Taselisib (GDC-0032) is an orally bioavailable inhibitor of the class I phosphatidylinositol 3-kinase (PI3K) alpha isoform (PIK3CA), with potential antineoplastic activity.

Characterization of Biochemical and Cellular Activity in Vitro: Enzymatic activity of the class I PI3K isoforms is measured using a fluorescence polarization assay that monitors formation of the product 3,4,5-inositoltriphosphate molecule as it competes with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidyl inositide-3-phosphate product results in a decrease in fluorescence polarization signal as the labeled fluorophore is displaced from the GRP-1 protein binding site. Class I PI3K isoforms are expressed and purified as heterodimeric recombinant proteins. Tetramethylrhodamine-labeled PIP3 (TAMRA-PIP3), di-C8-PIP2, and PIP3 detection reagents are purchased from Echelon Biosciences. PI3Kα is assayed under initial rate conditions in the presence of 10 mM Tris (pH 7.5), 25 μM ATP, 9.75 μM PIP2, 5% glycerol, 4 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 2% (v/v) DMSO at 60 ng/mL. After assay for 30 min at 25 °C, reactions are terminated with a final concentration of 9 mM EDTA, 4.5 nM TAMRA-PIP3, and 4.2 μg/mL GRP-1 detector protein before reading fluorescence polarization on an Envision plate reader. IC50 values are calculated from the fit of the dose-response curves to a 4-parameter equation. Each reported value is an average of three experiments, and all have a standard deviation less than one geometric mean.

GDC-0032 is dissolved in DMSO. Cells are seeded in replicates of 6 in 96-well plates with 500 to 5,000 cells/well overnight and then treated with GDC-0032. After 4 days, the media are removed and the cells are fixed with 4% glutaraldehyde for 30 minutes. Fixed cells are stained with 0.1% crystal violet for 2 minutes, then washed, and dissolved in 10% acetic acid.