WAY-600 is a potent, ATP-competitive and selective inhibitor of mTOR with IC50 of 9 nM; blocks mTORC1/P-S6K(T389) and mTORC2/P-AKT(S473) but not P-AKT(T308); selective for mTOR than PI3Kα (>100-fold) and PI3Kγ (>500-fold).

mTOR:9 nM

免疫复合物激酶分析表明，mTORC2特异性底物His6-AKT或mTORC1底物His6-S6K被WAY-600剂量依赖性抑制。WAY-600在细胞模型中剂量依赖性全面抑制mTOR信号通路和AKT功能。WAY-600在MDA361人类乳腺癌细胞中显著抑制帽依赖型和全局蛋白质合成。WAY-600在多种癌细胞系中展现出抗增殖效果，涉及G1细胞周期阻滞和选择性凋亡。WAY-600在U87 mg、MDA361和LNCap细胞中下调血管生成因子VEGF和HIF-1α。[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 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-S6K is 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. 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.

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(Only for Reference)