Samples were added to the test plate glass filter, scintillation and Z hlungen Harvested per minute were determined using a Packard TopCount. A3 receptor functional: This test measures the ability F antagonists inhibited by A3 AB MECA I GTPgS induced cell membranes. The test was carried out as described in the functional assay A1 Chinese Hamster Ovary cells stably transfected with the human receptor A3. Inhibition of PDE isoenzymes PDE1 nucleotide was purified from human lung from EX 527 patients operated on for cancer. PDE2 and 5 were purified from platelet concentrates obtained from the local blood transfusion center. Purification of human PDE3 and cloning and expression of human PDE4A, PDE4B 4D and rats were performed as previously described. PDE activity T is using cAMP or cGMP as substrate. A test kinase kinase phosphorylated form of human p38a His MAP was used to phosphorylate the substrate immobilized GST ATF 1, cold in the presence of 120 mM ATP.
The phosphorylated GST ATF 1 was polyclonal rabbit Antique Body, by biotin labeled goat anti-IgG, streptavidin-alkaline A66 phosphatase and the substrate is detected followed. Their phosphorylated form p38ba His p38d and MAP kinases JNK1 human origin were used to phosphorylate the immobilized substrate GST ATF 2 in the presence of cold ATP. Other kinase inhibition assays in conditions for each kinase and ATP concentrations similar Km of the enzyme with the respective performed optimized ATP: 8 mm, 1 mm, 13 mm, 2 mm, 20 mm, 30 mm and 7.5 mm. For tyrosine kinases, with filter binding assays GST recombinant kinase fused Dom NEN Receptors were used in baculovirus expressed and purified on glutathione-Sepharose.
ATP was used as a phosphate donor, and the peptide was used as an acceptor polyGluTyr. The human leukocyte-based assays All assays were performed as previously described with isolated cells from the blood of healthy volunteers. Neutrophils were stimulated with formyl Met-Leu-Phe and the F ability Of the cells to produce superoxide anions when oxidative metabolism was stimulated. Using a test of the reduction of cytochrome c The mononuclear Ren Cells were stimulated with either anti-CD3 monoclonal or LPS and interferon-gamma and TNF IFN g of a measurement. After incubation for 20 h at 371C, 5% CO2, the Cured Nde harvested and cytokine levels were measured by commercially available sandwich enzyme immunoassay. The crystal structures of molecular modeling of human p38 MAP kinase and phosphodiesterase PDE 4B2B were downloaded from the Brookhaven Protein Data Bank.
PDB figures 1WFC and 1F0J. The crystal structures were added in Sybyl loaded and hydrogen atoms. CGH2466 was bound hand in the pocket of the ATP binding of p38. The ligand and the Reset Nde sen were a gradient of 0.05 kcal mol 1 1A ˚ relaxed poor contact with the Tripos force field to L. The connection was established manually CGH2466 followed in the active site of PDE4 by a molecular dynamics simulation of 50 ps and the relaxation of the ligand residues and by 0.05 kcal mol 1A ˚ 1 using the Tripos force field. In vivo models of female BALB / usen cM Or C57BL / 6 Mice were purchased from Harlan. The animals were in plastic K Housed cages in a conditioned room at 241C.