Description

Cancer

Biochemical kinase assays

c-Met catalytic activity is quantitated using a continuous-coupled spectrophotometric assay in which the time-dependent production of ADP by c-Met is determined by analysis of the rate of consumption of NADH. NADH consumption is measured by a decrease in absorbance at 340 nm by spectrophotometry at designated time points. To determine K_i values, PF-2341066 is introduced into test wells at various concentrations in the presence of assay reagents and incubated for 10 minutes at 37 °C. The assay is initiated by the addition of the c-Met enzyme.

Cell Lines

GTL-16 gastric carcinoma cells and T47D breast carcinoma cells

Concentrations

0-256 nM

Incubation Time

1 hour

Methods

Cells including GTL-16 gastric carcinoma cells and T47D breast carcinoma cells are seeded in 96-well plates in media supplemented with 10% fetal bovine serum (FBS) and transferred to serum-free media [with 0.04% bovine serum albumin (BSA)] after 24 hours. In experiments investigating ligand-dependent RTK phosphorylation, corresponding growth factors are added for up to 20 minutes. After incubation of cells with PF-2341066 for 1 hour and/or appropriate ligands for the designated times, cells are washed once with HBSS supplemented with 1 mM Na₃VO₄, and protein lysates are generated from cells. Subsequently, phosphorylation of selected protein kinases is assessed by a sandwich ELISA method using specific capture antibodies used to coat 96-well plates and a detection antibody specific for phosphorylated tyrosine residues. Antibody-coated plates are (a) incubated in the presence of protein lysates at 4 °C overnight; (b) washed seven times in 1% Tween 20 in PBS; (c) incubated in a horseradish peroxidase–conjugated anti–total-phosphotyrosine (PY-20) antibody (1:500) for 30 min; (d) washed seven times again; (e) incubated in 3,3^′,5,5^′-tetramethyl benzidine peroxidase substrate to initiate a colorimetric reaction that is stopped by adding 0.09 N H₂SO₄; and (f) measured for absorbance in 450 nm using a spectrophotometer.

Animal Models

Female or male nu/nu mice bearing NCI-H441,or DLD-1, or MDA-MB-231

Formulation

Doses

12.5 mg/kg/day, 25 mg/kg/day, and 50 mg/kg/day

Administration

Administered via p.o.