Molecule

Description

Cancer

Description

WYE-687 is an ATP-competitive and selective inhibitor of mTOR with IC50 of 7 nM.

Targets

IC₅₀

In Vitro

Immune-complex kinase assay shows mTORC2-specific substrate His6-AKT or the mTORC1 substrate His6-S6K is dose dependently inhibited by WYE-687. WYE-687 globally inhibits mTOR signaling and AKT function in cellular models dose dependently. WYE-678 profoundly inhibits cap-dependent and global protein synthesis in MDA361, human breast cancer cells. WYE-687 shows antiproliferative effects in various cancer cell lines involving G1 cell cycle arrest and selective apoptosis. WYE-687 down-regulate angiogenic factors, VEGF and HIF-1α, in U87MG, MDA361 and LNCap cells. ^[1]

In Vivo

Clinical Trials

Features

DELFIA Format of Purified FLAG-TOR

The routine assays with purified FLAG-TOR (FL and 3.5) are performed in 96-well plates as follows. Enzymes are first diluted in kinase assay buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 50 mM β-glycerophosphate, 10 mM MnCl₂, 0.5 mM DTT, 0.25 lM microcystin LR, and 100 lg/mL BSA). To each well, 12 μL of the diluted enzyme is mixed briefly with 0.5 μL test inhibitor or control vehicle dimethyl sulfoxide (DMSO). The kinase reaction is initiated by adding 12.5 μL kinase assay buffer containing ATP and His6-S6K to give a final reaction volume of 25 μL containing 800 ng/mL FLAG-TOR, 100 μM ATP, and 1.25 μM His6-S6K. The reaction plate is incubated for 2 hours (linear at 1–6 hour) at room temperature with gentle shaking and then terminated by adding 25 μL Stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA).The DELFIA detection of the phosphorylated (Thr-389) His6-S6Kis performed at room temperature using a monoclonal anti-P(T389)- p70S6K antibody (1A5) labeled with Europium-N1- ITC (Eu) (10.4 Eu per antibody). Fortyfive microliters of the terminated kinase reaction mixture are transferred to a MaxiSorp plate containing 55 μL PBS. TheHis6-S6K is allowed to attach for 2 hour after which the wells are aspirated and washed once with PBS. One hundred microliters ofDELFIA buffer with 40 ng/mL Eu-P(T389)-S6K antibody is added.The antibody binding is continued for 1 hour with gentle agitation. Thewells are then aspirated and washed four times with PBS containing0.05% Tween 20 (PBST). One hundred microliters of DELFIAEnhancement solution is added to each well and the plates are read in a PerkinElmer Victor model plate reader. Data obtained are used to calculate enzymatic activity and enzyme inhibition by potential inhibitors.

Cell Lines

MDA361, U87MG, HCT116, LNCap and HT29 cells

Concentrations

0-100 μM

Incubation Time

24 or 48 hours

Methods

For cell cycle analysis, cells are seeded in 96-well culture plates at 10,000 cells per well for 24 hours, treated with various inhibitors for 24 hours or 48 hours. Following treatment, cells are harvested, washed with PBS and fixed overnight at -20 °C in 70% ethanol. Cells are washed, stained with propidium iodide and analyzed for cell cycle profile (acquired 5000 cells/well) on Guava PCA-96 instrument according to the Guava Cell Cycle Protocol.