AT7519

AT7519は、新しい小分子です。それは、マルチ・サイクリン依存的なキナーゼ阻害剤で、CDK1/cyclin B、 CDK2/Cyclin A、 CDK3/Cyclin E、 CDk4/Cyclin D1、 CDk5/p35、 CDK6/Cyclin D3に作用すると、IC50が それぞれ210 nM、47 nM、360 nM、100 nM、13 nM、170 nM になる。

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AT7519 化学構造
分子量: 382.24

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Quality Control & MSDS

製品説明

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製品の説明

生物活性

製品説明 AT7519は、新しい小分子です。それは、マルチ・サイクリン依存的なキナーゼ阻害剤で、CDK1/cyclin B、 CDK2/Cyclin A、 CDK3/Cyclin E、 CDk4/Cyclin D1、 CDk5/p35、 CDK6/Cyclin D3に作用すると、IC50が それぞれ210 nM、47 nM、360 nM、100 nM、13 nM、170 nM になる。
ターゲット CDK1/cyclin B CDK2/Cyclin A CDK3/Cyclin E CDK4/Cyclin D1 CDK5/p35 CDK6/Cyclin D3
IC50 210 nM 47 nM 360 nM 100 nM 13 nM 170 nM [1]
In vitro試験 AT7519 is an ATP competitive CDK inhibitor with a Ki value of 38 nM for CDK1. AT7519 is inactive against all non-CDK kinases with the exception of GSK3β (IC50 = 89 nM). AT7519 shows potent antiproliferative activity in a variety of human tumor cell lines with IC50 values ranging from 40 nM for MCF-7 to 940 nM for SW620 consistent with the inhibition of CDK1 and CDK2. [1] AT7519 induces dose-dependent cytotoxicity in multiple myeloma (MM) cell lines with IC50 values ranging from 0.5 to 2 μM at 48 hours, with the most sensitive cell lines being MM.1S (0.5 μM) and U266 (0.5 μM) and the most resistant MM.1R (>2 μM). It does not induce cytotoxicity in peripheral blood mononuclear cells (PBMNC). AT7519 partially overcomes the proliferative advantage conferred by IL6 and IGF-1 as well as the protective effect of bone marrow stromal cells (BMSCs). AT7519 induces rapid dephosphorylation of RNA pol II CTD at serine 2 and serine 5 sites, and leads to the inhibition of transcription, partially contributing to AT7519 induced cytotoxicity of MM cells. AT7519 induces activation of GSK-3β by down-regulating GSK-3β phosphorylation, which also contributes to AT7519 induced apoptosis independent of the inhibition of transcription. [2]
In vivo試験 A twice daily dosing of AT7519 (9.1 mg/kg) causes tumor regression of both early-stage and advanced-stage s.c. tumors in the HCT116 and HT29 colon cancer xenograft models. [1] AT7519 treatment (15 mg/kg) inhibits tumor growth and prolongs the median overall survival of mice in the human MM xenograft mouse model in association with increased caspase 3 activation. [2]
臨床試験 A Phase I/II study to determine whether AT7519M alone or AT7519M plus bortezomib are effective treatments in patients with previously treated multiple myeloma is currently recruiting participants.
特集

プロトコル (参考用のみ)

キナーゼアッセイ: [1]

In vitro Kinase Assays Kinase assays for CDK1, CDK2 and GSK3-β are all carried out in a radiometric filter binding format. Assays for CDK5 are in DELFIA format and for CDKs 4 and 6 in ELISA format. For CDKs 1 and 2, the relevant CDK and 0.12 μg/mL Histone H1 are incubated in 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mg/mL BSA, 45 μM ATP (0.78 Ci/mmol) and different concentrations of AT7519 for 2 or 4 hours respectively. For GSK3-β, the relevant enzyme and 5 μM glycogen synthase peptide 2 along with 10 mM MOPS pH 7.0, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, 0.2 mM EDTA, 10 mM MgCl2, 0.01% β-mercaptoethanol, 15 μM ATP (2.31 Ci/mmol) and different concentrations of AT7519 are incubated for 3 hours. Assay reactions are stopped by adding an excess of orthophosphoric acid and filtered using Millipore MAPH filter plates. The plates are then washed, scintillant added and radioactivity measured by scintillation counting on a Packard TopCount. For CDK5, CDK5/p35 and 1μM of a biotinylated Histone H1 peptide (Biotin-PKTPKKAKKL) are incubated in 25 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 0.025% Brij-35, 0.1 mg/mL BSA, 1 mM DTT, 15 μM ATP and different concentrations of AT7519 for 30 minutes. Assay reactions are stopped using EDTA, transferred to Neutravidin-coated plates and phosphorylated peptide quantified by means of a rabbit phospho-cdk1 substrate polyclonal antibody and DELFIA europium-labelled anti-rabbit IgG secondary antibody using time-resolved fluorescence at λex=335nm, λem=620nm. For CDK 4 and 6 assays, plates are coated with GST- pRb769-921 and blocked with Superblock. CDK4 or 6 is incubated with 15 mM MgCl2, 50 mM HEPES, pH 7.4, 1 mM DTT, 1 mM EGTA, pH 8.0, 0.02% Triton X-100, 2.5% DMSO and different concentrations of AT7519; the reaction is initiated by addition of ATP. After 30 minutes, reactions are stopped by the addition of 0.5 M EDTA pH 8.0. Plates are then washed and incubated for one hour with the primary antibody (anti- p-Rb Serine 780) diluted in Superblock followed by secondary antibody (alkaline phosphatase linked anti-rabbit) for a further hour. Plates are developed using the Attophos system and fluorescence read on a Spectramax Gemini plate reader at excitation 450 nm and emission 580 nm. In all cases, IC50 values are calculated from replicate curves, using GraphPad Prism software.

細胞アッセイ: [2]

細胞株 MM.1S, MM.1R, RPMI8226, U266, RPMI8266, RPMI-Dox40, OPM1 cells, primary MM cells and PBMNCs
濃度 Dissolved in DMSO at a concentration of 10 mM, final concentrations 0.25-4 μM
反応時間 24 or 48 hours
実験の流れ Cells are incubated with different concentrations of AT7519 for 24 or 48 hours at 37 °C. Cell viability is assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrasodium bromide (MTT) dye absorbance. DNA synthesis is measured by tritiated thymidine uptake (3H-TdR). Apoptosis is assessed by using Annexin V/PI staining. The percentage of cells undergoing apoptosis is defined as the sum of early apoptosis (Annexin V-positive cells) and late apoptosis (Annexin V-positive and PI-positive cells

動物実験: [2]

動物モデル Male SCID mice inoculated subcutaneously with MM.1S cells
製剤 Dissolved in 0.9% saline
投薬量 15 mg/kg/day
投与方法 Dosed i.p.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

SpeciesMouseRatRabbitGuinea pigHamsterDogMonkeyBaboon
Weight (kg)0.020.151.80.40.0810312
Body Surface Area (m2)0.0070.0250.150.050.020.50.240.6
Km factor361285201220
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×  mouse Km(3)  = 11.2 mg/kg
rat Km(6)
1

参考

化学情報

Download AT7519 SDF
分子量 382.24
化学式

C16H17Cl2N5O2

CAS No. 844442-38-2
保管 2年-20℃
6月-80℃in solvent
別名 N/A
溶解度 (25°C) * In vitro DMSO 10 mg/mL (26.16 mM)
<1 mg/mL (<1 mM)
エタノール <1 mg/mL (<1 mM)
In vivo 30% propylene glycol, 5% Tween 80, 65% D5W 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.
化学名 4-(2,6-dichlorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide

カスタマーフィードバック (3)


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Rating
Source AIDS, 2014, 28(15), 2213-22. AT7519 purchased from Selleck
Method Immunoblot
Cell Lines Monocyte-derived macrophages
Concentrations 0.5 mM
Incubation Time
Results AT7519 partially decreased SAMHD1 phosphorylation at Thr592 or reduced the appearance of slow migrating forms of SAMHD1, which are hallmarks of SAMHD1 phosphorylation.

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Rating
Source PLoS One, 2011, 6(9), e25683. AT7519 purchased from Selleck
Method Morphological analysis
Cell Lines Macrophages
Concentrations 30 mg/kg
Incubation Time 6 h
Results At 6 h post treatment typical morphology of pleuralcavity cells from vehicle treated animals demonstrating viable eosinophils and macrophages without apoptotic bodies (E) and AT7519 treated animals demonstrating apoptotic eosinophils as well as apoptotic eosinophils inside macrophages (F).

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Rating
Source Dr. Srinivas Narasipura of Rush University Medical Center. AT7519 purchased from Selleck
Method flow cytometry
Cell Lines U87MG astroglioma cells
Concentrations 0-400 nM
Incubation Time 0-3 d
Results Results indicated that AT7519 partially inhibited cell proliferation. Higher than 400 nM resulted in cell death.

文献中の引用 (7)

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description
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