Modification of CellSensor irf1-bla TF-1 and irf1-bla HEL assays for direct comparison of wild-type JAK2 and JAK2 V617F inhibition.
The Janus kinase (JAK)-signal transducer and activator of transcription pathway is an important therapeutic target because of its role in the regulation of cell growth. Aberrant, constitutive activation of JAK2 signaling has been implicated in myeloproliferative disorders with a single, activating somatic V617F mutation in the JH2 pseudokinase domain of JAK2 as the prevalent molecular lesion. Invitrogen has developed the CellSensor(®) cell lines interferon regulatory factor-1 (irf1)-beta-lactamase (bla) TF-1 and irf1-bla HEL for use in evaluating inhibitors of wild-type JAK2 and mutant JAK2 V617F, respectively. Both contain a bla reporter gene downstream of the irf1 response element stably integrated into either TF-1 or HEL cells. A fluorescence resonance energy transfer-based bla substrate is utilized to give a robust detection of JAK2 activity. Examination of Invitrogen's protocols for the two cell lines revealed significant differences that are not conducive to direct comparison of inhibitor activities against wild-type and mutant JAK2. Systematic changes to standardize the two assays were incorporated and evaluated for effects on assay response ratio, assay quality, and potency for a diverse series of inhibitors.
Lead Discovery and Profiling, Worldwide Discovery Research, Cephalon Inc., West Chester, Pennsylvania.
Jennifer L
JL
Mason
Beverly P
BP
Holskin
Kristen A
KA
Murray
Sheryl L
SL
Meyer
Kevin J
KJ
Wells-Knecht
Mark A
MA
Ator
Thelma S
TS
Angeles
D001681:Biological Assay / Q000379:methods*; D002460:Cell Line / Q000187:drug effects / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000378:metabolism*; D053614:Janus Kinase 2 / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*
C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D047428:Protein Kinase Inhibitors; D053614:Janus Kinase 2