MLN8054

Aurora A kinase (IC50 = 4 nM)

MLN8054 是一种可逆、ATP 竞争性重组 Aurora A 激酶抑制剂。当 MLN8054 与家族成员 Aurora B 相比时，它对 Aurora A 的选择性高出 40 倍以上。在体外培养的人类肿瘤细胞中，MLN8054 优先抑制 Aurora A 而非 Aurora B。使用 MLN8054 治疗会导致 G2/M 积累、纺锤体异常，并抑制几种类型的培养人类肿瘤细胞的增殖。 MLN8054 的 IC50 范围为 0.11 至 1.43 μM，可有效抑制各种组织来源的细胞增殖[1]。当用 MLN8054 培养人类肿瘤细胞时，观察到与衰老相关的某些形态和生化变化[2]。

体内 MLN8054 给药会导致细胞凋亡、有丝分裂细胞积聚以及 Aurora A 的抑制[1]。在 30 mg/kg 的剂量下，MLN8054 特异性抑制 Aurora A 激酶的活性。已证明，在此剂量下，MLN8054 可抑制 HCT116 肿瘤组织中的 Aurora A 自磷酸化，并增加 Aurora B 底物 pHisH3 的水平[2]。

酶测定。[1]
重组小鼠Aurora A和Aurora B蛋白在Sf9细胞中表达，并用GST亲和层析纯化。Aurora A的肽底物与生物素（生物素GLRRASLG）结合。使用Image FlashPlates在2μM的50 mM Hepes（pH 7.5）/10 mM MgCl2/5 mM DTT/0.05%吐温20/2μM肽底物/3.3μCi/ml[γ-33P]ATP中测定Aurora A激酶（5 nM）。Aurora B激酶（2 nM）用10μM生物素化肽生物素TKQTARKSTGGKAPR在50 mM Tricine（pH 8.0）/2.5 mM MgCl2/5 mM DTT/10%甘油/2%BSA/40μCi/ml[γ-33P]ATP中在250μM下进行测定。所有其他体外激酶测定的条件可应要求提供MLN8054在226激酶筛选中以1μM化合物浓度和10μM ATP浓度进行所有检测

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Aurora A激酶siRNA实验[2]
如前所述，在0小时和72小时分别转染悬浮的HCT-116肿瘤细胞（2×105）。转染后24、72和144小时收集细胞，并如前所示进行蛋白质印迹分析。使用针对Aurora A的单克隆抗体检测Aurora A蛋白表达（Anti-IAK1）。如上所述，对Aurora A激酶siRNA转染的细胞和打乱的siRNA对照细胞进行β-半乳糖苷酶染色。所示图像是使用20倍物镜如上所述拍摄的。

培养细胞中的免疫荧光。[1]
HCT-116人肿瘤细胞系在用DMSO稀释的MLN8054玻璃盖玻片上生长。用DMSO（0.2%）处理的细胞作为载体对照。对于免疫荧光染色，细胞用抗Aurora A pT288兔抗体（1:60）、抗IAK/Aurora A激酶小鼠单克隆抗体（1:100）、抗磷酸Ser/Thr-Pro、MPM2小鼠抗体（1:750）、pHisH3（Ser-10）小鼠单克隆抗体、磷酸PLK（Ser-137）兔抗体（1:120）或抗α-微管蛋白小鼠抗体（1:1000）的各种组合染色。使用适当的Alexa Fluor 594和488二抗。Hoescht（1:50000）用于突出显示DNA。用尼康TE 300荧光显微镜观察荧光标记的细胞，用数码相机（滨松）拍摄图像。有丝分裂细胞的百分比是通过用Discovery-1高含量成像系统对细胞进行成像，并使用Metamorph软件计算对MPM2呈阳性的Hoescht染色细胞的百分比来确定的。

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基于Aurora A和Aurora B细胞的检测。[1]
HeLa细胞在96孔细胞培养皿上生长1小时15分钟，用2倍连续稀释的DMSO稀释的MLN8054。MLN8054每次稀释时，在培养皿上以三到四行的重复方式加入。用DMSO处理的细胞（每板n=12-16孔；终浓度0.2%）作为载体对照。对于Aurora A测定，细胞用磷酸化Aurora 2/AIK（T288）兔抗体（1:60）和抗磷酸化Ser/Thr-Pro、MPM2小鼠抗体（1:750）染色，然后用Alexa Fluor 488偶联的山羊抗兔IgG（1:180）和Alexa Fluor 594偶联的鸡抗小鼠IgG（1:180:Molecular Probes）染色。然后用Alexa Fluor 488偶联的鸡抗山羊IgG（1:180）和Hoescht（1:50000）对细胞进行染色。对于Aurora B测定，用pHisH3（Ser-10）单克隆小鼠抗体（1:120）和磷酸化PLK（Ser-137）兔抗体（1:10）对细胞进行染色，然后用Alexa Fluor 488偶联的山羊抗兔IgG（1:180）和Alexa Fluor 594偶联的山羊反鼠IgG（1:1180）进行染色。然后用Hoescht（1:50000）对细胞进行染色。[1]
对于基于细胞的检测，使用Discovery-1高含量成像系统对免疫荧光细胞进行可视化。每孔9或16个位置的图像以×200的放大倍数拍摄。对于Aurora A测定，通过使用Metamorph软件测量MPM2免疫阳性（有丝分裂）细胞内的pT288（Aurora A自磷酸化）荧光强度来确定Aurora A的抑制作用。通过计算MLN8054处理的样品中pT288荧光强度相对于DMSO处理的对照的降低，生成浓度-反应曲线，并从这些曲线中确定生长抑制（IC50）值。对于Aurora B测定，通过使用Metamorph软件计数pHisH3染色阳性的pPLK137免疫阳性（有丝分裂）细胞的数量来确定Aurora B的抑制作用。如上所述，生成了浓度-响应曲线。

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培养细胞的β-Gal染色[2]
细胞被放置在12孔板中（1.4×105个细胞/孔），并在37°C下孵育过夜。第二天，用阿霉素（0.1μmol/L）或MLN8054（0.25、1或4μmol/L）处理细胞。在指定的日子里，根据制造商的说明，使用美国生物制品染色试剂盒固定细胞并染色以检测β-半乳糖苷酶的表达。细胞与β-半乳糖苷酶染色溶液在37°C下孵育48小时，以最大限度地提高β-半乳苷酶信号。取出染色液，将平板在4°C下储存在1×PBS中。为了鉴定细胞核，在成像前，用1×PBS的4′，6-二脒基-2-苯基吲哚（DAPI；100 ng/mL）对细胞进行染色30分钟。DAPI染色在β-半乳糖苷酶定量过程中可用于区分单个细胞。使用10×PlanFluor物镜、Spot Insight CCD相机和MetaMorph软件在尼康TE300显微镜上采集图像。对每个时间点和每个浓度的五个随机视场进行成像。将细胞手动评分为β-半乳糖苷酶阳性或阴性（根据可用性对每个区域50-100个细胞进行评分），并对每个治疗/时间点进行平均。

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体外结晶紫染色[2]
将HCT-116、A549、DLD-1、NCI-H460和SW480细胞铺在六孔板（600个细胞/孔）中，并在37°C下孵育过夜。第二天，用MLN8054（0.25、1或4μmol/L）处理细胞。细胞连续处理6或12天，然后用结晶紫染色，或12天后在无药物培养基中恢复6天，并在第18天染色。在指定的日子里，使用冰冷的甲醇固定细胞，然后用0.5%的结晶紫溶液染色，以鉴定细胞集落的存在。对每个孔进行成像，并使用Metamorph软件确定菌落数量，其中排除了含有少于20个细胞的菌落。结果以三个单独孔的菌落数±标准差的形式报告。

In Vivo Efficacy Studies.[1]
Female (HCT-116) and male (PC3) athymic nude NCR (nu/nu) mice were used in all in vivo studies. Animals had access to food and water ad libitum. All animals were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals and Millennium Institutional Animal Care and Use Committee guidelines. Eight-week-old nude mice were inoculated with either HCT-116 (1 × 106) or PC-3 (2 × 106) cells s.c. in the right flank. MLN8054 formulated in 10% hydroxypropyl-β-cyclodextri with 5% sodium bicarbonate was administered orally (100 μl). Tumor volumes were measured by using a vernier caliper and calculated with the formula L × W2 × 0.5. TGI was calculated with the formula (Δ control average volume − Δ treated average volume) × 100/(Δ control average volume).

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In Vivo Mechanism of Action Studies.[1]
Aurora A activity, mitotic index, and apoptosis were measured in frozen tissue sections of control and MLN8054-treated HCT-116 tumors by using immunofluorescent staining for pT288 (rabbit monoclonal generated in-house), pHisH3 (Ser-10) and cleaved caspase 3, respectively. Briefly, frozen sections were fixed with fresh 4% paraformaldehyde, and dual immunofluorescent staining was performed to measure Aurora A activity using the pT288 antibody (4 μg/ml final concentration) and pHisH3 (1.28 μg/ml final concentration). The expression of pT288 was detected by using Rhodamine red-X-conjugated goat anti-rabbit IgG; pHisH3 was detected by using Alexa Fluor 488-conjugated streptavidin. Cells expressing pT288, pHisH3, and/or caspase-3 were detected and imaged by using fluorescence microscopy and quantified with Image Pro Plus software.

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In vivo Efficacy Study[1]
NCr female nude mice bearing HCT-116 xenograft tumors were dosed orally (p.o.) with vehicle or MLN8054 (30 mg/kg) for 21 d (n = 10 animals/group) using a twice daily dosing schedule (0 and 8 h daily dosing). Tumor growth was measured using vernier calipers and tumor growth inhibition was calculated using the following formula: tumor growth inhibition = 100 − (MTV treated / MTV control) × 100. Additional details have been described previously. Statistical significance in the tumor growth trends over time between pairs of treatment groups were assessed using linear mixed effects regression models. These models account for the fact that each animal was measured at multiple time points. A separate model was fit for each comparison, and the areas under the curve for each treatment group were calculated using the predicted values from the model. The percentage of decrease in areas under the curve relative to the reference group were then calculated. A statistically significant P value suggests that the trends over time for the two treatment groups were different.

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In vivo Immunohistochemistry[1]
HCT-116 tumor-bearing NCr female nude mice were dosed with MLN8054 at 30 mg/kg using a twice daily dosing (0 and 8 h) schedule. Tumor tissue was harvested at the indicated times and placed in 10% neutral buffered formalin. Immunofluorescence was done on 5-μm paraffin-embedded tumor sections using the Discovery XT automated staining system. Sections were deparaffinized, followed by epitope unmasking with cell conditioning 1 solution for 20 min. Tumor sections were stained for pHisH3 as described previously. The DNA stain DAPI was used to estimate the total number of cells/field. One representative tissue section was used for each of the three animals in a treatment group. Images were acquired using a Leica DMLB microscope with a Photometrics Cool Snap HQ camera. Five images from each slide were captured using a 20× Leica Plan objective and analyzed on Metamorph image processing software using a custom image processing application module. The number of pHisH3-positive cells were counted and averaged in the five fields of view and DAPI staining was used to estimate the total number of cells in the fields. Anti-alpha tubulin antibody (0.18 μg/mL) was diluted in Dako diluent and incubated with tissue sections for 1 h at 37°C. Secondary goat anti-rabbit rhodamine red-X conjugate (30 μg/mL) was added for 30 min at room temperature. DAPI vectashield HardSet Medium was used as a chromatin counter stain. Images were captured with a Nikon Eclipse E800 (20× objective) and analyzed with Metamorph 6.3r7 software.

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Dissolved in 10% hydroxypropyl-β-cyclodextrin with 5% sodium bicarbonate; 30 mg/kg; Oral gavage

HCT-116 and PC-3 cells are injected s.c. into the right flank of nude mice.

[1]. Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase. Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):4106-11.

[2]. MLN8054, an inhibitor of Aurora A kinase, induces senescence in human tumor cells both in vitro and in vivo. Mol Cancer Res. 2010 Mar;8(3):373-84.

4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]benzoic acid is a benzazepine.
MLN8054 has been used in trials studying the treatment of Colon Neoplasm, Breast Neoplasm, Bladder Neoplasm, Pancreatic Neoplasm, and Advanced Malignancies.
Aurora Kinase Inhibitor MLN8054 is an orally bioavailable, highly selective small molecule inhibitor of the serine/threonine protein kinase Aurora A kinase with potential antineoplastic activity. Auora kinase inhibitor MLN8054 binds to and inhibits Aurora kinase A, resulting in disruption of the assembly of the mitotic spindle apparatus, disruption of chromosome segregation, and inhibition of cell proliferation. Aurora A localizes in mitosis to the spindle poles and to spindle microtubules and is thought to regulate spindle assembly. Aberrant expression of Aurora kinases occurs in a wide variety of cancers, including colon and breast cancers.

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Mechanism of Action
MLN8054 is a selective small-molecule Aurora A kinase inhibitor that has entered Phase I clinical trials for advanced solid tumors. MLN8054 inhibits recombinant Aurora A kinase activity in vitro and is selective for Aurora A over the family member Aurora B in cultured cells. MLN8054 treatment results in G2/M accumulation and spindle defects and inhibits proliferation in multiple cultured human tumor cells lines.

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Increased Aurora A expression occurs in a variety of human cancers and induces chromosomal abnormalities during mitosis associated with tumor initiation and progression. MLN8054 is a selective small-molecule Aurora A kinase inhibitor that has entered Phase I clinical trials for advanced solid tumors. MLN8054 inhibits recombinant Aurora A kinase activity in vitro and is selective for Aurora A over the family member Aurora B in cultured cells. MLN8054 treatment results in G(2)/M accumulation and spindle defects and inhibits proliferation in multiple cultured human tumor cells lines. Growth of human tumor xenografts in nude mice was dramatically inhibited after oral administration of MLN8054 at well tolerated doses. Moreover, the tumor growth inhibition was sustained after discontinuing MLN8054 treatment. In human tumor xenografts, MLN8054 induced mitotic accumulation and apoptosis, phenotypes consistent with inhibition of Aurora A. MLN8054 is a selective inhibitor of Aurora A kinase that robustly inhibits growth of human tumor xenografts and represents an attractive modality for therapeutic intervention of human cancers.[1]

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Aurora A kinase is a serine/threonine protein kinase responsible for regulating several mitotic processes including centrosome separation, spindle assembly, and chromosome segregation. Small molecule inhibitors of Aurora A kinase are being pursued as novel anticancer agents, some of which have entered clinical trials. Despite the progress in developing these agents, terminal outcomes associated with Aurora A inhibition are not fully understood. Although evidence exists that Aurora A inhibition leads to apoptosis, other therapeutically relevant cell fates have not been reported. Here, we used the small molecule inhibitor MLN8054 to show that inhibition of Aurora A induces tumor cell senescence both in vitro and in vivo. Treatment of human tumor cells grown in culture with MLN8054 showed a number of morphologic and biochemical changes associated with senescence. These include increased staining of senescence-associated beta-galactosidase, increased nuclear and cell body size, vacuolated cellular morphology, upregulation/stabilization of p53, p21, and hypophosphorylated pRb. To determine if Aurora A inhibition induces senescence in vivo, HCT-116 xenograft-bearing animals were dosed orally with MLN8054 for 3 weeks. In the MLN8054-treated animals, increased senescence-associated beta-galactosidase activity was detected in tissue sections starting on day 15. In addition, DNA and tubulin staining of tumor tissue showed a significant increase in nuclear and cell body area, consistent with a senescent phenotype. Taken together, this data shows that senescence is a terminal outcome of Aurora A inhibition and supports the evaluation of senescence biomarkers in clinic samples.[2]

C25H15CLF2N4O2

476.86

476.085

C, 62.97; H, 3.17; Cl, 7.43; F, 7.97; N, 11.75; O, 6.71

869363-13-3

11712649

White to yellow solid powder

1.2±0.1 g/cm3

429.5±45.0 °C at 760 mmHg

213.5±28.7 °C

0.0±1.0 mmHg at 25°C

1.524

2.02

64.63

2

8

4

34

755

0

HHFBDROWDBDFBR-UHFFFAOYSA-N

InChI=1S/C25H15ClF2N4O2/c26-15-6-9-17-18(10-15)23(21-19(27)2-1-3-20(21)28)29-11-14-12-30-25(32-22(14)17)31-16-7-4-13(5-8-16)24(33)34/h1-10,12H,11H2,(H,33,34)(H,30,31,32)

4-((9-chloro-7-(2,6-difluorophenyl)-5H-benzo[c]pyrimido[4,5-e]azepin-2-yl)amino)benzoic acid.

MLN 8054; MLN-8054; 4-[[9-Chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]benzoic acid; 4-((9-chloro-7-(2,6-difluorophenyl)-5H-benzo[c]pyrimido[4,5-e]azepin-2-yl)amino)benzoic acid; 4-(9-chloro-7-(2,6-difluorophenyl)-5H-benzo[e]pyrimido[5,4-c]azepin-2-ylamino)benzoic acid; BX854EHD63; MLN8054

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: 2.5 mg/mL (5.24 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.36 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.36 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 15% Captisol:30mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。