AT7519

PDF

S1524

5~200mg

Brand

Selleckchem

Description

AT7519

Catalog No.S1524

1 Reviews 3 Product Citations

AT7519 is a multi-CDK inhibitor for CDK1, 2, 4, 6 and 9 with IC50 of 10-210 nM. It is less potent to CDK3 and little active to CDK7. Phase 1.

Distributor Tel: +886-2-2827-1197jimmy@new.abscience.com.tw

Biological Activity

Description	AT7519 is a multi-CDK inhibitor for CDK1, 2, 4, 6 and 9 with IC50 of 10-210 nM. It is less potent to CDK3 and little active to CDK7. Phase 1.
Targets	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	AT7519 is an ATP competitive CDK inhibitor with a K_i 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	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]
Features

Protocol(Only for Reference)

Kinase Assay: ^[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 λ_ex=335nm, λ_em=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.

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 λ_ex=335nm, λ_em=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.

Cell Assay: ^[2]

Cell lines	MM.1S, MM.1R, RPMI8226, U266, RPMI8266, RPMI-Dox40, OPM1 cells, primary MM cells and PBMNCs
Concentrations	Dissolved in DMSO at a concentration of 10 mM, final concentrations 0.25-4 μM
Incubation Time	24 or 48 hours
Method	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

Animal Study: ^[2]

Animal Models	Male SCID mice inoculated subcutaneously with MM.1S cells
Dosages	15 mg/kg/day
Administration	Dosed i.p.
Solubility	30% propylene glycol, 5% Tween 80, 65% D5W, 30 mg/mL

References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT00390117	Completed	Lymphoma\|Unspecified Adult Solid Tumor, Protocol Specific	NCIC Clinical Trials Group	2006-08	Phase 1
NCT01183949	Active, not recruiting	Multiple Myeloma\|Relapsed Multiple Myeloma\|Refractory Multiple Myeloma	Astex Pharmaceuticals\|Multiple Myeloma Research Consortium	2010-11	Phase 1\|Phase 2
NCT01627054	Recruiting	Chronic Lymphocytic Leukemia	NCIC Clinical Trials Group\|Novartis Pharmaceuticals\|previously: Astex Pharmaceuticals	2012-08	Phase 2
NCT01652144	Recruiting	Mantle Cell Lymphoma	NCIC Clinical Trials Group\|Novartis Pharmaceuticals\|previously: Astex Pharmaceuticals	2012-08	Phase 2

Chemical Information

Download AT7519 SDF

Molecular Weight (MW)	382.24
Formula	C₁₆H₁₇Cl₂N₅O₂
CAS No.	844442-38-2

Storage	3 years -20â„ƒPowder
Storage	6 months-80â„ƒin DMSO
Syonnyms	N/A

Solubility (25°C) *	In vitro	DMSO	10 mg/mL (26 mM)
		Water	<1 mg/mL (<1 mM)
		Ethanol	<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.

Chemical Name	4-(2,6-dichlorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.38224	3.8224	7.6448	–

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Customer Reviews (1)

Click to enlarge	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.