KU-0063794

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, this compound 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 this compound is needed to inhibit the S6K1 activity by ~90%. This chemical at 100-300 nM also completely inhibits the amino-acid-induced phosphorylation of S6K1 and S6 protein. Similar to S6K1, it 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, this compound 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. This compound 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 it dose not inhibit phorbol ester induced ERK or RSK phosphorylation and RSK activation. Compared with rapamycin, this chemical exhibits more significant potency to induce the complete dephosphorylation of 4E-BP1 at Thr³⁷, Thr⁴⁶ and Ser⁶⁵. It inhibits cell growth of both wild-type and mLST8-deficient MEFs and induces a G1 cell cycle arrest, more significantly than rapamycin. [1]

Ku0063794 inhibits tumor growth and mTOR signaling in a preclinical renal cell carcinoma model. However, this compound was not more effective in the animal study. A possible explanation for lack of greater activity in vivo for this chemical[2].

• mTOR-complexen kinase-assays

  HEK-293 cellen worden vers gelyseerd in Hepes lysisbuffer. Lysaat (1-4 mg) wordt voorgeklaard door incubatie met 5-20 μL Proteïne G-Sepharose geconjugeerd aan pre-immuun IgG. De lysaatextracten worden vervolgens geïncubeerd met 5-20 μL Proteïne G-Sepharose geconjugeerd aan 5-20 μg van ofwel anti-Rictor of anti-Raptor antilichaam, of pre-immuun IgG. Alle antilichamen zijn covalent geconjugeerd aan Proteïne G-Sepharose. Immunoprecipitaties worden gedurende 1 uur bij 4 °C uitgevoerd op een trilplatform. De immunoprecipitaten worden vier keer gewassen met Hepes lysisbuffer, gevolgd door twee wassingen met Hepes kinasebuffer. Voor Raptor-immunoprecipitaten die worden gebruikt voor het fosforyleren van S6K1, bevat de buffer voor de eerste twee wasstappen 0,5 M NaCl om optimale kinase-activiteit te garanderen. GST-Akt1 wordt geïsoleerd uit serum-deprived HEK-293 cellen geïncubeerd met PI-103 (1 μM gedurende 1 uur). GST-S6K1 wordt gezuiverd uit serum-deprived HEK-293 cellen geïncubeerd met rapamycine (0,1 μM gedurende 1 uur). mTOR-reacties worden geïnitieerd door toevoeging van 0,1 mM ATP en 10 mM MgCl₂ in aanwezigheid van verschillende concentraties KU-0063794 en GST-Akt1 (0,5 μg) of GST-S6K1 (0,5 μg). De reacties worden gedurende 30 minuten bij 30 °C uitgevoerd op een trilplatform en gestopt door toevoeging van SDS-monsterbuffer. Reactiemengsels worden vervolgens gefilterd door een 0,22 μm poriegrootte Spin-X-filter en monsters worden onderworpen aan elektroforese en immunoblot-analyse met de aangegeven antilichamen.

• Cellijnen

  Wild-type and mLST8 deficient MEFs

• Concentraties

  Dissolved in DMSO, final concentration ~3 μM

• Incubatietijd

  24, 48, and 72 hours

• Methode

  Cells are treated with KU-0063794 for 24, 48, and 72 hours, and the medium is changed every 24 hours with freshly dissolved this compound. 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.

• Dierlijke modellen

  Nu/Nu nude mice

• Doseringen

  8 mg/kg

• Toediening

  i.p.