A receptor-independent, cell-based JAK activation assay for screening for JAK3-specific inhibitors.
New immunosuppressive compounds with less systemic toxicity that could replace calcineurin inhibitors are urgently needed. For identification of specific inhibitors of JAK3, a potential new drug target, from large chemical libraries we developed a cell-based screening system. TEL-JAK fusion proteins composed of an oligomerization domain of TEL and kinase and/or pseudokinase domains of JAKs provided constitutive activation of JAKs without receiving a signal from the cytokine receptors. These fusion proteins also induced STAT5b phosphorylation in the absence of cytokine receptors. Both the kinase and pseudokinase domains of JAKs were required for full activation of the JAKs, and four copies of STAT5 response elements provided the greatest luciferase activity. The sensitivity and specificity of the system was evaluated using specific JAK3, JAK2, or MEK inhibitors. Thus, we generated a receptor-independent, cell-based selective screening system for specific JAK3 inhibitors, which is easily convertible to a high-throughput screening platform.
Laboratory of Immunology, Department of Anatomy, Seoul National University College of Medicine, Republic of Korea.
Keunhee
K
Oh
Kyeung Min
KM
Joo
Yong Sik
YS
Jung
Jaehwan
J
Lee
Heonjoong
H
Kang
Hee-Yoon
HY
Lee
Dong-Sup
DS
Lee
D001665:Binding Sites; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D053616:Janus Kinase 3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D010880:Piperidines; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D050783:Proto-Oncogene Proteins c-ets / Q000235:genetics / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020218:Response Elements; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020032:Tyrphostins / Q000494:pharmacology; D000097764:ETS Translocation Variant 6 Protein
D002070:Butadienes; D007166:Immunosuppressive Agents; D053614:Janus Kinase 2; D053616:Janus Kinase 3; D020930:MAP Kinase Kinase Kinases; D009570:Nitriles; D010880:Piperidines; D047428:Protein Kinase Inhibitors; D050783:Proto-Oncogene Proteins c-ets; D011743:Pyrimidines; D011758:Pyrroles; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D050799:STAT5 Transcription Factor; D020032:Tyrphostins; D000097764:ETS Translocation Variant 6 Protein; C494095:STAT5B protein, human; C113580:U 0126; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; C479163:tofacitinib; C507933:JAK3 protein, human