.cp_wz table {border-top: твердое тело 1px #ccc; border-left: твердое тело 1px #ccc; } .cp_wz таблица td {border-right: 1px solid #ccc; нижняя граница: сплошной 1px #ccc; padding: 5px 0px 0px 5px;} .cp_wz table th {border-right: 1px solid #ccc; border-bottom: 1px solid #ccc; padding: 5px 0px 0px 5px;} \ n Молекулярный вес: 528,61 WYE-687 является АТФ-конкурентным и селективным ингибитором mTOR с IC50 7 нМ; блокирует mTORC1 / pS6K (T389) и mTORC2 / P-AKT (S473), но не влияет на P-AKT (T308). Селективность по mTOR выше, чем у PI3Kα (> 100 раз) и PI3Kγ (> 500 раз). Анализ киназы иммунного комплекса показывает, что специфический для mTORC2 субстрат His6-AKT или субстрат mTORC1 His6-S6K дозозависимо ингибируется WYE-687. WYE-687 глобально подавляет передачу сигналов mTOR и функцию AKT в клеточных моделях дозозависимо. WYE-678 глубоко ингибирует кэп-зависимый и глобальный синтез белка в MDA361, клетках рака молочной железы человека. WYE-687 проявляет антипролиферативные эффекты в различных линиях раковых клеток, включая остановку клеточного цикла G1 и селективный апоптоз. WYE-687 подавляет ангиогенные факторы, VEGF и HIF-1α, в клетках U87MG, MDA361 и LNCap. \ п
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DELFIA Format of Purified FLAG-TOR
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The routine assays with purified FLAG-TOR (FL and 3.5) are performed in 96-well plates as follows. Enzymes are first diluted in kinase assay buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 50 mM β-glycerophosphate, 10 mM MnCl2, 0.5 mM DTT, 0.25 lM microcystin LR, and 100 lg/mL BSA). To each well, 12 μL of the diluted enzyme is mixed briefly with 0.5 μL test inhibitor or control vehicle dimethyl sulfoxide (DMSO). The kinase reaction is initiated by adding 12.5 μL kinase assay buffer containing ATP and His6-S6K to give a final reaction volume of 25 μL containing 800 ng/mL FLAG-TOR, 100 μM ATP, and 1.25 μM His6-S6K. The reaction plate is incubated for 2 hours (linear at 1-6 hour) at room temperature with gentle shaking and then terminated by adding 25 μL Stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA).
The DELFIA detection of the phosphorylated (Thr-389) His6-S6K
is performed at room temperature using a monoclonal anti-P(T389)- p70S6K antibody (1A5) labeled with Europium-N1- ITC (Eu) (10.4 Eu per antibody). Fortyfive microliters of the terminated kinase reaction mixture are transferred to a MaxiSorp plate containing 55 μL PBS. The
His6-S6K is allowed to attach for 2 hour after which the wells are aspirated and washed once with PBS. One hundred microliters of
DELFIA buffer with 40 ng/mL Eu-P(T389)-S6K antibody is added.
The antibody binding is continued for 1 hour with gentle agitation. The
wells are then aspirated and washed four times with PBS containing
0.05% Tween 20 (PBST). One hundred microliters of DELFIA
Enhancement solution is added to each well and the plates are read in a PerkinElmer Victor model plate reader. Data obtained are used to calculate enzymatic activity and enzyme inhibition by potential inhibitors.
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Клеточный анализ: [1]
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Cell lines
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MDA361, U87MG, HCT116, LNCap and HT29 cells
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Concentrations
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0-100 μM
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Incubation Time
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24 or 48 hours
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Method
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For cell cycle analysis, cells are seeded in 96-well culture plates at 10,000 cells per well for 24 hours, treated with various inhibitors for 24 hours or 48 hours. Following treatment, cells are harvested, washed with PBS and fixed overnight at -20 °C in 70% ethanol. Cells are washed, stained with propidium iodide and analyzed for cell cycle profile (acquired 5000 cells/well) on Guava PCA-96 instrument according to the Guava Cell Cycle Protocol
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Конверсия различных модельных животных на основе BSA (значение на основе данных из проекта рекомендаций FDA)
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Species
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Baboon
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Dog
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Monkey
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Rabbit
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Guinea pig
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Rat
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Hamster
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Mouse
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Weight (kg)
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12
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10
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3
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1.8
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0.4
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0.15
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0.08
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0.02
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Body Surface Area (m2)
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0.6
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0.5
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0.24
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0.15
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0.05
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0.025
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0.02
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0.007
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Km factor
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20
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20
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12
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12
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8
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6
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5
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3
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Animal A (mg/kg) = Animal B (mg/kg) multiplied by
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Animal B Km
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Animal A Km
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Например, чтобы изменить дозу ресвератрола, используемую для мыши (22,4 мг / кг), на дозу, основанную на BSA для крысы, умножьте 22,4 мг / кг на коэффициент Km для мыши, а затем разделите на коэффициент Km для крыса. Этот расчет дает эквивалентную дозу ресвератрола для крыс 11,2 мг / кг.
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Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×
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mouse Km(3)
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= 11.2 mg/kg
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rat Km(6)
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\ n Химическая информация
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Molecular Weight (MW)
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528.61
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Formula
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C28H32N8O3
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CAS No.
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1062161-90-3
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Storage
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3 years -20℃Powder
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6 months-80℃in solvent (DMSO, water, etc.)
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Synonyms
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Solubility (25°C) *
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In vitro
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DMSO
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0.5 mg/mL
(
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Water
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<1 mg/mL
(
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Ethanol
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<1 mg/mL
(
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* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
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Chemical Name
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methyl 4-(4-morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenylcarbamate
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Калькулятор молярности Калькулятор разведения Калькулятор молекулярной массы
Группа Продуктов : PI3K / Akt / mTOR > mTOR ингибитор
