KU-0063794

(5-(2-((2R, 6S)-2, 6-dimethylmorpholino)-4-morpholinopyrido[2, 3-d]pyrimidin-7-yl)-2-methoxyphenyl)methanol

KU-0063794 is a potent and highly specific mTOR inhibitor for both mTORC1 and mTORC2 with IC50 ca.10 nM.

Compared with the mTOR inhibitor PP242, KU-0063794 exhibits higher specificity for mTOR, as being inactive against PI3Ks or 76 other kinases. In HEK-293 cells, KU-0063794 at 30 nM is sufficient to rapidly ablate S6K1 activity by blocking the phosphorylation of the hydrophobic motif (Thr389) and subsequently the phosphorylation of the T-loop residue (Thr229). In case of IGF1 stimulation of serum-starved HEK-293 cells, 300 nM of KU-0063794 is needed to inhibit the S6K1 activity by ca.90%. KU-0063794 at 100-300 nM also completely inhibits the amino-acid-induced phosphorylation of S6K1 and S6 protein. Similar to S6K1, KU-0063794 inhibits the phosphorylation of mTORC1 at Ser2448 and mTORC2 at Ser2481 in a dose-dependent and time-dependent manner. In the presence of serum or following IGF1 stimulation, KU-0063794 induces a dose-dependent inhibition of the activity and phosphorylation of Akt at Ser473 and unexpected Thr308 as well as the phosphorylation of the Akt substrates PRAS40 at Thr246, GSK3alpha/GSK3beta at Ser21/Ser9 and Foxo-1/3a at Thr24/Thr32. KU-0063794 but not rapamycin inhibits SGK1 activity and Ser422 phosphorylation as well as its physiological substrate NDGR1 in a dose-dependent manner, to the same extent as S6K1 and Akt phosphorylation, whereas KU-0063794 dose not inhibit phorbol ester induced ERK or RSK phosphorylation and RSK activation. Compared with rapamycin, KU-0063794 exhibits more significant potency to induce the complete dephosphorylation of 4E-BP1 at Thr37, Thr46 and Ser65. KU-0063794 inhibits cell growth of both wild-type and mLST8-deficient MEFs and induces a G1 cell cycle arrest, more significantly than rapamycin. [1]

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 uL of Protein G-Sepharose conjugated to pre-immune IgG. The lysate extracts are then incubated with 5-20 uL of Protein G-Sepharose conjugated to 5-20 ug 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 uM for 1 hour). GST-S6K1 is purified from serum-deprived HEK-293 cells incubated with rapamycin (0.1 uM 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 ug) or GST-S6K1 (0.5 ug). 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-um-poresize Spin-X filter and samples are subjected to electrophoresis and immunoblot analysis with the indicated antibodies.

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, , Dr. Yong-Weon Yi from Georgetown University Medical Center, Ku-0063794purchased from Selleck, For MTT assays, cells (2, 000 ca. 5, 000 cells/well) were subcultured into 96-well plates according to their growth properties. Cell proliferation was assayed at 72 hr after treatment of KU-0063794 by adding 20 l of 5 mg/ml 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) solution per 100 l of growth medium. After incubating for 3-4 h at 37C, the media were removed and 150 l/well of MTT solvent (either absolute DMSO or isopropanol containing 4 M HCl and 0.1% Nonidet-40) was added to dissolve the formazan. The absorbance of each well was measured by ELx808 (BioTek, Winooski, VT) or Wallac Victor2 (Perkin-Elmer Life Sciences, Boston, MA) Microplate Reader. Viable cells are presented as percent of control, vehicle-treated cells.

DMSO 16 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL