.cp_wz tabla {borde superior: 1px sólido #ccc; borde izquierdo: 1px sólido #ccc; } .cp_wz table td {borde derecho: 1px sólido #ccc; borde inferior: 1px sólido #ccc; padding: 5px 0px 0px 5px;} .cp_wz table th {border-right: 1px solid #ccc; border-bottom: 1px solid #ccc; relleno: 5px 0px 0px 5px;} \ n Peso molecular: 465.54 KU-0063794 es un inhibidor de mTOR dual potente y altamente específico de mTORC1 y mTORC2 con IC50 de ~ 10 nM; ningún efecto sobre PI3K. \ n En comparación con el inhibidor de mTOR PP242, KU-0063794 exhibe una mayor especificidad por mTOR, ya que es inactivo contra PI3K u otras 76 quinasas. En las células HEK-293, KU-0063794 a 30 nM es suficiente para eliminar rápidamente la actividad de S6K1 bloqueando la fosforilación del motivo hidrófobo (Thr389) y posteriormente la fosforilación del residuo del bucle T (Thr229). En caso de estimulación con IGF1 de células HEK-293 privadas de suero, se necesitan 300 nM de KU-0063794 para inhibir la actividad de S6K1 en ~ 90%. KU-0063794 a 100-300 nM también inhibe completamente la fosforilación inducida por aminoácidos de la proteína S6K1 y S6. Similar a S6K1, KU-0063794 inhibe la fosforilación de mTORC1 en Ser2448 y mTORC2 en Ser2481 de una manera dependiente de la dosis y dependiente del tiempo. En presencia de suero o después de la estimulación de IGF1, KU-0063794 induce una inhibición dependiente de la dosis de la actividad y fosforilación de Akt en Ser473 y Thr308 inesperado, así como la fosforilación de los sustratos de Akt PRAS40 en Thr246, GSK3α / GSK3β en Ser21 / Ser9 y Foxo-1 / 3a en Thr24 / Thr32. KU-0063794 pero no la rapamicina inhibe la actividad de SGK1 y la fosforilación de Ser422, así como su sustrato fisiológico NDGR1 de una manera dependiente de la dosis, en la misma medida que la fosforilación de S6K1 y Akt, mientras que la dosis de KU-0063794 no inhibe la fosforilación de ERK o RSK inducida por éster de forbol y activación de RSK. En comparación con la rapamicina, KU-0063794 exhibe una potencia más significativa para inducir la desfosforilación completa de 4E-BP1 en Thr37, Thr46 y Ser65. KU-0063794 inhibe el crecimiento celular de MEF de tipo salvaje y deficientes en mLST8 e induce una detención del ciclo celular G1, de manera más significativa que la rapamicina.
orte
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mTOR complexes kinase assays
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HEK-293 cells are freshly lysed in Hepes lysis buffer. Lysate (1-4 mg) is pre-cleared by incubating with 5-20 μL of Protein G-Sepharose conjugated to pre-immune IgG. The lysate extracts are 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 are covalently conjugated to Protein G-Sepharose. Immunoprecipitations are carried out for 1 hour at 4 °C on a vibrating platform. The immunoprecipitates are 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 includes 0.5 M NaCl to ensure optimal kinase activity. GST-Akt1 is isolated from serum-deprived HEK-293 cells incubated with PI-103 (1 μM for 1 hour). GST-S6K1 is purified from serum-deprived HEK-293 cells incubated with rapamycin (0.1 μM for 1 hour). mTOR reactions are initiated by adding 0.1 mM ATP and 10 mM MgCl2 in the presence of various concentrations of KU-0063794 and GST-Akt1 (0.5 μg) or GST-S6K1 (0.5 μg). Reaction are carried out for 30 minutes at 30 °C on a vibrating platform and stopped by addition of SDS sample buffer. Reaction mixtures are then filtered through a 0.22-μm-poresize Spin-X filter and samples are subjected to electrophoresis and immunoblot analysis with the indicated antibodies.
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Ensayo celular: [1]
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Cell lines
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Wild-type and mLST8 deficient MEFs
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Concentrations
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Dissolved in DMSO, final concentration ~3 μM
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Incubation Time
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24, 48, and 72 hours
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Method
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Cells are treated with KU-0063794 for 24, 48, and 72 hours, and the medium is changed every 24 hours with freshly dissolved KU-0063794. 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.
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Conversión de diferentes modelos de animales basados en BSA (valor basado en datos del Borrador de Directrices de la 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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Por ejemplo, para modificar la dosis de resveratrol utilizada para un ratón (22,4 mg / kg) a una dosis basada en el BSA para una rata, multiplique 22,4 mg / kg por el factor Km para un ratón y luego divida por el factor Km para una rata. Este cálculo da como resultado una dosis equivalente para ratas de resveratrol de 11,2 mg / kg.
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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 Información química
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Molecular Weight (MW)
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465.54
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Formula
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C25H31N5O4
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CAS No.
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938440-64-3
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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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16 mg/mL
(34.36 mM)
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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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In vivo
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30% PEG400/0.5% Tween80/5% propylene glycol
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30 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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(5-(2-((2R,6S)-2,6-dimethylmorpholino)-4-morpholinopyrido[2,3-d]pyrimidin-7-yl)-2-methoxyphenyl)methanol
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Calculadora de molaridad Calculadora de dilución Calculadora de peso molecular
Grupos de Producto : PI3K / Akt / mTOR > Inhibidor de mTOR
