| In Vitro | In vitro activity: Pacritinib is a potent inhibitor of both wild-type JAK2 and JAK2V617F mutant (IC50= 19 nM) that is present in high frequencies among patients with MPD. Relative to JAK2, Pacritinib is two-fold less potent against TYK2 (IC50= 50 nM), 23-fold less potent against JAK3 (IC50= 520 nM) and 56-fold less potent against JAK1 (IC50= 1280 nM). Pacritinib effectively permeates cells to modulate signaling pathways downstream of JAK2, whether agonist activated or mutationally activated. Pacritinib induces apoptosis, cell cycle arrest and antiproliferative effects in JAK2WT- and JAK2V617F-dependent cells. Pacritinib inhibits cell proliferation of Karpas 1106P and Ba/F3-JAK2V617F with IC50 of 348 and 160 nM, respectively. Pacritinib inhibits endogenous colony growth derived from erythroid and myeloid progenitors with IC50 of 63 and 53 nM , respectively. SB1518 also inhibits FLT3 and its mutant FLT3-D835Y(IC50= 6 nM ). Pacritinib inhibits FLT3 phosphorylation and downstream STAT, MAPK and PI3K signaling in FLT3-internal-tandem duplication (ITD), FLT3-wt cells and primary AML blast cells. Pacritinib treatment leads to a dose-dependent decrease of pFLT3, pSTAT5, pERK1/2 and pAkt in FLT3-ITD harboring MV4-11 cells with IC50 of 80, 40, 33 and 29 nM , respectively. Treatment of the primary AML blast cells with Pacritinib for 3 h leads to a dose-dependent decrease of pFLT3, pSTAT3 and pSTAT5 with an IC50 below 0.5 μM. Pacritinib induces apoptosis, cell cycle arrest and anti-proliferative effects in FLT3-mutant and FLT3-wt cells. Pacritinib inhibits cell proliferation of FLT3-ITD-harboring cells MV4-11 and primary AML blast cells with IC50s of 47 nM and 0.19-1.3 μM, respectively.
Kinase Assay: All assays are carried out in 384-well white microtiter plates. Compounds are 4-fold serially diluted in 8 steps, starting from 10 μM. The reaction mixture consisted of 25 μL assay buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 5 mM β-glycerol phosphate). For FLT3 assays, the reaction contains 2.0 μg/mL FLT3 enzyme, 5 μM of poly(Glu,Tyr) substrate and 4 μM of ATP. For JAK1 assays, the reaction contains 2.5 μg/mL of JAK1 enzyme, 10 μM of poly(Glu,Ala,Tyr) substrate and 1.0 μM of ATP. For JAK2 assays, the reaction contained 0.35 μg/mL of JAK2 enzyme, 10 μM of poly (Glu,Ala,Tyr) substrate and 0.15 μM of ATP. For JAK3 assays, the reaction contained 3.5 μg/mL of JAK3 enzyme, 10 μM of poly (Glu,Ala,Tyr) substrate and 6.0 μM of ATP. For TYK2 assays, the reaction contained 2.5 μg/mL of TYK2 enzyme, 10 μM of poly (Glu,Ala,Tyr) substrate and 0.15 μM of ATP. The reaction is incubated at room temperature for 2 h prior to addition of 13 μL PKLight® detection reagent. After 10 min incubation luminescent signals are read on a multi-label plate reader.
Cell Assay: Cells (Karpas 1106P cells) are seeded at 30-50% confluency in 96-well plates and are treated with different concentrations of compounds (in triplicate) for 48 h. Cell viability is monitored using the CellTiter-Glo assay. |
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