Biochemical and cellular characterization of a selective Janus kinase 2 (JAK2) inhibitor, CEP-33779. (A) Enzymatic inhibitory activities of CEP-33779 against JAK2 and family members. Inhibition of the kinase activity of each recombinant enzyme (JAK2 and JAK3) by CEP-33779 was evaluated using a plate-based, time-resolved fluorescence detection system. The effect in a cellular system was measured using irf-bla TF-1 cells (JAK2) and Ba/F3 JAK3 cells not tested for JAK1 and TYK2. Half maximal inhibitory concentration (IC50) values are reported as the averages ± SD of at least four independent determinations. FAK, focal adhesion kinase. (B) In vitro activity of CEP-33779 in HEL92 cells and potent JAK2 inhibition as determined by the inhibition of phosphorylation of downstream target signal transducer and activator of transcription 5 (pSTAT5), vehicle (veh). HEL92 cells were treated with increasing concentrations of CEP-33779 as indicated for 1 hour in serum-free media. Extracts were prepared in a Triton X-100-based lysis buffer, protein concentrations were determined, and equal amounts were resolved on sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gels and blotted. STAT5 and pSTAT5 were analyzed using specific antibodies (see Materials and methods). Blots were scanned, and signals for each group were determined using GelPro as phosphor/total. Prism software was used to calculate the IC50 values. (C) In vivo pharmacodynamic (PD) in which mice were dosed orally (p.o.) with 55 mg/kg CEP-33779 before the removal of HEL92 tumor extracts (2 hours postinjection) and quantitation of STAT5 and pSTAT5 levels using Western blot analysis. Each lane represents an individual sample. Profiling studies for CEP-33779 were repeated for HEL92 cells and performed in several tumor lines both in vitro and in vivo, reconfirming JAK2 inhibition (data not shown).