title: Identification by high-throughput screening of viridin analogs as biochemical and cell-based inhibitors of the cell cycle-regulated nek2 kinase.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Hayward
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton SM2 5NG, UK.
authors.forename: Yvette
authors.initials: Y
authors.lastname: Newbatt
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton SM2 5NG, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Pickard
authors.affiliation: Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Eilis
authors.initials: E
authors.lastname: Byrne
authors.affiliation: Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Guojie
authors.initials: G
authors.lastname: Mao
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton SM2 5NG, UK.
authors.forename: Samantha
authors.initials: S
authors.lastname: Burns
authors.affiliation: Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Navdeep K
authors.initials: NK
authors.lastname: Sahota
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton SM2 5NG, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Workman
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton SM2 5NG, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Collins
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton SM2 5NG, UK.
authors.forename: Wynne
authors.initials: W
authors.lastname: Aherne
authors.affiliation: Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Fry
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000736:Androstenes / Q000032:analysis / Q000737:chemistry* / Q000494:pharmacology; D001430:Bacteriocins / Q000032:analysis / Q000737:chemistry* / Q000494:pharmacology; D001669:Biochemical Phenomena / Q000187:drug effects; D002138:Calibration; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002477:Cells / Q000187:drug effects* / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards; D006801:Humans; D020128:Inhibitory Concentration 50; D008954:Models, Biological; D000072379:NIMA-Related Kinases; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000302:isolation & purification / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000736:Androstenes; D001430:Bacteriocins; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; C089441:NEK2 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases; C012097:viridin