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Research Area
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Description
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on-Hodgkin's lymphoma,cancer, Solid tumours |
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Biological Activity
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Description
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MLN8237 (Alisertib) is a selective Aurora A inhibitor with IC50 of 1.2 nM. |
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Targets
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Aurora A |
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IC50 |
1.2 nM [1] |
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In Vitro
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MLN8237 shows >200-fold higher selectivity for Aurora A than the structurally related Aurora B with an IC50 of 396.5 nM, and does not have any significant activity against 205 other kinases. [1] MLN8237 (0.5 μM) treatment inhibits the phosphorylation of Aurora A in MM1.S and OPM1 cells, without affecting the Aurora B mediated histone H3 phosphorylation. MLN8237 significantly inhibits cell proliferation in multiple myeloma (MM) cell lines with IC50 values of 0.003-1.71 μM. MLN8237 displays more potent anti-proliferation activity against primary MM cells and MM cell lines in the presence of BM stroma cells, as well as IL-6 and IGF-1 than against MM cells alone. MLN8237 (0.5 μM) induces 2- to 6-fold increase in G2/M phase in primary MM cells and cell lines, as well as significant apoptosis and senescence, involving the up-regulation of p53, p21 and p27, as well as PARP, caspase 3, and caspase 9 cleavage. In addition, MLN8237 shows strong synergistic anti-MM effect with dexamethasone, as well as additive effect with doxorubicin and bortezomib. [2] MLN8237 (0.5 μM) treatment causes the inhibition of colony formation of FLO-1, OE19, and OE33 esophageal adenocarinoma cell lines, and induces a significant increase in the percentage of polyploid cells, and subsequently an increase in the percentage of cells in the sub-G1 phase, which can be further enhanced in combination with cisplatin (2.5 μM), involving the higher induction of TAp73β, PUMA, NOXA, cleaved caspase-3, and cleaved PARP as compared with a single-agent treatment. [3] |
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In Vivo
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MLN8237 significantly reduces the tumor burden with tumor growth inhibition (TGI) of 42% and 80% at 15 mg/kg and 30 mg/kg, respectively, and prolongs the survival of mice compared with the control. [2] |
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Clinical Trials
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A Phase II study of MLN8237 for treatment of patients with ovarian, fallopian tube, or peritoneal carcinoma has been completed. |
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Features
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First orally available inhibitor of Aurora A |
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Combination Therapy
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Description
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MLN8237 (30 mg/kg) in combination with cisplatin (2 mg/kg) displays an enhanced anti-tumor activity against FLO-1xenografts, as compared with single-agent treatments, correlating with the suppressed Ki-67 expression, enhanced nuclear p73 protein and cleaved caspase 3 expression. [3] |
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Protocol
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Kinase Assay
[1]
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| Aurora A radioactive Flashplate enzyme assay |
Aurora A radioactive Flashplate enzyme assay is conducted to determine the nature and degree of MLN8237-mediated inhibition in vitro. Recombinant Aurora A is expressed in Sf9 cells and purified with GST affinity chromatography. The peptide substrate for Aurora A is conjugated with biotin (Biotin-GLRRASLG). Aurora A kinase (5 nM) is assayed in 50 mM Hepes (pH 7.5), 10 mM MgCl2, 5 mM DTT, 0.05% Tween 20, 2 μM peptide substrate, 3.3 μCi/mL [γ-33P]ATP at 2 μM, and increasing concentrations of MLN8237 by using Image FlashPlates. |
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Cell Assay
[2]
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| Cell Lines |
MM1.S, MM.1R, LR5, RPMI 8226, DOX40, OPM1, OPM2, INA6, and U266 |
| Concentrations |
Dissolved in DMSO, final concentrations ~10 μM |
| Incubation Time |
24, 48, and 72 hours |
| Methods |
Cells are exposed to various concentrations of MLN8237 for 24, 48, and 72 hours. Cells viability is measured using MTT assay, and cell proliferation is measured using 3[H]-thymidine incorporation. For cell cycle analysis, cells are permeabilized by 70% ethanol at -20 °C, and incubated with 50 μg/mL PI and 20 units/mL RNase-A. DNA content is analyzed by flow cytometry using BDFACS-Canto II and FlowJo software. For the detection of apoptosis and senescence, cells are stained with fluorescein isothiocyanate-annexin V and PI. Apoptotic cells are determined by flow cytometric analysis using BDFACS-Canto II and FlowJo software. |
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Animal Study
[2]
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| Animal Models |
Severe combined immune-deficient (SCID) mice inoculated subcutaneously with MM1.S cells |
| Formulation |
Formulated in 10% 2-hydroxypropyl-β-cyclodextrin/1% sodium bicarbonate |
| Doses |
~30 mg/kg/day |
| Administration |
Orally |
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References |
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[1] Manfredi MG, et al. Clin Cancer Res, 2011, 17(24), 7614-7624.
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[2] Görgün G, et al. Blood, 2010, 115(25), 5202-5213.
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[3] Sehdev V, et al. Mol Cancer Ther, 2012, 11(3), 763-774.
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