Twenty-five microliters of the bead suspension was added to each well of the enolase-coated 96-well high protein binding plate together with an appropriate concentration of compound and [γ-32P]ATP (25 μl/well of a 200 μCi/ml solution in kinase buffer). After incubation for 20 min at 20°C, 60 μl of boiling 2 × solubilization buffer containing 10 mM ATP was added to the assay wells to terminate the reactions. Thirty microliters of the samples was removed from the wells, boiled for 5 min, and run on a 7.5% SDS-polyacrylamide gel. The gels were subsequently dried and exposed to Kodak X-AR film (see Fig. 2A). For quantitation, films were scanned using a Molecular Dynamics laser scanner, and the optical density of the major substrate band, enolase p46, was determined. Concentrations of compound that caused 50% inhibition of enolase phosphorylation (IC50) were determined from a plot of the density versus concentration of compound (see Fig. 2B). In companion experiments for measuring the activity of compounds against Lck (see Fig. 2C), the assay plate was washed with two wash cycles on a Skatron harvester using 50 mM EDTA, 1 mM ATP. Scintillation fluid (100 μl) was then added to the wells, and 32P incorporation was measured using a Pharmacia Biotech micro-β-counter. Concentrations of compound that caused 50% inhibition of enzyme activity (IC50) were determined from a plot of the percent inhibition of enzyme activity versus concentration of compound. Since there was good correlation between the gel and plate assays, subsequent repeat experiments for both Lck and FynT were performed using scintillation counting (see Table 1). EGF-R activity was measured by immunoprecipitation of EGF-R from A-431 cells obtained from the American Type Culture Collection. Cell lysates were prepared by adding 4 ml of lysis buffer to a T-75 flask that contained a confluent layer of cells. The lysates were clarified by centrifugation as described above and then incubated with 10 μg/ml anti-EGF-R for 2 h at 4°C. Protein A-Sepharose beads were added to the antibody/lysate mixture at 250 μl/ml and allowed to incubate for 30 min at 4°C. The beads were then washed twice in 1.0 ml of lysis buffer and twice in 1.0 ml of kinase buffer (as described above) and finally resuspended to 50% (w/v) in kinase buffer. To each 1.5-ml assay tube was aliquoted 50 μl of bead suspension, which was then spun for 15 s at 14,000 rpm in an Eppendorf microcentrifuge, and the supernatant was discarded. To the bead pellet was then added 5 μl of the appropriate compound dilution, 5 μl of EGF (Upstate Biotechnology, Inc.) to a final concentration of 100 pM, and 5 μl of a 33 μCi [γ-32P]ATP/ml solution in kinase buffer. After incubation for 20 min at 20°C, the beads were washed once with 1.0 ml of lysis buffer and once with 1.0 ml of 1 × PBS. To the bead pellet was added 60 μl of boiling 2 × solubilization buffer (26) containing 10 mM ATP. Samples were run on 7.5% SDS-polyacrylamide gels, which were subsequently dried and exposed using BAS-III imaging plates. Labeled EGF-R protein bands were visualized, and 32P incorporation was quantitated using the BAS-2000 BioImaging analyzer. Concentrations of compound that caused 50% inhibition of enzyme activity (IC50) were determined from a plot of the percent inhibition of enzyme activity by different concentrations of compound. Murine JAK2 was produced in baculovirus and supplied as an immune complex bound to protein A-Sepharose beads (Upstate Biotechnology, Inc.). JAK2 beads (2.5 μl) were resuspended in 20 μl of kinase buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 0.1 mM Na3VO4, 0.25 mCi/ml [γ-32P]ATP) for 10 min at room temperature. The beads were then washed, and JAK2 autophosphorylation was measured by eluting the labeled proteins into SDS-PAGE buffer and was analyzed on a 7.5% polyacrylamide gel. Bands corresponding to JAK2 were quantitated using the Fuji BAS-1000 phosphoimager. IC50 values were determined as described above. Full-length ZAP-70 kinase was produced using baculovirus expression. Lysates from Sf9 cells infected 48 h previously with a human ZAP-70 recombinant virus were prepared as described above for Lck, and a 1:100 dilution was used in a soluble kinase assay. Briefly, kinase activity was quantitated by measuring the incorporation of γ-32P into the substrate p62, using SDS-PAGE to resolve phosphorylated p62 and a phosphoimager to quantitate radioactivity. ZAP-70-specific activity was assessed by subtracting p62 phosphorylation obtained using Sf9 cell lysates infected with nonrecombinant baculovirus. IC50 values were determined as described above.

Solubility in Formulation 3: ≥ 1.67 mg/mL (5.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.