A potent, selective JAK1/JAK2 inhibitor
Janus-
1.Leonard, W.J., and O'Shea, J.J.JAKS AND STATS: Biological implicationsAnnu. Rev. Immunol.16293-322(1998) 2.Yamaoka, K., Saharinen, P., Pesu, M., et al.The Janus kinases (Jaks)Genome Biol.5(12)253(2004) 3.Verstovsek, S.Therapeutic potential of JAK2 inhibitorsHematology Am. Soc. Hematol. Educ. Program636-642(2009) 4.Quintás-Cardama, A., Vaddi, K., Liu, P., et al.Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: Therapeutic implications for the treatment of myeloproliferative neoplasmsBlood115(15)3109-3117(2010)
Kinase experiment: | Recombinant proteins are expressed using Sf21 cells and baculovirus vectors and purified with affinity chromatography. JAK kinase assays use a homogeneous time-resolved fluorescence assay with the peptide substrate (-EQEDEPEGDYFEWLE). Each enzyme reaction is carried out with Ruxolitinib or control, JAK enzyme, 500 nM peptide, adenosine triphosphate (ATP; 1mM), and 2% dimethyl sulfoxide (DMSO) for 1 hour. The 50% inhibitory concentration (IC50) is calculated as Ruxolitinib concentration required for inhibition of 50% of the fluorescent signal[1]. |
Cell experiment: | Cells are seeded at 2 × 103/well of white bottom 96-well plates, treated with Ruxolitinib from DMSO stocks (0.2% final DMSO concentration), and incubated for 48 hours at 37°C with 5% CO2. Viability is measured by cellular ATP determination using the Cell-Titer Glo luciferase reagent or viable cell counting. Values are transformed to percent inhibition relative to vehicle control, and IC50 curves are fitted according to nonlinear regression analysis of the data using PRISM GraphPad[1]. |
Animal experiment: | Mice[1]Mice are fed standard rodent chow and provided with water ad libitum. Ba/F3-JAK2V617F cells (105 per mouse) are inoculated intravenously into 6- to 8-week-old female BALB/c mice. Survival is monitored daily, and moribund mice are humanely killed and considered deceased at time of death. Treatment with vehicle (5% dimethyl acetamide, 0.5% methocellulose) or Ruxolitinib begin within 24 hours of cell inoculation, twice daily by oral gavage. Hematologic parameters are measured using a Bayer Advia120 analyzed, and statistical significance is determined using Dunnett testing[1]. |
参考文献: [1]. Quintas-Cardama A, et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood, 2010, 115(15), 3109-3117. | |
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