NVP-BSK805 2HCl

The human JAK2 kinase domain (amino acids 840-1132) is contained in plasmid construct pAcG2TtevJAK2. The plasmid constructs for JAK3 (813-1124), TYK2 (888-1187), and JAK1(866-1154) are designed. The generation of the recombinant baculoviruses with BD BaculoGold^TM Bright linearized DNA, plaque assay, and virus amplification from single plaques is performed according to the manual (BD Biosciences Pharmingen). Janus kinase domains are expressed in Sf9 cells in 400 mL shake flasks with 100 mL ExCell420 culture medium (JRH Biosciences Ltd) with penicillin/streptomycin solution for 48 h at 27°C. Suspension culture cells are infected at a density of 1 × 10⁶ and the multiplicity of infection (MOI) for each virus is optimized for yield of soluble protein. The kinase domain of human JAK2 and of JAK1, JAK3, and TYK2 is expressed at an MOI of 1 and 0.5, respectively. Time of expression at 27°C is 48 hours for JAK2 and 48 hours or 72 hours for JAK1, JAK3, and TYK2. Forty-eight or seventy-two hours post-infection, the cells from a 100 mL expression culture are harvested by centrifugation at 3000 x g for 5 minutes and lysed with 12 mL lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 1 mM sodium orthovanadate, 1 % Triton X-100, 10 % glycerol, 1 x EDTA-free complete protease inhibitor cocktail (Roche Diagnostics), and 12.5 U/mL Benzonase for 30 minutes at 4°C, followed by centrifugation at 14,000 × g for 45 minutes to pellet insoluble material. For GST-tag affinity purification of kinase domain proteins all steps are performed at 4°C. The cleared lysates are incubated with 0.2 mL of a 50 % slurry of washed Glutathione Sepharose 4B for 2 hours at 4°C, followed by 5 washes with 1 mL of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 % Triton X-100, 1 mM DTT, and 10 % glycerol. Bound protein is eluted in 5 aliquots each starting with a 10 minutes incubation with 0.25 mL elution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 % Triton X-100, 1mM DTT, 10 % glycerol, 10 mM reduced L-glutathione). Eluates are concentrated about 5-fold with Amicon Ultra-4 spin columns. After addition of Brij35 to 0.1 % final concentration the protein is snap frozen in small aliquots and stored at -80 °C. In these conditions, kinase activities are stable for at least 6 months. The JAK kinasedomain enzymes are incubated for 30 minutes at room temperature in a medium containing 0.1 μM [γ³³P]-ATP, 1 mM MnCl₂, 5 mM MgCl₂, 30 μM of synthetic peptide substrate EQEDEPEGDYFEWLE, 1 mM DTT, 1 % DMSO, 50 μg/mL BSA, 0.01 % Brij35, and 50 mM Tris-HCl pH 7.5. The ATP concentration is below the Km for all proteins. Curves are fitted by non-linearregression using the logistic equation and the global fit function of XLfit® (model 205). Expression and characterization of full-length wild type and V617F mutant JAK2 as well as kinase assay conditions have been described elsewhere. Kinase selectivity of NVP-BSK805 is assessed in an internal kinase panel: In the Caliper assays, kinase reactions are carried out with peptide substrates that migrate with different velocities in an electrical field when phosphorylated. The peptides carry a fluorescent label in order to allow the detection and quantification of the peptides in a capillary system. Peptide fluorescence intensities are quantified using the LC3000 instrument (Caliper Life Sciences, Hopkinton, MA, USA). Kinase activity is measured by quantifying the amount of ATP remaining in solution following a kinase reaction. In the LanthaScreen™ TR-FRET kinase assays, terbium is used as the lanthanide chelate combined with an antibody directed against the phosphorylated substrate.