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 복합체 키나아제 분석

  HEK-293 세포를 Hepes 용해 완충액으로 즉시 용해합니다. 용해물(1-4 mg)을 사전 면역 IgG에 결합된 5-20 μL의 Protein G-Sepharose와 배양하여 전처리합니다. 그런 다음 용해물 추출물을 5-20 μg의 anti-Rictor 또는 anti-Raptor 항체 또는 사전 면역 IgG와 결합된 5-20 μL의 Protein G-Sepharose와 배양합니다. 모든 항체는 Protein G-Sepharose에 공유 결합되어 있습니다. 면역 침강은 진동 플랫폼에서 4 °C로 1시간 동안 수행합니다. 면역 침강물을 Hepes 용해 완충액으로 4회 세척한 후, Hepes 키나아제 완충액으로 2회 세척합니다. S6K1 인산화에 사용되는 Raptor 면역 침강물의 경우, 초기 두 번의 세척 단계에서 최적의 키나아제 활성을 보장하기 위해 완충액에 0.5 M NaCl을 포함합니다. GST-Akt1은 PI-103(1 μM, 1시간)으로 처리된 혈청 결핍 HEK-293 세포에서 분리합니다. GST-S6K1은 rapamycin(0.1 μM, 1시간)으로 처리된 혈청 결핍 HEK-293 세포에서 정제합니다. mTOR 반응은 다양한 농도의 KU-0063794 및 GST-Akt1(0.5 μg) 또는 GST-S6K1(0.5 μg)이 존재하는 상태에서 0.1 mM ATP와 10 mM MgCl₂를 첨가하여 시작합니다. 반응은 진동 플랫폼에서 30 °C로 30분 동안 수행하고 SDS 샘플 완충액을 첨가하여 중단합니다. 이후 반응 혼합물을 0.22-μm 공극 크기의 Spin-X 필터를 통해 여과하고, 샘플은 전기영동 및 해당 항체를 사용한 면역 블롯 분석을 거칩니다.

• 세포주

  Wild-type and mLST8 deficient MEFs

• 농도

  Dissolved in DMSO, final concentration ~3 μM

• 배양 시간

  24, 48, and 72 hours

• 방법

  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.

• 동물 모델

  Nu/Nu nude mice

• 용량

  8 mg/kg

• 투여

  i.p.