Alisertib (MLN8237)

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 Hexadecadrol, as well as additive effect with doxorubicin and LDP-341. ^[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 NSC 119875(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]

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]

• 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.

• 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

• Method

  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 Resorcinolphthalein isothiocyanate-annexin V and PI. Apoptotic cells are determined by flow cytometric analysis using BDFACS-Canto II and FlowJo software.

• Animal Models

  Severe combined immune-deficient (SCID) mice inoculated subcutaneously with MM1.S cells

• Dosages

  ~30 mg/kg/day

• Administration

  Orally