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 used. PI3K isoforms are 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, 2% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3β at 60 ng/mL; PI3Kγ at 8 ng/mL; PI3Kδ at 45 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. IC₅₀s are calculated from the fit of the dose?response curves to a 4-parameter equation^[1].

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

Mice^[1]
Human prostate cancer PC3 cells are resuspended in Hank’s Balanced Salt Solution and 3×10⁶ cells implanted subcutaneously into the right hind flank of athymic nu/nu (nude) mice. Tumors are monitored until they reached a mean tumor volume of 150-200 mm³ prior to the initiation of dosing. MCF7.1 cells resuspended in a 1:1 mixture of Hank’s Buffered Salt Solution and Matrigel Basement Membrane Matrix, were 5×10⁶ subcutaneously implanted into the right hind flank of athymic nu/nu (nude) mice. Prior to cell inoculation, 17 β-estradiol (0.36 mg/pellet, 60-day release) are implanted into the dorsal shoulder blade area of each nude mouse. After implantation of cells, tumors are monitored until they reached a mean tumor volume of 250-350 mm³ prior to initiating dosing. Female nude (nu/nu) mice that are 6-8 weeks old and weighed 20-30 g are used. Tumor bearing mice are dosed orally daily with 10 mg/kg of GNE-493 for 14 continuous days. Tumor volume is measured in two dimensions (length and width) and is analyzed using Excel version 11.2. Animal body weights are measured. Tumor sizes are recorded twice weekly over the course of the study (14-21 days)

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

• [1]. Sutherlin DP, et al. Discovery of (thienopyrimidin-2-yl)aminopyrimidines as potent, selective, and orally available pan-PI3-kinase and dual pan-PI3-kinase/mTOR inhibitors for the treatment of cancer. J Med Chem. 2010 Feb 11;53(3):1086-97.  [Content Brief]