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Identification of Small Molecule Inhibitors of the Mitotic Kinase Haspin by High-Throughput Screening Using a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assay

Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts

Jun Xian

Partners Center for Drug Discovery, Brigham and Women's Hospital, Harvard NeuroDiscovery Center, Cambridge, Massachusetts

Marcie A. Glicksman

Partners Center for Drug Discovery, Brigham and Women's Hospital, Harvard NeuroDiscovery Center, Cambridge, Massachusetts, Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women's Hospital, Harvard NeuroDiscovery Center, Cambridge, Massachusetts

Gregory D. Cuny

Partners Center for Drug Discovery, Brigham and Women's Hospital, Harvard NeuroDiscovery Center, Cambridge, Massachusetts, Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women's Hospital, Harvard NeuroDiscovery Center, Cambridge, Massachusetts

Ross L. Stein

Partners Center for Drug Discovery, Brigham and Women's Hospital, Harvard NeuroDiscovery Center, Cambridge, Massachusetts, Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women's Hospital, Harvard NeuroDiscovery Center, Cambridge, Massachusetts

Jonathan M.G. Higgins

Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, jhiggins{at}rics.bwh.harvard.edu

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at K_m concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC ₅₀ determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC ₅₀ values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics. (Journal of Biomolecular Screening 2008:1025-1034)

Key Words: antimitotics • chromatin • haspin • kinase • time-resolved fluorescence resonance energy transfer

This version was published on December 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 10, 1025-1034 (2008)
DOI: 10.1177/1087057108326081


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