored by Daniel G. Hayward, Yvette Newbatt, Lisa Pickard, Eilis Byrne, Guojie Mao, Samantha Burns, Navdeep K. Sahota, Paul Workman, Ian Collins, Wynne Aherne, Andrew M. Fry
Nek2 is a serine/threonine protein kinase that localizes to the centrosome and is implicated in mitotic regulation. Overexpression of Nek2 induces premature centrosome separation and nuclear defects indicative of mitotic errors, whereas depletion of Nek2 interferes with cell growth. As Nek2 expression is upregulated in a range of cancer cell lines and primary human tumors, inhibitors of Nek2 may have therapeutic value in cancer treatment. The authors used a radiometric proximity assay in a high-throughput screen to identify small-molecule inhibitors of Nek2 kinase activity. The assay was based on the measurement of the radiolabeled phosphorylated product of the kinase reaction brought into contact with the surface of wells of solid scintillant-coated microplates. Seventy nonaggregating hits were identified from approximately 73,000 compounds screened and included a number of toxoflavins and a series of viridin/wortmannin-like compounds. The viridin-like compounds were >70-fold selective for Nek2 over Nek6 and Nek7 and inhibited the growth of human tumor cell lines at concentrations consistent with their biochemical potencies. An automated mechanism-based microscopy assay in which centrosomes were visualized using pericentrin antibodies confirmed that 2 of the viridin inhibitors reduced centrosome separation in a human tumor cell line. The data presented show that pharmacological inhibition of Nek2 kinase results in the expected phenotype of disruption to centrosome function associated with growth inhibition and further supports Nek2 as a target for cancer drug discovery.


This work was supported by grants to AMF, WA, and PW from the Discovery Committee of Cancer Research UK. The Cancer Research UK Centre for Cancer Therapeutics is funded primarily by Cancer Research UK program grants C309/A2187 and C309/A8274. We acknowledge NHS funding to support the work of the NIHR Biomedical Research Centre, the ICR, and The Royal Marsden NHS Foundation Trust.



Journal of Biomolecular Screening, 2010, 15 (8), pp. 918-927


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