KU-0063794 is a potent and highly specific dual-mTOR inhibitor of mTORC1 and mTORC2.

mTORC2:10 nM, mTORC1:10 nM

KU-0063794但不是雷帕霉素以剂量依赖性方式抑制SGK1活性和Ser422磷酸化以及其生理学底物NDGR1,与S6K1和Akt磷酸化程度相同,而KU-0063794不抑制佛波酯诱导的ERK或RSK磷酸化和RSK激活。与雷帕霉素相比,KU-0063794在Thr37,Thr46和Ser65诱导4E-BP1完全去磷酸化方面表现出更显著的效力。KU-0063794抑制野生型和mLST8-缺陷型MEFs细胞生长,也诱导细胞周期停在G1期,比雷帕霉素效果更明显。与mTOR抑制剂PP242相比,KU-0063794对mTOR表现出更高的特异性,因为对PI3K或其他76种激酶无效。在HEK-293细胞中,30 nM的KU-0063794足以通过阻断疏水基序（Thr389）的磷酸化和随后T环残基（Thr229）的磷酸化来快速消除S6K1活性。100-300 nM的KU-0063794也完全抑制氨基酸诱导的S6K1和S6蛋白的磷酸化。类似于S6K1,KU-0063794以剂量依赖性和时间依赖性方式抑制Ser2448处的mTORC1和Ser2481处的mTORC2的磷酸化。

mTOR complexes kinase assays: HEK-293 cells are freshly lysed in Hepes lysis buffer. Lysate (1-4 mg) is pre-cleared by incubating with 5-20 μL of Protein G-Sepharose conjugated to pre-immune IgG. The lysate extracts are then incubated with 5-20 μL of Protein G-Sepharose conjugated to 5-20 μg of either anti-Rictor or anti-Raptor antibody, or pre-immune IgG. All antibodies are covalently conjugated to Protein G-Sepharose. Immunoprecipitations are carried out for 1 hour at 4 °C on a vibrating platform. The immunoprecipitates are washed four times with Hepes lysis buffer, followed by two washes with Hepes kinase buffer. For Raptor immunoprecipitates used for phosphorylating S6K1, for the initial two wash steps the buffer includes 0.5 M NaCl to ensure optimal kinase activity. GST-Akt1 is isolated from serum-deprived HEK-293 cells incubated with PI-103 (1 μM for 1 hour). GST-S6K1 is purified from serum-deprived HEK-293 cells incubated with rapamycin (0.1 μM for 1 hour). mTOR reactions are initiated by adding 0.1 mM ATP and 10 mM MgCl2 in the presence of various concentrations of KU-0063794 and GST-Akt1 (0.5 μg) or GST-S6K1 (0.5 μg). Reaction are carried out for 30 minutes at 30 °C on a vibrating platform and stopped by addition of SDS sample buffer. Reaction mixtures are then filtered through a 0.22-μm-poresize Spin-X filter and samples are subjected to electrophoresis and immunoblot analysis with the indicated antibodies.

Cells are treated with KU-0063794 for 24, 48, and 72 hours, and the medium is changed every 24 hours with freshly dissolved KU-0063794. For the measurement of cell growth, cells are washed once with PBS, and fixed in 4% (v/v) paraformaldehyde in PBS for 15 minutes. After washing once with water, the cells are stained with 0.1% Crystal Violet in 10% ethanol for 20 minutes and washed three times with water. Crystal Violet is extracted from cells with 0.5 mL of 10% (v/v) ethanoic (acetic) acid for 20 minutes. The eluate is then diluted 1:10 in water and absorbance at 590 nm is quantified. For the assessment of cell cycle distribution, cells are harvested by trypsinization, washed once in PBS, and re-suspended in ice-cold aq. 70% (v/v) ethanol. Cells are washed twice in PBS plus 1% (w/v) BSA and stained for 20 minutes in PBS plus 0.1% (v/v) Triton X-100 containing 50 g/mL propidium iodide and 50 g/mL RNase A. The DNA content of cells is determined using a FACSCalibur flow cytometer and CellQuest software. Red fluorescence (585 nm) is acquired on a linear scale, and pulse width analysis is used to exclude doublets. Cell-cycle distribution is determined using FlowJo software.(Only for Reference)