Comparison of Two Homogeneous Cell-Based Kinase Assays for Jak2 V617f: Surefire Pstat5 and Geneblazer Fluorescence Resonance Energy Transfer Assays

The Janus kinase (JAK)/Signal Transducers and Activators of Transcription (STAT) signaling pathway plays an important role in cellular responses to cytokines and growth factors. Aberrant, constitutive activation of JAK2 signaling has been implicated in myeloproliferative disorders (MPDs) with a single, activating somatic V617F mutation in the JH2 pseudokinase domain of JAK2 as the prevalent molecular lesion. In order to discover small molecule inhibitors for this target, cell‐based kinase assays are important for identifying potential inhibitors. Here, we present two homogeneous 384‐well plate cell‐based assays utilizing Invitrogen's CellSensor® JAK2 V617F irf1‐bla HEL cell line: (1) SureFire(tm) pSTAT5 AlphaScreen® assay from PerkinElmer; and (2) GeneBLAzer® fluorescence resonance energy transfer (FRET) assay from Invitrogen. The SureFire(tm) assay measures levels of phosphorylated STAT5 while the GeneBLAzer® assay monitors ?‐lactamase activity in cells. The S/B ratios for the SureFire(tm) and GeneBLAzer® assays were 10 and 3, respectively. The experimental procedures will be compared, as well as the potency of select compounds. In conclusion, both assays are suitable for compound profiling.