KU-0063794

For research use only.

Catalog No.S1226

65 publications

KU-0063794 Chemical Structure

CAS No. 938440-64-3

KU-0063794 is a potent and highly specific dual-mTOR inhibitor of mTORC1 and mTORC2 with IC50 of ~10 nM in cell-free assays; no effect on PI3Ks.

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Selleck's KU-0063794 has been cited by 65 publications

Purity & Quality Control

Choose Selective mTOR Inhibitors

Biological Activity

Description KU-0063794 is a potent and highly specific dual-mTOR inhibitor of mTORC1 and mTORC2 with IC50 of ~10 nM in cell-free assays; no effect on PI3Ks.
Targets
mTORC1 [1]
(Cell-free assay)
mTORC2 [1]
(Cell-free assay)
~10 nM ~10 nM
In vitro

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 ~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, GSK3α/GSK3β 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]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
HepG2  NIDXR21E\WyuIG\pZYJqdGm2eTDBd5NigQ>? MVKwMlHjiJN3MNMg{txO NGrtXGw4OiCq MnSw[IVkemWjc3XzJINmdGxidnnhZoltcXS7IHnuJIEh\G:|ZTDk[ZBmdmSnboSgcYFvdmW{ NFvnd3U9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{NkK3PFgyQSd-Mk[yO|g5OTl:L3G+
HepG2  M3rxR2NwdG:weTDGc5Ju[XSrb36gRZN{[Xl? MnXkNgKBmzVywrFOwG0> MWGxNEBl MYPk[YNz\WG|ZYOgeIhmKG63bXLldkBw\iC4aXHicIUhUGWyR{KgZ49td26rZYRCpJNq\26rZnnjZY51dHl? NFiwV5c9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{NkK3PFgyQSd-Mk[yO|g5OTl:L3G+
HepG2  M4rm[WFxd3C2b4Ppd{BCe3OjeR?= NVjCeFlHOC5z4pETOVDDqM7:TR?= NV7wTlBQPDhiaB?= NIPVcoVqdmS3Y3XzJIFxd3C2b4Ppd{BqdiCjIHTvd4Uh\GWyZX7k[Y51KG2jbn7ldi=> Mkj1QIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjZ{N{i4NVkoRjJ4Mke4PFE6RC:jPh?=
HepG2  M1PuRmZ2dmO2aX;uJGF{e2G7 MXK1M|ExKM7:TR?= NF;4TY8zPCCq NHfneGllemGvYYTpZ4FtdHliaX7obYJqfHNicHjvd5Bpd3K7bHH0bY9vKG:oIFHLWEBifCCVZYKtOFc{ MWW8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zPjJ5OEixPUc,OjZ{N{i4NVk9N2F-
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HepG3  M13vUWZ2dmO2aX;uJGF{e2G7 M4jyXlAvOeLCk{WwxsDPxE1? NIPPSZE1QCCq M2nod4lv\HWlZYOgZ4VtdCCjdYTvdIhi\3liaX6gZUBld3OnIHTldIVv\GWwdDDtZY5v\XJ? MkLQQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjZ{N{i4NVkoRjJ4Mke4PFE6RC:jPh?=
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H1650 M1fUUWZ2dmO2aX;uJGF{e2G7 M3vVXVcvPjFibl2= MVW3NkBp NFrJRodqdmirYnn0d{BuXE:UIIDoc5NxcG:{eXzheIlwdiC|dHH0eZPDqA>? MkDlQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjN6N{S4PFAoRjJ|OEe0PFgxRC:jPh?=
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PC9GR M{\XbGZ2dmO2aX;uJGF{e2G7 NGXkRoc3NjJzIH7N NGjLfJA4OiCq MV\pcohq[mm2czDwbI9{eGixconsZZRqd25ib3[gdFcxWz[N NHXIWoE9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{M{i3OFg5OCd-MkO4O|Q5QDB:L3G+
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PC-3 M2\uNWNmdGxiVnnhZoltcXS7IFHzd4F6 M2DCfVAuOTBizszN NYC0e5pIOjRiaNMg MUHk[YNz\WG|ZYOgZ4VtdCC4aXHibYxqfHliaX6gZUBld3OnIHTldIVv\GWwdDDtZY5v\XJ? MWe8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOzh2ME[wOUc,OjN6NEC2NFU9N2F-
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PC-3 NGTCcZpHfW6ldHnvckBCe3OjeR?= NX\Ocos1OjBy4pETPFAxKG6P NHLhdFkzPCCqwrC= MWLk[YNz\WG|ZYOgeIhmKHCqb4PwbI9zgWyjdHnvckBt\X[nbDDv[kBxPzCVNlugbY4h[SCmb4PlJIRmeGWwZHXueEBu[W6wZYK= NICx[Io9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{M{i0NFYxPSd-MkO4OFA3ODV:L3G+
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Caki-1  MmGwSpVv[3Srb36gRZN{[Xl? NFniXHQyODBvMkCwNEBvVQ>? NHm3clEyOC1zOECgcYlv NUOwbG9JTE2VTx?= NYnCUXJxcW6qaXLpeJMh[m:2aDDtWG9TSzFiYX7kJI1VV1KFMjDhd{BqdmSrY3H0[YQh[nlidHjlJIRm[3KnYYPlJIlvKHCqb4PwbI9zgWyjdHnvckBw\iCmb4fud5Rz\WGvIHXm[oVkfG:{cx?= MWS8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOzN2OUm4PUc,OjN|NEm5PFk9N2F-
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... Click to View More Cell Line Experimental Data

Assay
Methods Test Index PMID
Western blot
p-S6K / S6K / p-4E-BP1 / E7 / E6 / p53 ; 

PubMed: 28115701     


Normoxic HPV-positive cancer cells were treated for 24 h with 0.5, 1.0, or 5 μM KU-0063794 (KU) or 50 nM rapamycin. Shown are immunoblots of P-S6K, S6K, P-4E-BP1, 4E-BP1, HPV16/18 E6/E7, and p53 protein levels. DMSO served as a solvent control; β-actin, as a loading control.

p-mTOR; 

PubMed: 24262658     


Inhibition of mTOR pathway by mTOR kinase inhibitors. MCC-2 cells were treated with WYE354 (3 μM), PP242 (2.5 μM), and Ku-0063794 (5 μM) for 24h, respectively. Lysates were prepared and subjected to immunoblotting analysis with indicated antibodies. 

28115701 24262658
In vivo Ku0063794 inhibits tumor growth and mTOR signaling in a preclinical renal cell carcinoma model. However, Ku0063794 was not more effective than temsirolimus in the animal study. A possible explanation for lack of greater activity in vivo for Ku0063794 is that temsirolimus has important effects on the tumor microenvironment. Temsirolimus decreased angiogenesis in the xenograft tumors while Ku0063794 did not. Temsirolimus treated tumors expressed less VEGF and PDGF than Ku0063794 treated tumors, thus stimulating less angiogenesis[2].

Protocol

Kinase Assay:

[1]

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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.
Cell Research:

[1]

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  • Cell lines: Wild-type and mLST8 deficient MEFs
  • Concentrations: Dissolved in DMSO, final concentration ~3 μM
  • Incubation Time: 24, 48, and 72 hours
  • Method:

    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)
Animal Research:

[2]

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  • Animal Models: Nu/Nu nude mice
  • Dosages: 8 mg/kg
  • Administration: i.p.
    (Only for Reference)

Solubility (25°C)

In vitro DMSO 16 mg/mL (34.36 mM)
Water Insoluble
Ethanol Insoluble
In vivo Add solvents to the product individually and in order(Data is from Selleck tests instead of citations):
30% PEG400+0.5% Tween80+5% propylene glycol
For best results, use promptly after mixing.
30 mg/mL

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information

Molecular Weight 465.54
Formula

C25H31N5O4

CAS No. 938440-64-3
Storage powder
in solvent
Synonyms N/A
Smiles CC1CN(CC(O1)C)C2=NC3=C(C=CC(=N3)C4=CC(=C(C=C4)OC)CO)C(=N2)N5CCOCC5

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
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Step 2: Enter the in vivo formulation ()
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Molarity Calculator

Molarity Calculator

Calculate the mass, volume or concentration required for a solution. The Selleck molarity calculator is based on the following equation:

Mass (mg) = Concentration (mM) × Volume (mL) × Molecular Weight (g/mol)

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*When preparing stock solutions, please always use the batch-specific molecular weight of the product found on the via label and MSDS / COA (available on product pages).

Dilution Calculator

Dilution Calculator

Calculate the dilution required to prepare a stock solution. The Selleck dilution calculator is based on the following equation:

Concentration (start) x Volume (start) = Concentration (final) x Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2 ( Input Output )

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* When preparing stock solutions always use the batch-specific molecular weight of the product found on the vial label and MSDS / COA (available online).

The Serial Dilution Calculator Equation

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  • Computed Result

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Molarity Calculator

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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID