Browsing by Author "Patnaik, Debasis"

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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
(Journal of Biomolecular Screening, 2009-12) Patnaik, Debasis; Xian, Jun; Glicksman, Marcie A.; Cuny, Gregory D.; Stein, Ross L.; Higgins, Jonathan M.G.
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 Km 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
Structure–activity relationship study of acridine analogs as haspin and DYRK2 kinase inhibitors
(Bioorganic and Medicinal Chemistry Letters, 6/15/2011) Cuny, Gregory D.; Robin, Maxime; Ulyanova, Natalia P.; Patnaik, Debasis; Pique, Valerie; Casano, Gilles; Liu, Ji-Feng; Lin, Xiangjie; Xian, Jun; Glicksman, Marcie A.; Stein, Ross L.; Higgins, Jonathan M.G.
Haspin is a serine/threonine kinase required for completion of normal mitosis that is highly expressed during cell proliferation, including in a number of neoplasms. Consequently, it has emerged as a potential therapeutic target in oncology. A high throughput screen of approximately 140,000 compounds identified an acridine analog as a potent haspin kinase inhibitor. Profiling against a panel of 270 kinases revealed that the compound also exhibited potent inhibitory activity for DYRK2, another serine/threonine kinase. An optimization study of the acridine series revealed that the structure–activity relationship (SAR) of the acridine series for haspin and DYRK2 inhibition had many similarities. However, several structural differences were noted that allowed generation of a potent haspin kinase inhibitor (33, IC50 <60 nM) with 180-fold selectivity over DYRK2. In addition, a moderately potent DYRK2 inhibitor (41, IC50 <400 nM) with a 5.4-fold selectivity over haspin was also identified.
Structure–activity relationship study of beta-carboline derivatives as haspin kinase inhibitors
(Bioorganic and Medicinal Chemistry Letters, 2013-03) Cuny, Gregory D.; Ulyanova, Natalia P.; Patnaik, Debasis; Liu, Ji-Feng; Lin, Xiangjie; Auerbach, Ken; Ray, Soumya S.; Xian, Jun; Glicksman, Marcie A.; Stein, Ross L.; Higgins, Jonathan M.G.
Haspin is a serine/threonine kinase that phosphorylates Thr-3 of histone H3 in mitosis that has emerged as a possible cancer therapeutic target. High throughput screening of approximately 140,000 compounds identified the beta-carbolines harmine and harmol as moderately potent haspin kinase inhibitors. Based on information obtained from a structure–activity relationship study previously conducted for an acridine series of haspin inhibitors in conjunction with in silico docking using a recently disclosed crystal structure of the kinase, harmine analogs were designed that resulted in significantly increased haspin kinase inhibitory potency. The harmine derivatives also demonstrated less activity towards DYRK2 compared to the acridine series. In vitro mouse liver microsome stability and kinase profiling of a representative member of the harmine series (42, LDN-211898) are also presented.