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AT7519 HCl

AT7519 HCl is a multi-CDK inhibitor for CDK1, 2, 4, 6 and 9 with IC50 of 10-210 nM in cell-free assays. It is less potent to CDK3 and little active to CDK7. Phase 2.

AT7519 HCl化学構造

CAS No. 902135-91-5

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現在のバッチを見る： S780801 DMSO] 52 mg/mL] false] Water] 43 mg/mL] false] Ethanol] 28 mg/mL] false 純度： 99.44%
99.44

AT7519 HCl関連製品

シグナル伝達経路

CDK Signaling Pathways

CDKシグナル伝達経路

CDK阻害剤の選択性比較

生物活性

製品説明 AT7519 HCl is a multi-CDK inhibitor for CDK1, 2, 4, 6 and 9 with IC50 of 10-210 nM in cell-free assays. It is less potent to CDK3 and little active to CDK7. Phase 2.
Targets
CDK9/CyclinT [1]
(Cell-free assay)		 CDK5/p35 [1]
(Cell-free assay)		 CDK2/CyclinA [1]
(Cell-free assay)		 GSK-3β [1]
(Cell-free assay)		 CDK4/CyclinD1 [1]
(Cell-free assay)		 もっとクリックする
<10 nM 13 nM 47 nM 89 nM 100 nM
In Vitro
In vitro 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]
Kinase Assay 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.
細胞実験 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
In Vivo
In Vivo 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]
動物実験 動物モデル Male SCID mice inoculated subcutaneously with MM.1S cells
投与量 15 mg/kg/day
投与経路 Dosed i.p.
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT01183949 Completed
Multiple Myeloma
Astex Pharmaceuticals Inc.|Multiple Myeloma Research Consortium
 November 2010 Phase 1|Phase 2

化学情報

分子量 418.71 化学式

C16H18Cl3N5O2
CAS No. 902135-91-5 SDF Download AT7519 HCl SDFをダウンロードする
Smiles C1CNCCC1NC(=O)C2=C(C=NN2)NC(=O)C3=C(C=CC=C3Cl)Cl.Cl
保管

In vitro
Batch:

DMSO : 52 mg/mL ( (124.19 mM)； 吸湿したDMSOは溶解度を減少させます。新しいDMSOをご使用ください。)

Water : 43 mg/mL

Ethanol : 28 mg/mL

モル濃度計算器

in vivo
Batch:

Add solvents to the product individually and in order.

投与溶液組成計算機

実験計算

モル濃度計算器

質量 濃度 体積 分子量

投与溶液組成計算機(クリア溶液)

ステップ１：実験データを入力してください。（実験操作によるロスを考慮し、動物数を1匹分多くして計算・調製することを推奨します）

mg/kg g μL 匹

ステップ２：投与溶媒の組成を入力してください。（ロット毎に適した溶解組成が異なる場合があります。詳細については弊社までお問い合わせください）

% DMSO % % Tween 80 % ddH2O
%DMSO %

計算結果：

投与溶媒濃度： mg/ml;

DMSOストック溶液調製方法： mg 試薬を μL DMSOに溶解する（濃度 mg/mL, 注：濃度が当該ロットのDMSO溶解度を超える場合はご連絡ください。 )

投与溶媒調製方法：Take μL DMSOストック溶液に μL PEG300，を加え、完全溶解後μL Tween 80，を加えて完全溶解させた後 μL ddH2O，を加え完全に溶解させます。

投与溶媒調製方法：μL DMSOストック溶液に μL Corn oil，を加え、完全溶解。

注意：１.ストック溶液に沈殿、混濁などがないことをご確認ください；
２.順番通りに溶剤を加えてください。次のステップに進む前に溶液に沈殿、混濁などがないことを確認してから加えてください。ボルテックス、ソニケーション、水浴加熱など物理的な方法で溶解を早めることは可能です。

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