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Biological Activity
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Description
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TG101209 is a potent inhibitor of JAK2, Flt3 and RET with IC50 of 6 nM, 25 nM and 17 nM, respectively. |
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Targets
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JAK2 |
Flt3 |
RET |
JAK3 |
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IC50 |
6 nM |
25 nM |
17 nM |
169 nM [1] |
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In Vitro
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TG101209 is an orally bioavailable, small molecule, ATP-competitive inhibitor towards several tyrosine kinases. TG101209 inhibits growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC50 of B200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209 inducs cell cycle arrest and apoptosis, and inhibits phosphorylation of JAK2V617F, STAT5 and STAT3. TG101209 suppresses growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations. [1]TG101209 significantly reduces STAT5 phosphorylation without affecting the total amount of STAT5 protein. [2]TG101209 inhibits survivin and reduces phosphorylation of STAT3 in HCC2429 and H460 lung cancer cells. TG101209 results in radio sensitization of HCC2429 and H460 lung cancer cells in vitro. [3] A recent study indicates TG101209 abrogates BCR-JAK2 and STAT5 phosphorylation, decreases Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. [4] |
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In Vivo
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100 mg/kg of TG101209 effectively prolongs the survival in JAK2V617F-induced disease (10 days). Compared with placebo-treated animals, TG101209-treated animals exhibit statistically significant, dose-dependent reduction in the circulating tumor cell burden at day +11 to 20%. [1] |
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Clinical Trials
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Features
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Combination Therapy
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Description
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Combined TG101209 and radiation extends tumor growth delay, reduces tumor proliferation and vascular density and increases apoptosis in lung cancer mouse xenografts. [3]Cotreatment with TG101209 and panobinostat attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells. [5] |
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Protocol
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Kinase Assay
[1]
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| Cell-free Kinase Activity Assays |
IC50 values for TG101209 are determined using a luminescence-based kinase assay with recombinant JAK2, VEGFR2/KDR, and JAK3 obtained from Upstate Cell Signaling Solutions. Kinase reactions are carried out in a buffer consisting of 40mM Tris buffer (pH 7.4), 50mM MgCl2, 800?M EGTA, 350?M Triton X-100, 2?M ?-mercaptoethanol, 100?M peptide substrate, and an appropriate amount of JAK2, VEGFR2/KDR or JAK3 such that the assay is linear over 60 minutes. The reaction is initiated by the addition of 10?L of ATP to a final concentration of 3mM and terminated by the addition of Kinase-Glo reagent after 60 minutes. Luciferase activity is quantified using an Ultra 384 instrument set for luminosity measurements. IC50 values are derived from experimental data using the non-linear curve fitting capabilities of the GraphPad Prism 4.0 software. The single concentration inhibition data for a panel of 63 kinases is determined using the SelectScreen TM service. |
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Cell Assay
[1]
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| Cell Lines |
Ba/F3 cells expressing JAK2V617F (Ba/F3-EpoR-V617F) and MPLW515L (Ba/F3-W515L) mutants. |
| Concentrations |
4.6–38 400 nM |
| Incubation Time |
28-30 hours |
| Methods |
In brief, approximately 2 × 103 cells are plated into microtiterplate wells in 100 ml RPMI-1640 growth media with indicated concentrations of TG101209. The relative growth of cells is quantified at 24-hour intervals using Cell Proliferation Kit II (XTT) as per manufacturer’s guidelines. After incubation, 20 mL of XTT is added to the wells and allowed to incubate for 4–6 hours. The colored formazan product is measured spectrophotometrically at 450 nm with correction at 650 nm, and IC50 values are determined using the GraphPad Prism 4.0 software. Data are subjected to a non-linear regression-fit analysis and IC50 values are determined as the concentration that inhibited proliferation by 50%. All experiments are done in triplicate and the results normalized to growth of untreated cells. |
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Animal Study
[1]
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| Animal Models |
Ba/F3-V617F-GFP cells are injected into immunodeficient SCID mice to induce a rapidly fatal, fully penetrant hematopoietic disease. |
| Formulation |
TG101209 is dissolved in DMSO. |
| Doses |
100 mg/kg |
| Administration |
TG101209 is administered via oral gavage at the indicated doses beginning day +3 after tumor cell infusion and ending on day +20. |
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References |
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[1] Pardanani A, et al, Leukemia, 2007, 21(8), 1658-1668.
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[2] Ma AC, et al, Exp Hematol, 2009, 37(12), 1379-1386.
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[3] Sun Y, et al, J Thorac Oncol, 2011, 6(4), 699-706.
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[4] Cuesta-Dominquez A, et al, PLoS One, 2012, 7(2), e32451.
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[5] Wang Y, et al, Blood, 2009, 114(24), 5024-5033.
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