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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 |
Specificity kinase panel[1]
All assays were performed at The National Centre for Protein Kinase Profiling (http://www.kinase-screen.mrc.ac.uk/) as previously described [34]. Briefly, all assays were carried out robotically at room temperature (21 °C) and were linear with respect to time and enzyme concentration under the conditions used. Assays were performed for 30 min using Multidrop Micro reagent dispensers (Thermo Electron Corporation, Waltham, MA, U.S.A.) in a 96-well format. The abbreviations for each kinase are defined in legend to Table 1. The concentration of magnesium acetate in the assays was 10 mM and [γ-33P]ATP (∼800 c.p.m./pmol) was used at 5 μM for CK2α, DYRK3, EF2K, ERK1, ERK8, GSK3β, HIPK2, IGF1R, IRR, MARK3, MKK1, p38γ MAPK (mitogen-activated protein kinase), p38δ MAPK, PAK4, PIM2, Akt1, PLK1, PKCζ and PRK2; 20 μM for CaMKKβ, CDK2/cyclin A, CHK1, CHK2, CK1δ, CSK, EPH-B3, FGF-R1, IR, JNK1α1, JNK2α2, MAPKAP-K2, MSK1, MST2, MST4, p38β MAPK, PKA, PAK5, PAK6, PDK1, PIM1, PIM3, PKCα, ROCKII, PRAK, S6K1, SGK1, SYK, VEGFR and YES1; or 50 μM for AMPK, BRSK2, BTK, CaMK1, DYRK1a, DYRK2, EPH-A2, ERK2, IKKε, LCK, MELK, NEK2A, NEK6, p38α, PhKγ1, Akt2, PKD1, RSK1, RSK2, SRPK1 and TBK1, in order to be at or below the Km for ATP for each enzyme. Lipid kinase panel[1] SPHK1 (sphingosine kinase 1) was assayed as follows: SPHK1 [diluted in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM EGTA and 1 mM DTT (dithiothreitol)] was assayed against sphingosine in a final volume of 50 μl containing 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 10 μM sphingosine, 10 μM ATP and 1 mM DTT and incubated for 30 min at room temperature. SPHK2 was assayed as follows: SPHK2 (diluted in 50 mM Tris/HCl, pH 7.5, 200 mM KCl, 5 mM MgCl2, 1 mM EGTA and 1 mM DTT) was assayed against sphingosine in a final volume of 50 μl containing 50 mM Tris/HCl, pH 7.5, 200 mM KCl, 5 mM MgCl2, 1 mM EGTA, 10 μM sphingosine, 1 μM ATP and 1 mM DTT and incubated for 30 min at room temperature. View More
Choline kinase was assayed as follows: choline kinase (diluted in 25 mM glycine/NaOH, pH 8.5, 67 mM KCl and 5 mM MgCl2) was assayed against choline in a final volume of 50 μl containing 25 mM glycine/NaOH, pH 8.5, 67 mM KCl, 5 mM MgCl2, 1 mM choline, 1 μM ATP and 1 mM DTT and incubated for 30 min at room temperature. These three assays were stopped by addition of 50 μl Kinase Glo Plus Reagent, incubated for 10 min at room temperature and read for 1 s/well. mTOR complexes kinase assays[1] HEK-293 cells were freshly lysed in Hepes lysis buffer. Lysate (1–4 mg) was pre-cleared by incubating with 5–20 μl of Protein G–Sepharose conjugated to pre-immune IgG. The lysate extracts were 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 were covalently conjugated to Protein G–Sepharose. Immunoprecipitations were carried out for 1 h at 4 °C on a vibrating platform. The immunoprecipitates were 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 included 0.5 M NaCl to ensure optimal kinase activity [7]. GST–Akt1 was isolated from serum-deprived HEK-293 cells incubated with PI-103 (1 μM for 1 h) [24]. GST–S6K1 was purified from serum-deprived HEK-293 cells incubated with rapamycin (0.1 μM for 1 h) [25]. mTOR reactions were initiated by adding 0.1 mM ATP and 10 mM MgCl2 in the presence or absence of Ku-0063794 and GST–Akt1 (0.5 μg) or GST–S6K1 (0.5 μg). Reaction were carried out for 30 min at 30 °C on a vibrating platform and stopped by addition of SDS sample buffer. Reaction mixtures were then filtered through a 0.22-μm-pore-size Spin-X filter and samples were subjected to electrophoresis and immunoblot analysis. Kinase assays[1] HEK-293 were lysed in Tris lysis buffer. In order to perform Akt and S6K assays, 500 μg of lysate was incubated with 5 μg of the corresponding antibody conjugated to Protein G–Sepharose. To perform SGK1 activity assays, 50 μg of transfected lysate was incubated with 5 μg of glutathione–Sepharose. All the incubations were performed for 1 h at 4 °C on a vibrating platform. Kinase activity was assayed exactly as described previously [35] using the Crosstide peptide (GRPRTSSFAEG) at 30 μM. Incorporation of [32P]phosphate into the peptide substrate was determined by applying the reaction mixture to P81 phosphocellulose paper and liquid-scintillation counting of radioactivity after washing the papers in phosphoric acid. One unit of activity was defined as that which catalysed the incorporation of 1 nmol of [32P]phosphate into the substrate.[1] 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. |
| 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.
Cell Viability Assay[2] The cell viability assay was performed with the CellTiter-Glo® Luminescent Cell Viability Assay Kit in 96-well clear-bottom tissue-culture plates as recommended by the manufacturer. The Caki-1, 786-O or HUVEC cells were plated at densities low enough to ensure that cells never reach full confluency. A day after plating the cells, drug (Ku-0063794 or temsirolimus) or vehicle (DMSO) was added at the indicated concentrations in triplicate wells. Cell viability was measure after 24, 48, 72 and 96 hours of treatment. Luminescence was measured with the Wallac 1420 VICTOR2™ plate reader. Cell viability is presented as the percentage of the corresponding negative control at each time point. Inhibitory concentrations (e.g. IC50, IC30 and IC20) were calculated using Graphpad Prism (version 6.0). Flow Cytometric Analysis of Cell Cycle Distribution[2] Caki-1 and 786-O cells were plated in 10 cm cell culture dishes to allow the untreated control to reach 50% confluency by the end of the experiment. A day after plating the cells, the drug (Ku-0063794 or temsirolimus) or vehicle (DMSO) was added at the indicated concentrations in triplicate wells. After 72 hours of treatment, live cells in each dish were counted. To assess cell-cycle distribution, cells were resuspended in 70% ethanol (v/v). The cells were stained for 1 hour in the dark with PBS containing 50 µg/ml propidium iodide and 50 µg/ml RNase A. The DNA content of the cells was measured with the FACS Calibur flow cytometer and the CellQuest software. The cell-cycle distribution was determined using Modfit LT software. |
| Animal Protocol |
Nu/Nu nude mice
8 mg/kg i.p. Xenograft Model[2] Six-week-old female, Nu/Nu nude mice were purchased from Charles River Laboratories. Approximately 5×106 786-O cells were injected subcutaneously into the flank, and the tumors were allowed to reach 5 mm in diameter before starting treatment. The mice were randomly divided into three groups and treated once daily (five days a week) by intraperitoneal (IP) injection with DMSO (vehicle control), temsirolimus (0.6 mg/kg), or Ku0063794 (8 mg/kg). The tumor size and body weight were measured at least twice weekly. Tumor volume was estimated using the standard formula: (length×width2)/2. The mice were sacrificed after 46 days of treatment and the tumors were excised. Tumors were divided and either flash frozen in liquid nitrogen or placed in 10% buffered formalin and paraffin embedded (PE). The flash frozen tumors were homogenized in detergent lysis buffer with tissue homogenizer. The supernatant was used for western blotting. To prepare drugs for injection, temsirolimus was solubilized as a 5 mM stock solution in DMSO. Prior to IP injection, temsirolimus was diluted (15 µg/100 µl) in PEG1500 (50% (w/v) in 75 mM Hepes, pH 8.0). Ku0063794 was solubilized in one part DMSO and then diluted (200 µg/100 µl) with 4 parts PEG1500 (50% (w/v) in 75 mM Hepes, pH 8.0)[2]. |
| References | |
| Additional Infomation |
Ku-0063794 is a member of the class of pyridopyrimidines that is an mTOR inhibitor and shows anti-tumour properties. It has a role as a mTOR inhibitor and an antineoplastic agent. It is a member of morpholines, a pyridopyrimidine, a monomethoxybenzene, a tertiary amino compound and a member of benzyl alcohols.
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| Molecular Formula |
C25H31N5O4
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|---|---|
| Molecular Weight |
465.5447
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| Exact Mass |
465.237
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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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| PubChem CID |
16736978
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
694.3±65.0 °C at 760 mmHg
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| Flash Point |
373.7±34.3 °C
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| Vapour Pressure |
0.0±2.3 mmHg at 25°C
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| Index of Refraction |
1.609
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| LogP |
0.27
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
34
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| Complexity |
643
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C[C@@H]1O[C@H](C)CN(C2N=C3N=C(C4C=CC(OC)=C(CO)C=4)C=CC3=C(N3CCOCC3)N=2)C1
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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+
|
| 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; 81HJG228AB; CHEMBL1078983; 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 |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (3.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1.67 mg/mL (3.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 1.67 mg/mL (3.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 13mg/mL |
| 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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