AT7519

Male SCID mice inoculated subcutaneously with MM.1S cells

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

Dissolved in DMSO at a concentration of 10 mM, final concentrations 0.25-4 uM

15 mg/kg/day

210 nM, 210 nM, 210 nM, 210 nM, 210 nM, 210 nM

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]

In vitro Kinase Assays, Kinase assays for CDK1, CDK2 and GSK3-beta 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 ug/mL Histone H1 are incubated in 20 mM MOPS, pH 7.2, 25 mM beta-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mg/mL BSA, , 45 uM ATP (0.78 Ci/mmol) and different concentrations of AT7519 for 2 or 4 hours respectively. For GSK3-beta, the relevant enzyme and 5 uM 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% beta-mercaptoethanol, 15 uM 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 1uM 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 uM 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 lambdaex=335nm, lambdaem=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.

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, , Dr Srinivas Narasipura of Rush University Medical Center, AT7519purchased from Selleck, U87MG astroglioma cells were loaded with CFSE (5 M) and treated with 0-400 nM of AT7519. Data acquired on day 0 and day 3 on a FACS Caliber flow cytometer and analysed by FlowJo. Results indicated that AT7519 partially inhibited cell proliferation. Higher than 400 nM resulted in cell death.

2 years -20CPowder, 2 weeks4Cin DMSO, 2 months-80Cin DMSO