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].

• Ensayos de quinasas de complejos mTOR

  Las células HEK-293 se lisan frescas en tampón de lisis Hepes. El lisado (1-4 mg) se pre-clarifica incubando con 5-20 μL de Proteína G-Sepharose conjugada a IgG preinmune. Los extractos de lisado se incuban luego con 5-20 μL de Proteína G-Sepharose conjugada a 5-20 μg de anticuerpo anti-Rictor o anti-Raptor, o IgG preinmune. Todos los anticuerpos están conjugados covalentemente a Proteína G-Sepharose. Las inmunoprecipitaciones se realizan durante 1 hora a 4 °C en una plataforma vibratoria. Los inmunoprecipitados se lavan cuatro veces con tampón de lisis Hepes, seguido de dos lavados con tampón de quinasa Hepes. Para los inmunoprecipitados de Raptor utilizados para fosforilar S6K1, para los dos pasos de lavado iniciales el tampón incluye 0.5 M de NaCl para asegurar una actividad quinasa óptima. La GST-Akt1 se aísla de células HEK-293 privadas de suero incubadas con PI-103 (1 μM durante 1 hora). La GST-S6K1 se purifica de células HEK-293 privadas de suero incubadas con rapamicina (0.1 μM durante 1 hora). Las reacciones de mTOR se inician añadiendo 0.1 mM de ATP y 10 mM de MgCl₂ en presencia de varias concentraciones de KU-0063794 y GST-Akt1 (0.5 μg) o GST-S6K1 (0.5 μg). Las reacciones se realizan durante 30 minutos a 30 °C en una plataforma vibratoria y se detienen añadiendo tampón de muestra SDS. Las mezclas de reacción se filtran a través de un filtro Spin-X de 0.22 μm de tamaño de poro y las muestras se someten a electroforesis y análisis de inmunotransferencia con los anticuerpos indicados.

• Líneas celulares

  Wild-type and mLST8 deficient MEFs

• Concentraciones

  Dissolved in DMSO, final concentration ~3 μM

• Tiempo de incubación

  24, 48, and 72 hours

• Método

  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.

• Modelos animales

  Nu/Nu nude mice

• Dosificaciones

  8 mg/kg

• Administración

  i.p.