| Description |
AZD-1480 is a novel ATP-competitive JAK2 inhibitor with IC50 of < 0.4 nM, selectively against JAK3 and Tyk2, and to a smaller extent against JAK1.
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| Related Catalog |
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| Target |
JAK2:0.4 nM (IC50)
JAK1:1.3 nM (IC50)
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| In Vitro |
AZD1480 (5μM) induces G2/M arrest and cell death by inhibiting Aurora kinases[1]. AZD1480 is a potent JAK2 inhibitor that can suppress growth, survival, as well as FGFR3 and STAT3 signaling and downstream targets including Cyclin D2 in human multiple myeloma cells. At low micromolar concentrations, AZD1480 blocks cell proliferation and induces apoptosis of myeloma cell lines[2]. AZD1480 effectively blocks constitutive and stimulus-induced JAK1, JAK2, and STAT-3 phosphorylation in both human and murine glioma cells, and leads to a decrease in cell proliferation and induction of apoptosis[3]. AZD1480 is a potent, competitive small-molecule inhibitor of JAK1/2 kinase, and that it is capable of inhibiting STAT3 phosphorylation and tumor growth in a STAT3-dependent manner. AZD1480 inhibits tumor angiogenesis and metastasis in part by affecting the tumor microenvironment[4].
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| In Vivo |
AZD1480 inhibits the STAT3 phosphorylation in an xenograft model of human solid tumors and multiple myeloma[1]. In vivo, AZD1480 inhibits the growth of subcutaneous tumors and increases survival of mice bearing intracranial glioblastoma (GBM) tumors by inhibiting STAT-3 activity, indicating that pharmacologic inhibition of the JAK/STAT-3 pathway by AZD1480 should be considered for study in the treatment of patients with GBM tumors[3]. AZD1480 blocks lung infiltration of myeloid cells and formation of pulmonary metastases in both mouse syngeneic experimental and spontaneous metastatic models. Furthermore, AZD1480 reduces angiogenesis and metastasis in a human xenograft tumor model[4]. AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity[5].
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| Kinase Assay |
Inhibition studies of AZD1480 are performed using recombinant Jak1, Jak2, or Jak3 under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween-20, 50 mM/mL BSA, and 10 mM MgCl2. Jak3 enzyme is expressed as N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes are assayed in the presence of AZD1480 (10 point dose response, in triplicate, from 8.3 μM to 0.3 nM in half-log dilution steps) using 1.5 μM peptide substrate (Jak1: FITC-C6-KKHTDDGYMPMSPGVA-NH2, Jak2 and Jak3: FAM-SRCtide) and screened under their respective ATP Km (Jak1: 55 μM, Jak2: 15 μM, Jak3: 3 μM) and approximated physiological ATP concentration of 5 mM. Phosphorylated and unphosphorylated peptides are separated and quantified by a Caliper LC3000 system for calculating percent inhibition.
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| Cell Assay |
Renca or 786-O cells are suspended in DMEM medium with 5% FBS, and seeded in 96-well plates (5×103 per well) to allow adhesion and then treated with DMSO or AZD1480 for 48 hours. Cell viability is determined by MTS assay. Absorbance at 490 nm is measured with Mikrotek Laborsysteme. Mouse endothelial cells and splenic CD11b+/c- myeloid cells are enriched from tumor-bearing mice,and cultured in 5% FBS RPMI-1640 medium. HUVECs are cultured on collagen 1-coated plates in complete medium. All cells are treated with DMSO and AZD1480 at various doses for 24 hours. Cell viability is determined by counting cell number manually. All the experiments are repeated 3 times.
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| Animal Admin |
For subcutaneous (s.c.) tumor model, 2.5×106 Renca or 786-O cells suspended in 100 μL PBS are injected into the flank of BALB/c or nude mice, respectively. When average tumor volume reaches approximately 100-150 mm3, AZD1480 or vehicle is administered by oral gavage either once a day at the dose of 50 mg/kg, or twice daily at 30 mg/kg, as indicated. Tumor size is measured by caliper every other day. For experimental lung metastasis model, 0.1×106 Renca or 1×106 786-O cells suspended in 500 μL PBS are injected via tail vein to BALB/c or nude mice, respectively. Three days later, mice are orally treated with AZD1480 (50 mg/kg/d) or vehicle for 21 days for Renca tumors and 60 days for 786-O tumors respectively. For the Calu-6 model, 3×106 tumor cells in matrigel are implanted s.c. into the flanks of nude mice, randomized into vehicle (twice daily, BID) and drug treatment (AZD1480, 30 mg/kg BID) groups, and dosed orally daily for 19 days. For spontaneous lung metastasis model, 2×105 4T1 cells suspended in 100 μL PBS are injected in the mammary gland of female BALB/c mice by gently penetrating the skin. AZD1480 (50 mg/kg/d) or vehicle is given orally for 21 days.
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| References |
[1]. Derenzini E, et al. The JAK inhibitor AZD1480 regulates proliferation and immunity in Hodgkin lymphoma. Blood Cancer J. 2011 Dec;1(12):e46. [2]. Scuto A, et al. The novel JAK inhibitor AZD1480 blocks STAT3 and FGFR3 signaling, resulting in suppression of human myeloma cell growth and survival. Leukemia. 2011 Mar;25(3):538-50. [3]. McFarland BC, et al. Therapeutic potential of AZD1480 for the treatment of human glioblastoma. Mol Cancer Ther. 2011 Dec;10(12):2384-93. [4]. Xin H, et al. Antiangiogenic and antimetastatic activity of JAK inhibitor AZD1480. Cancer Res. 2011 Nov 1;71(21):6601-10. [5]. Hedvat M, et al. The JAK2 inhibitor AZD1480 potently blocks Stat3 signaling and oncogenesis in solid tumors. Cancer Cell. 2009 Dec 8;16(6):487-9 [6]. Ni J, et al. Tyrosine receptor kinase B is a drug target in astrocytomas. Neuro Oncol. 2017 Jan;19(1):22-30.
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