In Vitro

Kinase Assay: In vitro mTOR (FRAP1) kinase assay: Recombinant mTOR is incubated with PP242 at 2-fold dilutions over a concentration range of 50-0.001 μM in an assay containing 50 mM HEPES, pH 7.5, 1 mM EGTA, 10 mM MgCl2, 0.01% Tween, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Rat recombinant PHAS-1/4EBP1 (2 mg/mL) is used as a substrate. Reactions are terminated by spotting onto nitrocellulose, which is washed with 1 M NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging. IC50 value is calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package.

Cell Assay: Cells (MEFs) are treated with increasing concentrations of PP242 for 72 hours in 96-well plates. After 72 hours of treatment, 10 μL of 440 μM resazurin sodium salt is added to each well, and after 18 hours, the florescence intensity in each well is measured using a top-reading florescent plate reader with excitation at 530 nm and emission at 590 nm.

PP242 exhibits potent selectivity for mTOR over other PI3K family kinases such as p110α, p110β, p110γ, p110δ, and DNA-PK with IC50 of 1.96 μM, 2.2 μM, 1.27 μM, 0.102 μM, and 0.408 μM, respectively. PP242 displays some inhibitory activity against Ret, PKCα, PKCβ, and JAK2, while exhibits remarkable selectivity against 215 other protein kinases. Unlike rapamycin, PP242 inhibits both mTORC1 and mTORC2. In BT549 cells, PP242 treatment (0.04-10 μM) inhibits the phosphorylation of Akt, the mTOR substrate p70S6K, and its downstream target S6 in a dose-dependent manner. PP242 potently inhibits PKCα with IC50 of 49 nM. Low concentrations of PP242 inhibit the phosphorylation of Akt S473 and higher concentrations partially inhibit Akt T308-P in addition to S473-P. As PP242 is a more effective mTORC1 inhibitor than rapamycin, PP242 inhibits the proliferation of primary MEFs, and the phosphorylation of 4EBP1 at T36/45 and S65, more potently than rapamycin. PP242 but not rapamycin potently inhibits cap-dependent translation, by causing a higher level of binding between 4EBP1 and eIF4E than rapamycin. PP242 potently inhibits the proliferation of p190-transformed murine BM, SUP-B15, and K562 cells with GI50 of 12 nM, 90 nM, and 85 nM, respectively. PP242 also inhibits the growth of solid tumor cell lines such as SKOV3, PC3, 786-O, and U87 with GI50 of 0.49 μM, 0.19 μM, 2.13 μM, and 1.57 μM, respectively. PP242 is also more effective than rapamycin in achieving cytoreduction and apoptosis in multiple myeloma (MM) cells.