Chine KU-0063794 938440-64-3 Fabricants

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KU-0063794 938440-64-3

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Description du produit .cp_wz table {border-top : 1px solide #ccc;border-left:1px solide #ccc ; } .cp_wz table td{border-right : 1px solide #ccc ; border-bottom : 1px solide #ccc ; remplissage : 5px 0px 0px 5px;} .cp_wz table th {border-right : 1px solid #ccc;border-bottom : 1px solid #ccc ; rembourrage : 5px 0px 0px 5px ;}
Poids moléculaire : 465,54 KU-0063794 est un inhibiteur double mTOR puissant et hautement spécifique de mTORC1 et mTORC2 avec une IC50 d'environ 10 nM ; aucun effet sur les PI3K.
Comparé à l'inhibiteur de mTOR PP242, le KU-0063794 présente une spécificité plus élevée pour mTOR, car il est inactif contre les PI3K ou 76 autres kinases. Dans les cellules HEK-293, le KU-0063794 à 30 nM est suffisant pour éliminer rapidement l'activité S6K1 en bloquant la phosphorylation du motif hydrophobe (Thr389) et par la suite la phosphorylation du résidu de la boucle T (Thr229). En cas de stimulation par IGF1 de cellules HEK-293 affamées de sérum, 300 nM de KU-0063794 sont nécessaires pour inhiber l'activité S6K1 d'environ 90 %. KU-0063794 à 100-300 nM inhibe également complètement la phosphorylation induite par les acides aminés des protéines S6K1 et S6. Semblable à S6K1, KU-0063794 inhibe la phosphorylation de mTORC1 à Ser2448 et mTORC2 à Ser2481 d'une manière dépendante de la dose et du temps. En présence de sérum ou suite à une stimulation d'IGF1, le KU-0063794 induit une inhibition dose-dépendante de l'activité et de la phosphorylation d'Akt à Ser473 et inattendue de Thr308 ainsi que la phosphorylation des substrats d'Akt PRAS40 à Thr246, GSK3α/GSK3β à Ser21/ Ser9 et Foxo-1/3a à Thr24/Thr32. Le KU-0063794, mais pas la rapamycine, inhibe l'activité de SGK1 et la phosphorylation de Ser422 ainsi que son substrat physiologique NDGR1 de manière dose-dépendante, dans la même mesure que la phosphorylation de S6K1 et d'Akt, alors que la dose de KU-0063794 n'inhibe pas la phosphorylation d'ERK ou de RSK induite par l'ester de phorbol. et activation RSK. Comparé à la rapamycine, le KU-0063794 présente une puissance plus importante pour induire la déphosphorylation complète de 4E-BP1 à Thr37, Thr46 et Ser65. Le KU-0063794 inhibe la croissance cellulaire des MEF de type sauvage et déficients en mLST8 et induit un arrêt du cycle cellulaire G1, plus significativement que la rapamycine.

mTOR complexes kinase assays	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.

mTOR complexes kinase assays

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.

Dosage cellulaire : [1]

Cell lines	Wild-type and mLST8 deficient MEFs
Concentrations	Dissolved in DMSO, final concentration ~3 μM
Incubation Time	24, 48, and 72 hours
Method	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.

Conversion de différents animaux modèles sur la base de la BSA (valeur basée sur les données des lignes directrices provisoires de la FDA)

Species	Baboon	Dog	Monkey	Rabbit	Guinea pig	Rat	Hamster	Mouse
Weight (kg)	12	10	3	1.8	0.4	0.15	0.08	0.02
Body Surface Area (m2)	0.6	0.5	0.24	0.15	0.05	0.025	0.02	0.007
Km factor	20	20	12	12	8	6	5	3

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

Par exemple, pour modifier la dose de resvératrol utilisée pour une souris (22,4 mg/kg) à une dose basée sur la BSA pour un rat, multiplier 22,4 mg/kg par le facteur Km pour une souris puis diviser par le facteur Km pour un rat. Ce calcul donne une dose équivalente chez le rat pour le resvératrol de 11,2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×	mouse Km(3)	= 11.2 mg/kg
	rat Km(6)

Informations chimiques

Molecular Weight (MW)	465.54
Formula	C25H31N5O4
CAS No.	938440-64-3

Storage	3 years -20℃Powder
Storage	6 months-80℃in solvent (DMSO, water, etc.)
Synonyms

Solubility (25°C) *	In vitro	DMSO	16 mg/mL (34.36 mM)
		Water	<1 mg/mL (
		Ethanol	<1 mg/mL (
	In vivo	30% PEG400/0.5% Tween80/5% propylene glycol	30 mg/mL
* <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.