.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; padding: 5px 0px 0px 5px;} \ n Peso molecular: 470.52 ETP-46464 es un inhibidor potente y selectivo de ATR con IC50 de 25 nM. \ n ETP-46464 muestra una actividad casi completa contra ATR a 100 nM. ETP-46464 inhibe la actividad de PI3Kα, mTOR y DNA-PKcs con IC50 de 170, 0,6 y 36 nM. ETP-46464 puede promover la rotura de horquillas de replicación estancadas. La exposición a ETP-46464 conduce a la generación de cantidades sustanciales de daño en el ADN en las células en replicación. ETP-46464 1 μM anula el punto de control G2 / M inducido por radiación ionizante y conduce a la presencia de micronúcleos o núcleos completamente fragmentados en las células. ETP-46464 es particularmente tóxico para las células deficientes en p53, lo que se ve agravado por las condiciones que generan estrés replicativo, como la sobreexpresión de ciclina E. \ n Protocolo (solo como referencia) Ensayo de quinasa: [1]
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ATR kinase activity assay
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For the in vitro kinase assay flag-tagged ATR kinase is immunoprecipitated from 293T cells using Anti-flag M2 affinity gel. As a substrate, p53/1-101 peptide fused to GST is purified from IPTG stimulated BL21 bacteria by conventional GST purification methods. Briefly, pBJF-flag-ATR plasmids (wild type and kinase dead versions) is transfected into 293T cells. 48 hours after transfection, cells are lysed in TGN buffer (50 mM Tris (pH 7.5), 50 mM glycerophosphate, 150 mM NaCl, 10% glycerol, 1% tween-20 and protease inhibitors cocktail) for 15 minutes on ice and, after centrifugation, the supernatant is pre-cleared for 1h with unspecific rabbit IgG and protein A on a rotator at 4 degrees. Next, the supernatant is incubated with M2-slurry for 2 h at 4 degrees. For a confluent p100 plate of 293T cells, 400 μL of buffer, 10 μg of IgG, 30 μL of protein A slurry, and 25 μL of M2 beads are used. Anti flag M2 beads are then washed 5 times in ice cold TNG, 3 times in ice cold LiCl buffer (100mM Tris pH 7.5, 0.5 M LiCl), and 5 times in ice cold Kinase buffer (10 mM Hepes (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 10 mM Mn2, 1 mM DTT). After the last wash, IPs are splitted in the proper number of tubes for the assay (approximately one confluent 293T p100 is used per condition). For the kinase reaction, 100 μM `cold` ATP is added to the buffer. Only fresh IPs are used. Prior to every experiment a mix is prepared containing 2 μg of GST-p53 and 10 μ Ci of 32P-γATP per condition. Inhibitor are first added to the beads. Last, 25μL of the mix is added to each tube. Reactions are performed for 20 minutes at 30 degrees and stopped by adding 15 microliters of 4 × NuPAGE SDS buffer and boiling for 5 minutes. Beads are pelleted and supernatants are resolved by SDS-PAGE on a polyacrylamide gel. Radioactive gels are slightly washed and directly exposed to Phospor-Imager screens for 2h to 16h. Radioactivity is then detected using a STORM scanner. Finally, gels are stained with coomasie to check for protein levels.
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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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470.52
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Formula
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C30H22N4O2
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CAS No.
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1345675-02-6
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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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N/A
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Solubility (25°C) *
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In vitro
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DMSO
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6 mg/mL
(12.75 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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* <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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Benzeneacetonitrile, α,α-dimethyl-4-[2-oxo-9-(3-quinolinyl)-2H-[1,3]oxazino[5,4-c]quinolin-1(4H)-yl]-
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Calculadora de molaridad Calculadora de dilución Calculadora de peso molecular
Grupos de Producto : PI3K / Akt / mTOR > Inhibidor ATM / ATR
