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. 2009 Jun 12;421(1):29-42.
doi: 10.1042/BJ20090489.

Ku-0063794 is a specific inhibitor of the mammalian _target of rapamycin (mTOR)

Juan M García-Martínez  1 , Jennifer Moran, Rosemary G Clarke, Alex Gray, Sabina C Cosulich, Christine M Chresta, Dario R Alessi
Affiliations

Ku-0063794 is a specific inhibitor of the mammalian _target of rapamycin (mTOR)

Juan M García-Martínez et al. Biochem J. .

Abstract

mTOR (mammalian _target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of approximately 10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.

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Figures

Figure 1
Figure 1. Ku-0063794 inhibits both mTORC1 and mTORC2 complexes in vitro
(A) Structure of Ku-0063794. (B and C) HEK-293 cell extracts were subjected to immunoprecipitation (IP) with pre-immune (IgG), anti-Raptor (B) or anti-Rictor antibody (C). The immunoprecipitates were incubated with dephosphorylated GST–S6K1 (B) or GST–Akt1 (C) in the presence of the indicated concentrations of Ku-0063794 or vehicle (DMSO). Kinases assays were performed for 30 min in the presence of MgATP and then subjected to immunoblot analysis with the indicated antibodies. Similar results were obtained in three independent experiments. T389-P, phosphorylated Thr389; S473-P, phosphorylated Ser473.
Figure 2
Figure 2. Ku-0063794 inhibits mTORC1 activity in vivo
(A and B) HEK-293 cells cultured in the presence of 10% foetal bovine serum, were treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 (A) or with 1 μM Ku-0063794 for the indicated time (B). Cells were lysed and S6K1 immunoprecipitated and catalytic activity assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from three different samples, with each sample assayed in duplicate. Cell lysates were also analysed by immunoblotting with the indicated antibodies. (C) As in (A), except that cells were deprived of serum for 16 h prior to stimulation with 50 ng/ml of IGF-1 for 20 min. (D) As in (C), except that, after 16 h of serum deprivation, cells were incubated for 1 h in amino-acid-free EBSS (Earle's Balanced Salt Solution) medium containing 10% dialysed serum. Cells were then incubated in the absence or presence or the indicated amount of Ku-0063794 for 30 min prior to re-addition of physiological levels of amino acids for an additional 30 min. Immunoblots are representative of three different experiments.
Figure 3
Figure 3. Ku-0063794 ablates mTORC2 in vivo
HEK-293 cells were cultured in serum, and stimulated with IGF-1 in the absence or presence of Ku-0063794 as described in the legend to Figure 2. Akt1 was immunoprecipitated and catalytic activity assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from three different samples, with each sample assayed in duplicate. Cell lysates were analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. In (D), medium containing freshly dissolved Ku-0063794 was replaced every 12 h. P, phosphorylated.
Figure 4
Figure 4. Further investigation of the effect of Ku-0063794 on Akt Thr308 phosphorylation
(A) HEK-293 cells were deprived of serum for 16 h, treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 or PI-103, then stimulated with 50 ng/ml of IGF-1 for 10 min. Cell were lysed with 0.5 M trichloroacetic acid and amounts of PtdIns(3,4,5)P3 were determined. Each bar represents the means±S.E.M. from three independent samples. (B) HEK-293 cells were transfected with a DNA construct encoding wild-type or mutant GST–Akt1. At 24 h post-transfection, cells were deprived of serum for 16 h and stimulated with IGF-1 for 20 min, in the presence or absence of inhibitors as described in (A). Cell lysates were analysed by immunoblotting with the indicated antibodies and immunoblots shown are representative of three separate experiments. (C–E) The indicated wild-type (wt) or knockout (ko) MEFs were deprived and stimulated with IGF-1 for 20 min, in the presence or absence of inhibitors as described in (A). Cell lysates were analysed by immunoblotting with the indicated antibodies. Similar results were obtained in three independent experiments. P, phosphorylated.
Figure 5
Figure 5. Ku-0063794 suppresses hydrophobic motif phosphorylation and activation of SGK1 but not RSK
(A) HEK-293 cells were transfected with a DNA construct encoding GST–SGK1 (full-length enzyme). Cells were cultured in the presence of 10% foetal bovine serum in order to maintain PI3K pathway activity. At 36 h post-transfection, cells were treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 or PI-103. Cells were lysed, SGK1 was affinity-purified on glutathione–Sepharose and catalytic activity was assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from three different samples, with each sample assayed in duplicate. Affinity purified SGK1 was also subjected to immunoblotting with an anti-GST antibody (SGK1-Total) as well as an anti-Ser422 phosphospecific antibody (S422-P). Cell lysates were also analysed by immunoblotting with the indicated non-SGK antibodies. (B and C) HeLa cells or the indicated wild-type (wt) or knockout (ko) MEFs were cultured in the presence of 10% serum, then treated for 30 min in the absence or presence of the indicated concentrations of inhibitors. Cells were lysed and extracts were analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. (D) HEK-293 cells were deprived of serum for 16 h, treated for 30 min in the absence or presence of 1 μM Ku-0063794 or 0.2 μM PD 0325901 then stimulated with 400 ng/ml of PMA for 15 min. RSK was immunoprecipitated with an antibody recognizing all isoforms and catalytic activity assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from two different samples, with each sample assayed in duplicate. Cell lysates were also analysed by immunoblotting with the indicated antibodies. P, phosphorylated.
Figure 6
Figure 6. Ku-0063794 induces a marked dephosphorylation of 4E-BP1
(A and B) The indicated wild-type (wt) or knockout (ko) MEFs (A) or HEK-293 cells (B) were cultured in the presence of 10% serum, and treated for 30 min in the absence or presence of the indicated concentrations of inhibitors. Cells were lysed and extracts analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. P, phosphorylated.
Figure 7
Figure 7. Ku-0063794 inhibits cell growth and induces a G1 cell cycle arrest
The indicated wild-type (wt) or knockout (ko) MEFs were passaged at low density (20000 cells per 1.91 cm2 dish) and cells were cultured for 1, 2 or 3 days in the absence (DMSO) or presence of 3 μM Ku-0063794 or 100 nM rapamycin. (A and C) Cell numbers were measured in quadruplicate using a Crystal Violet staining as described in the Materials and methods section. The results are presented as means±S.E.M. of the percentage of cells relative to the DMSO control. Flow cytometric analysis was also undertaken as described in the Materials and methods section. (B and D) DNA was stained with propidium iodide, and cellular content was analysed. The percentage of cells in G1, S, or G2 phases were determined from triplicate dishes for each condition using CellQuest software. Two-way ANOVA and Bonferroni posttests were performed, *P<0.001 compared with DMSO control. #P<0.001 compared with rapamycin.
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