WYE-687

MTOR

528, 61

0-100 uM

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-1alpha, in U87MG, MDA361 and LNCap cells. [1]

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 beta-glycerophosphate, 10 mM MnCl2, 0.5 mM DTT, 0.25 lM microcystin LR, and 100 lg/mL BSA). To each well, 12 uL of the diluted enzyme is mixed briefly with 0.5 uL test inhibitor or control vehicle dimethyl sulfoxide (DMSO). The kinase reaction is initiated by adding 12.5 uL kinase assay buffer containing ATP and His6-S6K to give a final reaction volume of 25 uL containing 800 ng/mL FLAG-TOR, 100 uM ATP, and 1.25 uM 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 uL Stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA). The DELFIA detection of the phosphorylated (Thr-389) His6-S6K is performed at room temperature using a monoclonal anti-P(T389)- p70S6K antibody (1A5) labeled witEuropium-N1- ITC (Eu) (10.4 Eu per antibody). Fortyfive microliters of the terminated kinase reaction mixture are transferred to a MaxiSorp plate containing 55 uL PBS. The His6-S6K is allowed to attach for 2 hour after which the wells are aspirated and washed once with PBS. One hundred microliters of DELFIA buffer with 40 ng/mL Eu-P(T389)-S6K antibody is added. The antibody binding is continued for 1 hour with gentle agitation. The wells are then aspirated and washed four times with PBS containing 0.05% Tween 20 (PBST). One hundred microliters of DELFIA Enhancement 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.

DMSO 0.5 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL

methyl 4-(4-morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3, 4-d]pyrimidin-6-yl)phenylcarbamate