Protein kinase inhibitor activity Concentration dependent inhibition of protein kinase activity was done essentially as previously described. Active p38 SAPK2 enzyme was obtained from Milli pore, Veliparib ABT-888 and bovine myelin basic protein sub strate was obtained from Sigma Aldrich. Enzyme activity assays were done in a final volume of 50l and each point was tested in duplicate. Assays were done by incubation with 0. 33 mg ml myelin basic protein, 100M ATP and ATP in assay buffer. Reactions were initiated by addition of the active p38 kinase at a final concentration of 2g ml, and incu bated for 10 min at 30 C. Reactions were stopped by transfer of a 35l aliquot of the assay mixture onto P81 paper, washes with 75 mM phos phoric acid and 95% ethanol, and quantification by scin tillation counting.
When compounds were tested for kinase inhibitory activity, 10X stock solutions of com pounds were prepared in DMSO or water, and control samples contained the same final concentration Inhibitors,Modulators,Libraries of solvent as the samples containing compound. Data are expressed as percent of the maximal enzyme activity, where enzyme activity in the absence of compound is taken as 100%. IC50 values were Inhibitors,Modulators,Libraries calculated by a nonlinear regression data analysis using Microsoft Excel. Selectivity against pathway or structurally related protein kinases Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries was done using purified kinases and standard in vitro assays described pre viously, and included use of the Millipore kinase screening service at 20M final concentration of 069A and 90M final concentration of ATP.
Oral bioavailability and brain uptake analysis Analysis of oral bioavailability and brain uptake was done by a modification of that previously described. Briefly, 069A was administered to C57Bl 6 mice by oral gavage in a 0. 5% carboxymethylcellulose suspension. At 5, 15, 30, 60, and 120 min after compound administration, Inhibitors,Modulators,Libraries blood was collected in heparinized tubes from anesthe tized animals and plasma obtained by centrifugation. After perfusion, brains were harvested, homogenized in 0. 1% formic acid and deproteinized with ice cold ace tonitrile. After centrifugation to remove precipitated pro tein, the brain homogenate supernatants were further diluted with 0. 1% formic acid. The plasma samples were acidified by diluting with 0. 1% formic acid. Solid phase extraction followed by HPLC analysis was used to quantify the amount of compound in the plasma and brain supernatants, with compound MW01 6 189WH used as an internal standard.
Briefly, 30 mg car tridges were conditioned with 1 mL of acetonitrile and equilibrated with 1 mL of water. Acid ified samples were loaded onto the cartridge followed by a 1 mL wash with 5% acetonitrile. Compound was eluted from the cartridge using selleckchem Idelalisib 100% acetonitrile. The eluate was evaporated to dryness, reconstituted in 60% acetonitrile 0. 1% formic acid water and analyzed by HPLC. Recovery of internal standard and compound 069a was approxi mately 70%.