Thirty three patients were enrolled in the phase 2 portion; all patients had re

The search model was a crystal structure of Jak2 reported previously, Coordinates were generated tumor based on the molecu lar replacement solution. The refinement of the Tyk2 Compound 1 complex crystal structure began with the molecular replacement solution coordinates. Rigid body refinement was conducted by the program REFMAC in the CCP4 suite of programs, which resulted in the fol lowing statistics at 2. 6 : R 0. 39, Experimen tal Tyk2 and inhibitor electron density was observed. Manual building of Compound 1 into the density was attempted using the molecular graphics program O and examination of 2Fo Fc and Fo Fc electron density maps. The refinement program REFMAC was used for it erative rounds of restrained refinement, Final rounds of refinement were conducted using AUTOBUSTER, which added water molecules to the final model, resulting in the following statistics: R 0.<br><br> 199, Final refinement statistics Lenalidomide ic50 are shown in Table 3. The quality of all models was evaluated using COOT, The co crystal structure of Compound 2 complexed to Tyk2 was solved by molecular replacement using the Tyk2 Compound 1 structure as a probe. An ori gin shift of was applied to match the Compound 1 coordinates. DETWIN was used with a twinning frac tion of 0. 24 to improve refinement statistics. Final rounds of refinement were conducted using AUTOBUSTER, Final refinement statistics are listed in Table 3. Time resolved fluorescence resonance energy transfer kinase activity assays Tyk2 6 nM purified human Tyk2 enzyme was mixed with 2 uM peptide substrate GAEEEIYAAFFA COOH at varying concentrations of inhibitor in reaction buffer: 50 mM MOPSO pH 6.<br><br> 5, 10 mM MgCl2, 2 mM MnCl2, LY2603618 臨床試験 1 uM ATP, 2. 5 mM DTT, 0. 01% BSA, and 0. 1 mM Na3VO4. After 60 min incubation at room temperature, the reaction was quenched by addition of EDTA and developed by addition of revelation reagents and 3. 12 ug mL SAXL, The developed reaction was incubated in the dark either at 4 C over night or at room temperature for 1 h, then read with a time resolved fluorescence detector using a 337 nm laser for excitation and emis sion wavelengths of 620 nm and 665 nm. Within the lin ear range of the assay, this signal is directly related to phosphorylated product and was used to calculate IC50 values. Typically, seven point inhibitor dilutions were used.<br><br> IC50 values were calculated by fitting the following equation: where is total inhibitor concentration, Y is the per centage of activity at a given inhibitor concentration, and Ymax is the maximum activity generated in the absence of inhibitor. Jak1, Jak2 and Jak3 Purified Jak2 and Jak3 were purchased from Upstate Millipore, Jak1 was expressed in Sf9 cells as a GST fusion protein and purified in house, and used in trFRET kinase assays in the reaction buffer described above. Jak1 and Jak3 assays used substrate peptide biotin TYR2 AEEEYFFLFA amide while the Jak2 assay used biotin TYR1 GAEEEIYAAFFA COOH. Mouse Tyk2 kinase domain was incubated for 0 to 90 min utes with thermolysin at room temperature in 50 mM HEPES pH 6. 7, 150 mM NaCl, 5% glycerol, and 2. 5 mM CaCl2 in the presence and absence of 30 uM Compound 2. EDTA was used as stop solution to quench the proteolysis reactions.