GW843682X  (Synonyms: GW843682)

PLK1 and PLK3 proteins are prepared from baculovirus-infected Trichoplusia ni cells. Enzyme activity for PLK1 and PLK3 is determined as follows. All measurements are obtained under conditions where signal production increased linearly with time and enzyme. Test compounds are added to white 384-well assay plates (0.1 μL for 10 μL and some 20 μL assays, 1 μL for some 20 μL assays) at variable known concentrations in 100% DMSO. DMSO (1-5% final) and EDTA (65 mM) are used as controls. Reaction Mix containes the following components at 22°C: 25 mM HEPES (pH 7.2); 15 mM MgCl₂; 1 μM ATP; 0.05 μCi/well [γ-³³P]ATP (10 Ci/mmol); 1 μM substrate peptide (Biotin-Ahx-SFNDTLDFD); 0.15 mg/mL bovine serum albumin; 1 mM DTT; and 2 nM PLK1 kinase domain or 5 nM full-length PLK3. Reaction Mix (10 or 20 μL) is quickly added to each well immediately following addition of enzyme via automated liquid handlers and incubated for 1 to 1.5 h at 22°C. The 20 μL enzymatic reactions are stopped with 50 μL of stop mix [50 mM EDTA, 4.0 mg/mL streptavidin SPA beads in Dulbecco's PBS (without Mg²⁺ and Ca²⁺), 50 μM ATP] per well. The 10 μL reactions are stopped with 10 μL of stop mix [50 mM EDTA, 3.0 mg/mL streptavidin-coupled SPA Imaging Beadsin Dulbecco's PBS (without Mg²⁺ and Ca²⁺), 50 μM ATP] per well. Plates are sealed, spun at 500 × g for 1 min or settled overnight, and counted in Packard TopCount for 30 s/well or imaged with a Viewlux imager. Signal above background (EDTA controls) is converted to percent inhibition relative to that obtained in control (DMSO-only) wells^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Assays are carried out and data analyzed. In these assays, H460 cells are plated at a density of 2,000 per well, HDF cells are plated at 5,000 per well, and the drug-resistant cell line MES-SA/DX5 and its sensitive parent line MES-SA are plated at 7,000 and 6,000 per well, respectively, in a 96-well plate. These densities allowed vehicle controls to grow logarithmically during the course of the 3-day assay. All cells are exposed to 3-fold dilutions of the compound (30-0.00152 μM) in low-glucose DMEM containing 5% FBS, 50 μg/mL gentamicin, and 0.3% (v/v) DMSO (HDF cells); RPMI 1640 containing 5% FBS, 50 μg/mL gentamicin, and 0.3% (v/v) DMSO (H460); or McCoy's 5A containing 5% FBS, 50 μg/mL gentamicin, and 0.3% (v/v) DMSO (MES-SA and MES-SA/DX5)^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Lansing TJ, et al. In vitro biological activity of a novel small-molecule inhibitor of polo-like kinase 1. Mol Cancer Ther. 2007 Feb;6(2):450-9. Epub 2007 Jan 31.  [Content Brief]

  [2]. Didier C, et al. Evaluation of Polo-like Kinase 1 inhibition on the G2/M checkpoint in Acute Myelocytic Leukaemia. Eur J Pharmacol. 2008 Sep 4;591(1-3):102-5.  [Content Brief]

  [3]. Ikezoe T, et al. A novel treatment strategy targeting polo-like kinase 1 in hematological malignancies. Leukemia. 2009 Sep;23(9):1564-76.  [Content Brief]