Aurora A kinase (IC50 = 4 nM)

MLN8054 is a reversible, ATP-competitive inhibitor of recombinant Aurora A kinase. When MLN8054 is compared to family member Aurora B, it is >40-fold more selective for Aurora A. In human tumor cells cultivated in vitro, MLN8054 preferentially inhibits Aurora A over Aurora B. Treatment with MLN8054 causes G2/M accumulation, spindle abnormalities, and suppresses proliferation in several types of cultured human tumor cells. With an IC50 range of 0.11 to 1.43 μM, MLN8054 efficiently suppresses the proliferation of cells originating from various tissue origins[1]. When human tumor cells are cultured with MLN8054, certain morphologic and biochemical alterations linked to senescence are observed[2].

In vivo MLN8054 administration causes apoptosis, accumulation of mitotic cells, and inhibition of Aurora A[1]. At a dosage of 30 mg/kg, MLN8054 specifically inhibits the activity of Aurora A kinase. It has been demonstrated that MLN8054 inhibits Aurora A autophosphorylation in HCT116 tumor tissue at this dose and increases the levels of the Aurora B substrate, pHisH3[2].

Enzyme Assays.[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.

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Aurora A Kinase siRNA Experiments[2]
Suspended HCT-116 tumor cells (2 × 105) were transfected as described previously at 0 h and again at 72 h. Cells were harvested at 24, 72, and 144 h after transfection and processed for Western blot analysis as described previously. A monoclonal antibody directed against Aurora A was used to detect Aurora A protein expression (Anti-IAK1). Aurora A Kinase siRNA transfected cells and scrambled siRNA control cells were stained for the presence of β-galactosidase as described above. Images shown were taken as described above using a 20× objective.

Immunofluorescence in Cultured Cells.[1]
HCT-116 human tumor cell lines were grown on glass coverslips with MLN8054 diluted in DMSO. Cells treated with DMSO (0.2%) served as the vehicle control. For immunofluorescent staining, the cells were stained with various combinations of anti-Aurora A pT288 rabbit antibody (1:60), anti-IAK/Aurora A kinase mouse monoclonal antibody (1:100), anti-phospho-Ser/Thr-Pro, MPM2 mouse antibody (1:750), pHisH3 (Ser-10) mouse monoclonal antibody (1:120,), phospho-PLK (Ser-137) rabbit antibody (1:120), or anti-α-tubulin mouse antibody (1:1,000). Appropriate Alexa Fluor 594 and 488 secondary antibodies were used. Hoescht (1:50,000) was used to highlight DNA. Fluorescently labeled cells were visualized with a Nikon TE 300 fluorescent microscope, and images were captured with a digital camera (Hamamatsu). The percentage of mitotic cells was determined by imaging cells with a Discovery-1 High Content Imaging System and by calculating the percentage of Hoescht-stained cells that were positive for MPM2 using Metamorph software.

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Aurora A and Aurora B Cell-Based Assays.[1]
HeLa cells were grown on 96-well cell culture dishes for 1 h and 15 min with MLN8054 diluted in DMSO in 2-fold serial dilutions.MLN8054 at each dilution was added as replicates in three to four rows on the dish. Cells treated with DMSO (n = 12–16 wells per plate; 0.2% final concentration) served as the vehicle control. For the Aurora A assay, cells were stained with phospho-Aurora 2/AIK (T288) rabbit antibody (1:60) and anti-phospho-Ser/Thr-Pro, MPM2 mouse antibody (1:750) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:180) and Alexa Fluor 594-conjugated chicken anti-mouse IgG (1:180; Molecular Probes). The cells were then stained with Alexa Fluor 488-conjugated chicken anti-goat IgG (1:180) and Hoescht (1:50,000). For the Aurora B assay, cells were stained with pHisH3 (Ser-10) monoclonal mouse antibody (1:120) and phospho-PLK (Ser-137) rabbit antibody (1:120) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:180) and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:180). The cells were then stained with Hoescht (1:50,000).[1]
For cell-based assays, immunofluorescent cells were visualized by using a Discovery-1 High Content Imaging System. Images from 9 or 16 sites per well were captured at ×200 magnification. For the Aurora A assay, inhibition of Aurora A was determined by measuring pT288 (Aurora A autophosphorylation) fluorescent intensity within MPM2-immunopositive (mitotic) cells by using Metamorph software. Concentration–response curves were generated by calculating the decrease of pT288 fluorescent intensity in MLN8054-treated samples relative to the DMSO-treated controls, and growth inhibition (IC50) values were determined from those curves. For the Aurora B assay, inhibition of Aurora B was determined by counting the number of pPLK137-immunopositive (mitotic) cells that stained positive for pHisH3 by using Metamorph software. Concentration–response curves were generated as described above.

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β-Gal Staining of Cultured Cells[2]
Cells were plated in 12-well plates (1.4 × 105 cells/well) and incubated overnight at 37°C. The next day, cells were treated with either doxorubicin (0.1 μmol/L) or MLN8054 (0.25, 1, or 4 μmol/L). On the indicated days, cells were fixed and stained for β-galactosidase expression using the U.S. Biologicals staining kit according to the instructions of the manufacturer. Cells were incubated with the β-galactosidase staining solution for 48 h at 37°C to maximize the β-galactosidase signal. The staining solution was removed and the plates were stored at 4°C in 1× PBS. To identify cell nuclei, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; 100 ng/mL) in 1× PBS for 30 min before imaging. The DAPI staining was useful in distinguishing individual cells during the β-galactosidase quantification. Images were acquired on a Nikon TE300 microscope using a 10× PlanFluor objective lens, Spot Insight CCD camera, and MetaMorph software. Five random fields of view were imaged for each time point and each concentration. Cells were hand-scored as either β-galactosidase positive or negative (50-100 cells per field were scored based on availability) and averaged for each treatment/time point.

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In vitro Crystal Violet Staining[2]
HCT-116, A549, DLD-1, NCI-H460, and SW480 cells were plated in six-well plates (600 cells/well) and incubated overnight at 37°C. The next day, cells were treated with MLN8054 (0.25, 1, or 4 μmol/L). Cells were treated continuously for 6 or 12 d and then stained with crystal violet, or for 12 d and then allowed to recover for 6 d in drug-free medium and stained on day 18. On the indicated days, cells were fixed using ice-cold methanol and then stained with a 0.5% crystal violet solution to identify the presence of cell colonies. Each well was imaged and the number of colonies was determined using Metamorph software, in which colonies containing less than 20 cells were excluded. Results are reported as the number of colonies ±SD of three separate wells.

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

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)

Solubility in Formulation 1: 2.5 mg/mL (5.24 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.36 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 15% Captisol:30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)