title: Comparison of two homogeneous cell-based kinase assays for JAK2 V617F: SureFire pSTAT5 and GeneBLAzer fluorescence resonance energy transfer assays.
abstract: The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays an important role in cellular responses to cytokines and growth factors. Recent studies have identified a recurrent somatic activating mutation (JAK2 V617F) in majority of patients with myeloproliferative disorders (MPDs). Development of drugs that target JAK2 V617F is, therefore, of therapeutic relevance. To discover small molecule inhibitors for this target, robust and reliable cell-based assays are important. Here, we present a comparison of two homogeneous, 384-well plate-based cellular assays using Invitrogen's CellSensor® JAK2 V617F interferon regulatory factor-1 (irf1)-beta-lactamase (bla) human erythroleukemia line (HEL): (1) SureFire® pSTAT5 AlphaScreen® assay from PerkinElmer; and (2) GeneBLAzer® fluorescence resonance energy transfer assay from Invitrogen. HEL cells are growth factor-independent due to JAK2 V617F mutation that causes constitutive STAT5 activation. The SureFire assay measures levels of phosphorylated STAT5 downstream of JAKs, while the GeneBLAzer assay is a reporter assay that monitors bla activity further downstream of STAT5. Evaluation of a number of chemically diverse JAK2 inhibitors in the two cellular assays yielded comparable half-maximal inhibitory concentration (IC₅₀) values, boding well for the utility of these assay formats in compound profiling.
authors: Worldwide Discovery Research, Cephalon, Inc., West Chester, Pennsylvania 19380, USA.
authors: Jie
authors: J
authors: Qian
authors: Jennifer L
authors: JL
authors: Mason
authors: Beverly P
authors: BP
authors: Holskin
authors: Kristen A
authors: KA
authors: Murray
authors: Sheryl L
authors: SL
authors: Meyer
authors: Mark A
authors: MA
authors: Ator
authors: Thelma S
authors: TS
authors: Angeles
mesh_terms: D002460:Cell Line; D055808:Drug Discovery / Q000379:methods; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics*; D009154:Mutation / Q000502:physiology; D009196:Myeloproliferative Disorders / Q000201:enzymology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D050799:STAT5 Transcription Factor / Q000235:genetics*; D054852:Small Molecule Libraries; D012984:Software
chemical_list: D047428:Protein Kinase Inhibitors; D050799:STAT5 Transcription Factor; D054852:Small Molecule Libraries; C507924:JAK2 protein, human; D053614:Janus Kinase 2