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| 25mg |
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Purity: ≥98%
KU-0063794 is a novel, potent, cell permeable and selective inhibitor of mTOR (mammalian target of rapamycin) with potential anticancer activity. With an IC50 of less than 10 nM, it inhibits both mTORC1 and mTORC2. 76 additional protein kinases and seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations, are not inhibited by it. Additionally, Ku-0063794 prevented PDK1 from phosphorylating Akt's T-loop Thr308 residue. Additionally, Ku-0063794 inhibited cell growth and caused a G1 cell cycle arrest. Determining the physiological functions of mTOR will be helpful, and Ku-0063794 may be helpful in treating cancers where this pathway is unnecessarily activated.
| Targets |
mTORC1 (IC50 = 10 nM); mTORC2 (IC50 = 10 nM)
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| ln 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). t takes 300 nM of KU-0063794 to completely inhibit the S6K1 activity in serum-starved HEK-293 cells after IGF1 stimulation. Additionally, S6K1 and S6 protein phosphorylation brought on by amino acids is completely inhibited by KU-0063794 at concentrations of 100–300 nM. Similar to S6K1, KU-0063794 blocks mTORC1 at Ser2448 and mTORC2 at Ser2481 in a dose- and time-dependent manner. KU-0063794 causes 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, GSK3/GSK3 at Ser21/Ser9, and Foxo-1/3a at Thr24/Thr32 in the presence of serum or after IGF1 stimulation. In a dose-dependent manner, KU-0063794 but not rapamycin inhibits SGK1 activity and Ser422 phosphorylation as well as its physiological substrate NDGR1 to the same extent as S6K1 and Akt phosphorylation. However, KU-0063794 does not inhibit phorbol ester-induced ERK or RSK phosphorylation or RSK activation. KU-0063794 has a significantly higher potency to cause the complete dephosphorylation of 4E-BP1 at Thr37, Thr46, and Ser65 when compared to rapamycin. More significantly than rapamycin, KU-0063794 inhibits cell growth in MEFs with wild-type and mLST8-deficient mutations and causes a G1 cell cycle arrest. [1]
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| ln Vivo |
Ku0063794 inhibits tumor growth and mTOR signaling in a preclinical renal cell carcinoma model. In the animal study, Ku0063794 was not, however, more efficient than temsirolimus. The fact that temsirolimus has significant effects on the tumor microenvironment may be the cause of Ku0063794's lack of increased activity in vivo. While Ku0063794 had no effect on the xenograft tumors' angiogenesis, temsirolimus did. Tumors treated with temsirolimus expressed lower levels of VEGF and PDGF than tumors treated with Ku0063794 did, which reduced angiogenesis[2].
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| Enzyme Assay |
HEK-293 cells are freshly lysed in Hepes lysis buffer. By incubating lysate (1-4 mg) with 5-20 L of Protein G-Sepharose conjugated to pre-immune IgG, lysate is pre-cleared. After that, 5–20 L of Protein G–Sepharose coupled to 5–20 g of either anti–Rictor or anti–Raptor antibody, or pre–immune IgG, is incubated with the lysate extracts. Protein G-Sepharose is covalently coupled to every antibody. Immunoprecipitations are performed on a vibrating platform for 1 hour at 4 °C. Hepes lysis buffer is used to wash the immunoprecipitates four times, then Hepes kinase buffer is used to wash them twice. To ensure optimal kinase activity, the buffer for the first two wash steps of Raptor immunoprecipitates used for phosphorylating S6K1 contains 0.5 M NaCl. GST-Akt1 is purified from serum-starved HEK-293 cells after an hour of PI-103 (1 μM for 1 hour) incubation. GST-S6K1 is isolated from HEK-293 cells depleted of serum and rapamycin (0.1 μM for 1 hour). In order to start mTOR reactions, 0.1 mM ATP and 10 mM MgCl2 are added while various concentrations of KU-0063794 and GST-Akt1 (0.5 μg) or GST-S6K1 (0.5 μg) are also present. The addition of SDS sample buffer stops the reaction after it has been running for 30 minutes at 30 °C on a vibrating platform. After that, reaction mixtures are filtered through a Spin-X filter with a 0.22-m pore size, and samples are put through electrophoresis and an immunoblot analysis using the designated antibodies.
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| Cell Assay |
Every 24 hours, a new batch of freshly dissolved KU-0063794 is added to the medium as the cells are treated with it for 24, 48, and 72 hours. Cells are fixed in 4% (v/v) paraformaldehyde in PBS for 15 minutes in order to measure cell growth. The cells are washed once with water, stained for 20 minutes with 0.1% Crystal Violet in 10% ethanol, and then washed three times with water. Crystal Violet is extracted from cells using 0.5 mL of 10% (v/v) ethanoic (acetic) acid for 20 minutes. Then, the eluate is diluted 1:10 in water, and the absorbance at 590 nm is measured.
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| Animal Protocol |
Nu/Nu nude mice
8 mg/kg i.p. |
| References |
| Molecular Formula |
C25H31N5O4
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|---|---|
| Molecular Weight |
465.5447
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| Exact Mass |
465.5447
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| Elemental Analysis |
C, 64.50; H, 6.71; N, 15.04; O, 13.75
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| CAS # |
938440-64-3
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| Related CAS # |
938440-64-3
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| Appearance |
Solid powder
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| SMILES |
C[C@@H]1CN(C[C@@H](O1)C)C2=NC3=C(C=CC(=N3)C4=CC(=C(C=C4)OC)CO)C(=N2)N5CCOCC5
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| InChi Key |
RFSMUFRPPYDYRD-CALCHBBNSA-N
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| InChi Code |
InChI=1S/C25H31N5O4/c1-16-13-30(14-17(2)34-16)25-27-23-20(24(28-25)29-8-10-33-11-9-29)5-6-21(26-23)18-4-7-22(32-3)19(12-18)15-31/h4-7,12,16-17,31H,8-11,13-15H2,1-3H3/t16-,17+
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| Chemical Name |
[5-[2-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-4-morpholin-4-ylpyrido[2,3-d]pyrimidin-7-yl]-2-methoxyphenyl]methanol
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| Synonyms |
Ku0063794; Ku 0063794; KU0063794; Ku-0063794; KU-0063794; KU 0063794; KU 63794; KU-63794; KU63794
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~16 mg/mL (~34.4 mM)
Water: <1 mg/mL Ethanol: <1 mg/mL |
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| Solubility (In Vivo) |
30% PEG400+0.5% Tween80+5% propylene glycol: 13mg/mL (Please use freshly prepared in vivo formulations for optimal results.)
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1480 mL | 10.7402 mL | 21.4804 mL | |
| 5 mM | 0.4296 mL | 2.1480 mL | 4.2961 mL | |
| 10 mM | 0.2148 mL | 1.0740 mL | 2.1480 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Ku-0063794 inhibits mTORC1 activity in vivo.Biochem J.2009 Jun 12;421(1):29-42. |
Ku-0063794 inhibits both mTORC1 and mTORC2 complexesin vitro.Biochem J.2009 Jun 12;421(1):29-42. |
Ku-0063794 ablates mTORC2in vivo.Biochem J.2009 Jun 12;421(1):29-42. |
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