| In Vitro | In vitro activity: GDC-0879 also inhibits cellular pERK with IC50 of 63 nM. GDC-0879 shows comparable potency in A375 melanoma and Colo205 colorectal carcinoma cell lines, both of which are B-RafV600E mutant, with IC50 of 59 nM and 29 nM for pMEK1 inhibition respectively. GDC-0879 potently inhibits B-RafV600E in Malme3M cells with IC50 of 0.75 μM. GDC-0879 also shows EC50 values < 0.5 μM in many tumor cells (A375, 624, SK-MEL-28, Malme3M, C32, 928, 888, G-361, Colo205, Colo206, SW1417, CL34, and Colo201).
Kinase Assay: GDC-0879 is a potent and selective B-Raf inhibitor with an IC50 of 0.13 nM.
Cell Assay: GDC-0879 in vitro IC50 estimates for pMEK inhibition are determined using A375 and Colo205 cells. In brief, A375 or Colo205 cells are incubated with a range of GDC-0879 concentrations (from 0.5 nM to 6.75 μM) for 25 min. Cells are lysed, and the lysates are subjected to centrifugation at 16,100g for 30 min, and the level of total protein is determined. Enzyme-linked immunosorbent assay kits are used to determine pMEK1 and total MEK1 protein levels in a 96-well format. Samples are analyzed in duplicate at 20 μg of protein per well. The optical densities obtained at 450 nm are converted to units per milliliter (for pMEK1) or nanograms per milliliter (for total MEK1) using a standard curve determined with recombinant pMEK1 or MEK1. The pMEK1/total MEK1 ratios are then calculated as units per nanogram. The IC50 estimates for pMEK1 inhibition are estimated by nonlinear regression using GraphPad Prism version 4.02. |
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