.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: 384.48 ML323 es un inhibidor de USP1-UAF1 altamente potente y selectivo con IC50 de 76 nM. \ n ML323 inhibe la desubiquitinación de PCNA y FANCD2 al inhibir la actividad de USP1-UAF1 en células H596. Además, ML323 potencia la citotoxicidad del cisplatino en células NSCLC H596 y células de osteosarcoma U2OS al dirigirse a dos vías principales de respuesta al daño del ADN (TLS y FA). \ n Protocolo (solo como referencia) Ensayo de quinasa: [1]
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High-throughput screening
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For HTS, USP1-UAF1 activity is monitored using ubiquitin-rhodamine 110 as a substrate, where hydrolysis of the amide bond between the C-terminal glycine of ubiquitin and rhodamine results in an increase in fluorescence. The assay is miniaturized to a 4 μL volume in a 1,536-well format and is used to screen approximately 402,701 compounds in quantitative HTS mode, with each compound tested over a range of four to five concentrations. The assay shows robust performance with an average Z`factor of 0.8 throughout the screen.
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Ensayo celular: [1]
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Cell lines
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H596 cells
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Concentrations
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~30 μM
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Incubation Time
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7-12 days
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Method
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For the colony-forming assay, cells are seeded at a density of 300–500 cells per well in six-well plates and grown overnight. Cells are then treated with ML323 alone, cisplatin alone or a combination of cisplatin and ML323 (1:1 or 1:4) at the indicated concentrations. Cells treated with an equal volume of DMSO and saline are used as control. After 48 h of treatment, fresh growth medium is added, and cells are incubated for an additional 5-10 d to allow for colony formation. For UV combination treatment, the cells are treated with ML323 at the indicated concentrations or an equal volume of DMSO. After 48 h, the medium is removed, and cells are irradiated at 254 nm at the indicated dosage. Fresh growth medium is added, and the cells are incubated for an additional 5-10 d to allow for colony formation. The cells without UV irradiation but treated with ML323 or an equal volume of DMSO are used as controls and designated as 100%. After the formation of the colonies, cells are fixed with methanol and stained with 0.5% crystal violet. Colonies consisting of >50 cells are scored. The number of colonies is determined from triplicate plates. The dose-response curves are generated using GraphPad Prism and analyzed by using CalcuSyn to calculate the combination index, which is determined for the fraction of cells affected after the addition of fixed ratios of cisplatin and the USP1-UAF1 inhibitor.
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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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384.48
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Formula
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C23H24N6
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CAS No.
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1572414-83-5
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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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76 mg/mL
(197.66 mM)
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Water
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<1 mg/mL
(
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Ethanol
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38 mg/mL
(98.83 mM)
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Chemical Name
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4-Pyrimidinamine, 5-methyl-2-[2-(1-methylethyl)phenyl]-N-[[4-(1H-1,2,3-triazol-1-yl)phenyl]methyl]-
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Grupos de Producto : Ubiquitina > Inhibidor DUB
