GSK 2830371

Kinase experiment:

The primary in vitro Wip1 enzymatic assay measured fluorescence generated by Wip-1 (2-420) hydrolysis of fluorescein diphosphate (FDP). 50 μM FDP substrate is added with GSK 2830371 or DMSO at room temperature before addition of 10 nM Wip1 in assay buffer (50 mM TRIS, pH 7.5, 30 mM MgCl2, 0.8 mM CHAPS, 0.05 mg/mL BSA). Fluorescent signal is detected on a Spectramax microplate reader (485/530 nm)[1].

Cell experiment:

Cells are seeded into 96 well plates at 200-400 cells per well and treated with GSK 2830371 dilution series on day 1. After 7 d, we used the CellTiter-Glo cell viability assay to determine effects on cell growth. Luminescent signal is detected on an EnVision 2104. For clonogenic assays, cells are seeded in 12-well tissue culture plates at 2,000 cells per well. Cells are treated with a compound dilution series on day 1 and again on day 7. After 14 d, cells are washed with 1× PBS, stained with 1 mL of Coomassie Brilliant Blue R-250, and colonies are quantitated with the Optomax Sorcerer colony counter[1].

Animal experiment:

Mice[1] Female SCID mice are subcutaneously inoculated with 1×107 DOHH2 cells and tumor growth is monitored with electronic calipers. When tumors reached 100-200 mm3, animals are treated orally twice (BID) or thrice (TID) daily with vehicle (2% DMSO and 40% Captisol, pH 4.0) or GSK 2830371. For efficacy studies, eight mice per group are administered with GSK 2830371 at 75 or 150 mg per kg body weight BID or 150 mg per kg body weight TID. Tumor growth inhibition (% difference in tumor growth compared to control) is calculated when tumors for vehicle-control mice exceeded 1,000 mm3 (day 11). For pharmacodynamic biomarker analysis, DOHH2 tumors from three mice are harvested 2 or 4 h after final dose following 14 d of treatment with either vehicle or GSK 2830371 at 75 or 150 mg per kg body weight, BID; tumors are frozen in liquid nitrogen for subsequent lysis and western blot analysis.

References:

[1]. Gilmartin AG, et al. Allosteric Wip1 phosphatase inhibition through flap-subdomain interaction. Nat Chem Biol. 2014 Mar;10(3):181-7.
[2]. Esfandiari A, et al. Chemical Inhibition of Wild-Type p53-Induced Phosphatase 1 (WIP1/PPM1D) by GSK2830371 Potentiates the Sensitivity to MDM2 Inhibitors in a p53-Dependent Manner. Mol Cancer Ther. 2016 Mar;15(3):379-91.