title: Differential selectivity of JAK2 inhibitors in enzymatic and cellular settings.
abstract: Small molecule inhibitors of Janus kinase (JAK) family members (JAK1, JAK2, JAK3, and Tyk2) are currently being pursued as potential new modes of therapy for a variety of diseases, including the inhibition of JAK2 for the treatment of myeloproliferative disorders. Selective inhibition within the JAK family can be beneficial in avoiding undesirable side effects (e.g., immunosuppression) caused by parallel inhibition of other JAK members. In an effort to design an assay paradigm for the development of JAK2 selective inhibitors, we investigated whether compound selectivity differed between cellular and purified enzyme environments. A set of JAK2 inhibitors was tested in a high-throughput JAK family cell assay suite and in corresponding purified enzyme assays. The high-throughput JAK cell assay suite comprises Ba/F3 cells individually expressing translocated ETS leukemia (TEL) fusions of each JAK family member (TEL-JAK Ba/F3) and an AlphaScreen phosphorylated-STAT5 (pSTAT5) immunoassay. Compound potencies from the TEL-JAK Ba/F3 pSTAT5 assays were similar to those determined in downstream cell proliferation measurements and more physiologically relevant cytokine-induced pSTAT5 PBMC assays. However, compound selectivity data between cell and purified enzyme assays were discrepant because of different potency shifts between cell and purified enzyme values for each JAK family member. For any JAK small molecule development program, our results suggest that relying solely on enzyme potency and selectivity data may be misleading. Adopting the high-throughput TEL-JAK Ba/F3 pSTAT5 cell assay suite in lead development paradigms should provide a more meaningful understanding of selectivity and facilitate the development of more selective JAK inhibitors.
authors: Amgen, Thousand Oaks, California, USA. vyu@amgen.com
authors: Violeta
authors: V
authors: Yu
authors: Jeanne
authors: J
authors: Pistillo
authors: Ivonne
authors: I
authors: Archibeque
authors: Josie
authors: J
authors: Han Lee
authors: Bee-Chun
authors: BC
authors: Sun
authors: Laurie B
authors: LB
authors: Schenkel
authors: Stephanie
authors: S
authors: Geuns-Meyer
authors: Liqin
authors: L
authors: Liu
authors: Renee
authors: R
authors: Emkey
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D054448:Precursor Cells, B-Lymphoid / Q000187:drug effects* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D015203:Reproducibility of Results; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D000097764:ETS Translocation Variant 6 Protein
chemical_list: D053614:Janus Kinase 2; D015514:Oncogene Proteins, Fusion; D047428:Protein Kinase Inhibitors; D050799:STAT5 Transcription Factor; C491568:TEL-JAK2 fusion protein, mouse; D000097764:ETS Translocation Variant 6 Protein