Potent inhibitor of JAK2
LY2784544 is a potent, selective inhibitor of JAK2 with IC50 value of 3 nM.[1]
Janus kinase 2 (JAK2) belongs to the Janus kinase family and is a member of the type II cytokine receptor family. It is a non-receptor tyrosine kinase. The JAK2 mutation (V617F) within the autoinhibitory pseudokinase domain results in a gain-of-function activation of JAK2.The JAK2V617F mutant results in subsequent phosphorylation of signal transducer and activator of transcription 5 (STAT5). Then, STAT5 regulates the transcription of genes associated with cell growth, death and differentiation. The JAK2V617F mutant is oncogenic in cells, and sufficient for generating the myeloproliferative neoplasms (MPN) phenotypes in murine models. Mutant JAK2 kinase is an attractive therapeutic target for the therapy of MPNs. To selectively inhibit JAK2V617F but minimizing inhibition of wild-type JAK2 would be an optimal treatment approach.
LY2784544 is a potent and ATP-competitive inhibitor of JAK2 tyrosine kinase. LY2784544 has 8- and 16-fold selectivity for JAK2 than JAK1 and JAK3, respectively. LY2784544 more effective on inhibiting the phosphorylation of STAT5 in Ba/F3-TEL-JAK2 cells than in Ba/F3-TEL- JAK3 and –JAK1 cells with IC 50 of 0.191 (JAK2), 2.904 (JAK1) and 4.744 nM (JAK3). LY2784544 significantly inhibited STAT5 phosphorylation at a concentration range from 50 nM–20 nM in Ba/F3 cells expressing JAK2V617F. On the other hand, LY2784544 showed minimal inhibition of STAT5 phosphorylation at 50 and 200 nM concentrations in IL-3-stimulated Ba/F3 cells which express wild-type JAK2. LY2784544 significantly inhibited the cell proliferation of JAK2V617F-expressing cells with IC50 ＝55 nM. In JAK2V617F-expressing cells, LY2784544 induced apoptosis with EC50±s.d.of 113±0.023 nM measured by Caspase3/7-glo assays.[1]
LY2784544 with a TED50 (threshold effective Dose 50) of 12.7 mg/kg, potently inhibits STAT5 phosphorylation in SCID mice bearing Ba/F3-JAK2V617F-GFP. In mouse hematologic disease model, LY2784544 also dose-dependly reduces Ba/F3-JAK2V617F-GFP tumor burden.[1]
参考文献:
1.Ma L, Clayton JR, Walgren RA, Zhao B, Evans RJ, Smith MC, Heinz-Taheny KM, Kreklau EL, Bloem L, Pitou C et al: Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F. Blood Cancer J 2013, 3:e109.
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Cell experiment: |
Ba/F3 cells expressing JAK2V617F are placed in RPMI-1640-containing vehicle (DMSO) or Gandotinib (LY2784544) (concentration range, 0.001-20 μM) (1×104 cells/96-well). Ba/F3 cells expressing wild-type JAK2 are treated similarly except IL-3 (2 ng/mL) is added. After a 72-hour incubation, cell proliferation is assessed by adding Cell Titer 96 Aqueous One Solution Reagent (20 μL/well). The IC50 for inhibition of cell proliferation is calculated using the GraphPad Prism 4 software[1]. |
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Animal experiment: |
Mice[1]Dose- and time-dependent in vivo inhibition of JAK2V617F signaling is assessed by measuring inhibition of STAT5 phosphorylation in a mouse ascitic tumor model. Ba/F3-JAK2V617F-GFP cells (1×107) are implanted in the intraperitoneal cavity of severe combined immunodeficiency mice (SCID mice) and allowed to develop into ascitic tumors for 7 days. For dose-response studies (six animals/group), Gandotinib (LY2784544) is administered once by oral gavage (2.5, 5, 10, 20, 40, or 80 mg/kg), then 30 min later, ascitic tumor cells are collected, fixed, incubated for 2 h with Mouse-anti-pSTAT5 (pY694) Alexa Fluor 647 (1:10 dilution), and analyzed by flow cytometry. Time course studies are performed similarly, except the animals are treated with Gandotinib (LY2784544) at 20, 40 or 80 mg/kg and ascitic tumor cells collected at prespecified intervals of 0.25-6 h after dosing. Data are analyzed by the one-way analysis of variance, and Dunnett's test (α=0.05). Dose response data are analyzed with a four-parameter logistic curve-fitting program. |
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参考文献: [1]. Ma L, et al. Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F. Blood Cancer J. 2013, 3, e109. |
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