Alisertib (MLN 8237) is an Aurora A kinase inhibitor (IC50=1.2 nM) with oral activity and selectivity. Alisertib has antitumor activity, induces apoptosis and autophagy, and induces cell cycle arrest.

Aurora A:1.2 nM (cell free)

方法：从 MM 患者获得的一组 MM 细胞系和肿瘤细胞用 Alisertib (0.0001-4 µM) 处理 48-72 h，使用 MTT assay 检测细胞活力。
结果：尽管 Alisertib 的细胞毒性活性早在暴露于低于 0.1 µM/L 的 24-48 h 就在几种细胞系中检测到了，但在所有细胞系中，在 72 h 时都发生了更强烈的细胞毒性。[1]
方法：人结直肠癌细胞 HCT-116 用 Alisertib (0.05-1 µmol/L) 处理 24-48 h，使用 Flow cytometry 检测细胞周期。
结果：在浓度为 0.050 µmol/L 时，在 24 和 48 h 时，G2/M 期的细胞增加，表型与 Aurora A 抑制一致。在 0.250 和 1.000 µmol/L 的较高浓度下，Alisertib 处理的细胞显示出与 Aurora B 抑制一致的表型，显示 8N DNA 含量的细胞数量增加。[2]

方法：为研究抗肿瘤活性，将 Alisertib (7.5-30 mg/kg，10% 2-hydroxypropyl-β-cyclodextrin/1% sodium bicarbonate) 口服给药给携带 MM1.S 异种移植物的 SCID 小鼠，每天一次，持续二十一天。
结果：用 30 mg/kg Alisertib 治疗的动物的肿瘤负荷显著降低。与对照相比，15 mg/kg (TGI=42%) 和 30 mg/kg (TGI=80%) 处理的动物的 TGI 显著。[1]

Recombinant murine Aurora A and Aurora B protein were expressed in Sf9 cells and purified with GST affinity chromatography. The peptide substrate for Aurora A was conjugated with biotin (Biotin-GLRRASLG). Aurora A kinase (5 nM) was 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 by using Image FlashPlates. Aurora B kinase (2 nM) was assayed with 10 μM biotinylated peptide Biotin-TKQTARKSTGGKAPR in 50 mM Tricine (pH 8.0)/2.5 mM MgCl2/5 mM DTT/10% glycerol/2% BSA/40 μCi/ml [γ-33P]ATP at 250 μM. The conditions for all other in vitro kinase assays are available upon request. MLN8054 was run in a 226 kinase screen at a 1 μM compound concentration with an ATP concentration of 10 μM for all assays [2].

HCT-116 colorectal carcinoma cells were plated on 6-well dishes (2 × 10^5 per well) and propagated in McCoy's 5A media supplemented with 10% FBS. After 18 hours, alisertib at a final concentration of 0.050, 0.250, or 1.000 μmol/L was added, and the cells were grown for an additional 24 hours. Cells treated with dimethyl sulfoxide (DMSO; 0.2%) served as the untreated vehicle control. The cells were harvested with trypsin EDTA 1×, washed once with PBS, fixed in 70% ethanol, and stored at 4°C for 1 hour. The cells were resuspended in propidium iodide (1:40) and RNAse A (1:5,000) in PBS for 30 minutes at 4°C. Cell-cycle distributions were determined by measuring DNA content using flow cytometry, and samples were analyzed using Winlist 5.0 software [2].

Mice were irradiated (200 cGy), and then 5 × 106 MM1.S cells were inoculated subcutaneously in the right flank. When tumor growth was measurable (～ 2 weeks after the injection), mice were assigned into 4 groups (10 mice each) receiving vehicle orally (100 μL of 10% 2-hydroxypropyl-β-cyclodextrin/1% sodium bicarbonate) or MLN8237 (7.5 mg/kg, 15 mg/kg, and 30 mg/kg in a final formulation in 10% 2-hydroxypropyl-β-cyclodextrin/1% sodium bicarbonate) for 21 consecutive days. The maximal tolerated dose of MLN8237 in most mouse strains (continuous dosing for 21 days) is approximately 20 mg/kg twice a day (40 mg/kg per day). Tumor volumes were measured by a Vernier caliper every alternate day and calculated using the following formula: length × width2 × 0.5. Tumor growth inhibition (TGI) was calculated using the formula (Δcontrol average volume ? Δtreated average volume) × 100/(Δcontrol average volume). Mice were killed at the end of the treatment, 2 hours after the last treatment, or when tumor reached 2 cm^3; tumors were immediately collected from mice and evaluated for induction of apoptosis and cell death by TdT-mediated dUTP nick end labeling (TUNEL) assay [1].