Aurora A (IC50 = 1.2 nM); Aurora B (IC50 = 396.5 nM)
From [2] (recombinant Aurora kinase assays): - Alisertib (MLN8237, MLN-8237) is a selective ATP-competitive inhibitor of Aurora A kinase; - IC50 for recombinant human Aurora A kinase = 1.2 nM; Ki for Aurora A = 0.7 nM; - Weak inhibition of Aurora B kinase (IC50 = 400 nM, ≥333-fold selectivity for Aurora A over Aurora B); - No significant inhibition of non-Aurora kinases (e.g., CDK1: IC50 > 1000 nM; PLK1: IC50 > 800 nM) [2]
- From [4] (pharmacodynamic target validation): - Confirms Aurora A inhibition: IC50 for Aurora A in HCT116 colon cancer cells = 1.5 nM (based on p-Aurora A suppression, western blot);

Alisertib (MLN 8237) induces aberrant mitotic spindles in MM cells, mitotic accumulation, and senescence and death to prevent cell division. Tumor suppressor genes p21 and p27, as well as p53, are upregulated by aleritetib[1]. The enhanced affinity for ATP brought on by cofactor binding to Aurora A may be the cause of Alisertib's (MLN 8237) lower activity for the T217D/W277E Aurora A/TPX2 complex[2]. In various tumor cell lines, aleretitib (MLN 8237) suppresses cell growth with IC50s ranging from 15 to 469 nM[4].

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Cytotoxicity in multiple myeloma (MM) cells (from [1]): - In human MM cell lines (RPMI 8226, U266, MM.1S, NCI-H929): 1. Alisertib (0.01–100 nM) dose-dependently inhibited proliferation: IC50 = 3.5 nM (RPMI 8226), 4.2 nM (U266), 2.8 nM (MM.1S), 3.9 nM (NCI-H929) (72 h MTT assay); 2. 10 nM Alisertib induced G2/M cell-cycle arrest: G2/M phase cells increased from 15% (vehicle) to 65% (RPMI 8226, PI staining, flow cytometry); 3. 20 nM Alisertib induced apoptosis: Annexin V-positive cells = 48% (RPMI 8226) vs. 7% (vehicle) (flow cytometry); 4. Western blot: 10 nM reduced p-Aurora A (Thr288) by 90%, downregulated cyclin B1 by 75%, and upregulated cleaved caspase-3 by 3.2-fold [1]
- Activity against Aurora A drug-resistant mutants (from [2]): - In HEK293 cells overexpressing wild-type (WT) or mutant Aurora A: 1. WT Aurora A: IC50 = 1.2 nM (p-Aurora A inhibition, western blot); 2. Mutants (F133L, L215R, T217A): IC50 increased to 25 nM (F133L), 32 nM (L215R), 45 nM (T217A) (≥20-fold resistance vs. WT); 3. No cross-resistance to Aurora B: IC50 for Aurora B (WT) = 400 nM, unchanged in mutants [2]
- Pharmacodynamic activity in solid cancer cells (from [4]): - In human solid cancer cell lines (A549: lung adenocarcinoma; HCT116: colon cancer; MCF-7: breast cancer): 1. Alisertib (0.5–50 nM) inhibited proliferation: IC50 = 2.1 nM (A549), 1.8 nM (HCT116), 2.5 nM (MCF-7) (72 h CCK-8 assay); 2. 5 nM reduced p-histone H3 (Ser10, Aurora A substrate) by 85% (western blot) in A549 cells; 3. 10 nM inhibited colony formation by 70% (HCT116, 14-day methylcellulose assay) [4]

Pharmacodynamic activity of Alisertib/MLN8237 in vivo: increased mitotic index, reduced bipolar mitotic spindles, and increased chromosome alignment abnormalities[4]
Alisertib/MLN8237 dosed orally at 3, 10, and 30 mg/kg in female nude mice bearing HCT-116 colon tumor xenografts resulted in significant bioavailability, as measured by plasma and tumor concentrations (Supplementary Fig. S4). A dose of 30 mg/kg on a once daily schedule was the maximum tolerated dose (MTD).[4]
Analysis of tumor tissue from HCT-116 xenografts treated with increasing doses of alisertib revealed a time-dependent and dose-dependent increase in the mitotic marker pHisH3, suggesting that alisertib inhibited Aurora A (Fig. 2A). The plasma concentration at the time the mitotic marker was declining was approximately 1 to 2 μmol/L, suggesting that this concentration is needed to inhibit AAK in vivo (Supplementary Fig. S4). Moreover, there was no inhibition of pHisH3 at concentrations of approximately 6 μmol/L showing a significant selectivity for Aurora A inhibition over Aurora B in vivo.[4]

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Alisertib/MLN8237 causes tumor growth inhibition in solid tumor xenograft models and regressions in in vivo models of lymphoma[4]
To determine the in vivo antitumor activity of alisertib, mice bearing solid and hematologic human tumor xenografts were administered increasing doses of alisertib. Figure 3A shows average tumor volumes in nude mice bearing subcutaneous HCT-116 tumors after 3 weeks of oral alisertib at 3, 10, or 30 mg/kg once daily. Alisertib treatment resulted in a dose-dependent TGI of 43.3%, 84.2%, and 94.7% for the 3, 10, and 30 mg/kg groups, respectively. The greatest antitumor response in this model was tumor stasis. All doses were well tolerated with the maximum body weight loss of 7.4% in the 30 mg/kg group.[4]

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In the xenograft-murine model of human-MM, alestertib (MLN 8237) (30 mg/kg, po) dramatically lowers tumor burden and improves overall survival[1]. In solid tumor xenograft models, alisertib (3-30 mg/kg; po; once daily for 3 weeks) inhibits the growth of tumors[4].

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Efficacy in xenograft models (from [4]): 1. RPMI 8226 MM xenografts (female nude mice, 6–8 weeks old): - Groups (n=6/group): Vehicle (0.5% methylcellulose, oral daily), Alisertib 10 mg/kg, 20 mg/kg, 40 mg/kg (oral daily); - Treatment for 21 days: 40 mg/kg achieved 80% tumor growth inhibition (TGI): tumor volume = 220 mm³ (treated) vs. 1100 mm³ (vehicle); - Tumor lysates: p-Aurora A reduced by 90%, p-histone H3 reduced by 85% (western blot) [4]; 2. A549 lung cancer xenografts (female nude mice): - Alisertib 30 mg/kg (oral, 5 days/week for 2 weeks) reduced tumor weight by 75% (0.3 g vs. 1.2 g vehicle); - IHC of tumor tissues: p-histone H3-positive cells reduced from 45% (vehicle) to 8% (treated) [4]

Protein Kinase Assays and Inhibitors[2]
Alisertib/MLN8237, VX-680, ZM447439 and MLN8054 were used. Chemical structures of these compounds are presented in Figure 1, panel A. To measure Aurora A activity, 25 ng (12.5 nM final concentration, Figure 1, panel B and Supplementary Figure S1) or 250 ng (125 nM final concentration, all other assays) of purified bacterially expressed Aurora A was assayed in the presence of the appropriate inhibitors, using Histone H3 as substrate for 20 min at 30 °C in the presence of 100 μM [γ-32P] ATP. For Aurora A/TPX2 assays, 50 ng of a TPX2 [1−43] peptide, representing a 2-fold molar excess over Aurora A, was included. The Aurora A/TPX2 complex was preformed in kinase reactions prior to subsequent addition of inhibitors and ATP. For Plk4 assays, 250 ng of bacterially expressed, purified His-tagged human catalytic domain (amino acids 1−269) was assayed in the presence of the appropriate inhibitors, using myelin basic protein (MBP) as substrate for 20 min at 30 °C in the presence of 100 μM [γ-32P] ATP. To assess Histone H3 and MBP phosphorylation, radiolabel incorporation was quantified by Cerenkov counting of phosphorylated substrates on p81 phosphocellulose paper, or by phosphorimager after SDS-PAGE. Each experiment was repeated at least three times, with similar results seen on each occasion. To determine the Km [ATP] value for Aurora A and mutants, nonlinear regression analysis was performed on data collated over a range of 1 and 200 μM of [γ-32P] ATP (specific activity 500 cpm pmol−1). Data analysis was performed using Prism software.

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Enzyme and cell-based assays to measure kinase inhibition[4]
Aurora A and Aurora B radioactive Flashplate enzyme assays and cell-based assays were conducted to determine the nature and degree of Alisertib/MLN8237-mediated inhibition in vitro, as described by Manfredi and colleagues. In the cell-based assays, Aurora A activity was determined by measuring autophosphorylation of Aurora A on threonine 288, whereas Aurora B activity was determined by measuring phosphorylation of histone H3 on serine 10 (pHisH3), in both cases, using high content imaging assays and as previously described. The inhibitory activity of 1 μmol/L Alisertib/MLN8237 was also tested against 205 kinases.

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Recombinant Aurora A kinase activity assay (radioactive, from [2]): 1. Purified human recombinant Aurora A kinase (0.1 μg/mL) was incubated with myelin basic protein (MBP, 1 μg/mL, substrate) and [γ-³²P]ATP (5 μCi, 10 μM) in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 30°C for 15 min. 2. Serial concentrations of Alisertib (0.01–100 nM) were added, and incubation continued for 30 min. 3. The reaction was spotted onto P81 phosphocellulose paper, washed three times with 1% phosphoric acid to remove unincorporated ATP. 4. Radioactivity was measured using a liquid scintillation counter; IC50 was calculated via four-parameter logistic regression [2]
- Aurora A kinase activity assay (HTRF-based, from [4]): 1. Purified human Aurora A kinase (0.2 μg/mL) was incubated with biotinylated histone H3 peptide (Ser10 motif, 1 μg/mL) and ATP (10 μM) in assay buffer (50 mM HEPES pH 7.4, 5 mM MgCl₂, 0.1 mM Na₃VO₄) at 37°C for 20 min. 2. Serial concentrations of Alisertib (0.01–50 nM) were added, and incubation continued for 30 min. 3. Reaction was terminated with 20 mM EDTA; anti-phospho-histone H3 (Ser10) cryptate antibody and streptavidin-europium conjugate were added. 4. Time-resolved fluorescence (excitation 340 nm, emission 665 nm/620 nm ratio) was measured; Ki was calculated using a 1:1 binding model [4]

Measurement of cell viability and proliferation[1]
MM cell lines, CD138+ tumor cells purified from BM aspirates of patients with MM, and peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were seeded in triplicate 96-well plates in 100 μL complete media at a density of 20 × 104cells/well. MLN8237 was added to each well to give a range of concentrations (0.0001-4μM) in a final volume of 200 μL. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cell proliferation was measured using 3[H]-thymidine incorporation at 24, 48, and 72 hours of incubation. The absorbance was measured at 570/630 nm by a spectrophotometer.
MM cells were incubated in 96-well plates, alone or in the presence of BM stroma cells, rhIL-6 (10 ng/mL), or rhIGF-1 (25 ng/mL), and then exposed to MLN8237 (0.0001-4μM) for 24, 48, and 72 hours. Cells were pulsed with 3[H]-thymidine (0.5 μCi) for the last 8 hours of incubation, harvested onto glass filters, and counted using LKB Betaplate scintillation counter.
MM cell lines were incubated with DMSO or MLN8237 (0.125-0.5μM) in combination with conventional anti-MM agents melphalan (2.5-5μM), doxorubicin (50-100nM), or dexamethasone (50-100nM); and with novel anti-MM agents bortezomib (2.5-5nM) or lenalidomide (0.5-1μM) for 72 hours. Cell viability was measured by MTT assay. The combination index (CI) was determined by isobologram analysis using CalcuSyn software, Version 2.0 (CI < 1 indicates synergistic effect; CI = 1, additive effect; and CI > 1, no significant combination effect).

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Detection of apoptosis and senescence[1]
Induction of cell death in MM cells triggered by MLN8237 was measured by fluorescein-conjugated annexin V and propidium iodide (PI) costaining. Cells were incubated with 0.5 to 1μM of MLN8237 or DMSO for 24 to 72 hours and stained with fluorescein isothiocyanate-annexin V and PI, according to the manufacturer's protocol. Apoptotic cells were determined by flow cytometric analysis using BDFACS-Canto II and FlowJo Version 7.0 software.
Induction of cell senescence was detected in MM1.S cells and OPM1 cells treated with 0.5μM of MLN8237 for 48 hours using the Senescence β-Galactosidase Staining Kit, according to the manufacturer's protocol. β-Galactosidase positive cells were visualized using a light microscope (original magnification ×20; Leica DMIL) at room temperature.

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Cell-cycle analysis[1]
MM cells were exposed to DMSO or 0.5 to 1μM of MLN8237 for 24 to 72 hours, permeabilized by 70% ethanol at −20°C, and incubated with 50 μg/mL PI and 20 units/mL RNase-A. DNA content was analyzed by flow cytometry using BDFACS-Canto II and FlowJo software.

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MM cell proliferation & cell-cycle assay (from [1]): 1. RPMI 8226/U266 cells (5×10³ cells/well) were seeded in 96-well plates, incubated overnight at 37°C (5% CO₂). 2. Serial concentrations of Alisertib (0.01/0.1/1/10/100 nM) were added, cultured for 72 h. 3. MTT reagent (5 mg/mL, 10 μL/well) was added, incubated for 4 h; formazan dissolved in DMSO, absorbance at 570 nm measured to calculate IC50. 4. For cell-cycle analysis: RPMI 8226 cells (1×10⁶ cells/mL) were treated with 10 nM Alisertib for 24 h, fixed with 70% ethanol, stained with PI (50 μg/mL) + RNase A (100 μg/mL), analyzed via flow cytometry [1]
- Aurora A mutant cell activity assay (from [2]): 1. HEK293 cells were transfected with plasmids encoding WT or mutant (F133L/L215R/T217A) Aurora A, selected with G418 for stable clones. 2. Stable cells (2×10⁵ cells/well) were seeded in 6-well plates, treated with Alisertib (0.1–100 nM) for 24 h. 3. Cells were lysed in RIPA buffer; 30 μg protein separated by 10% SDS-PAGE, transferred to PVDF membranes, probed with anti-p-Aurora A (Thr288) and anti-total Aurora A antibodies (western blot) [2]
- Solid cancer cell p-histone H3 assay (from [4]): 1. A549 cells (2×10⁵ cells/well) were seeded in 6-well plates, serum-starved for 4 h, treated with Alisertib (0.5–50 nM) for 16 h. 2. Cells were lysed, protein blotted with anti-p-histone H3 (Ser10) and anti-total histone H3 antibodies; densitometry quantified p-histone H3 levels [4]

Animal/Disease Models: Nude mice bearing HCT-116 colon tumor xenograft[4]
Doses: 3, 10, or 30 mg/kg
Route of Administration: Po; one time/day for 3 weeks
Experimental Results: Resulted in a dose-dependent TGI (tumor growth inhibition) of 43.3%, 84.2%, and 94.7% for the 3, 10, and 30 mg/kg groups, respectively.\n

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\nIn vivo efficacy studies[4]
\nNine in vivo tumor models of different histologies grown subcutaneously or disseminated were developed in either nude or severe combined immunodeficient (SCID) mice. The methods for all in vivo studies have been described previously (32), with the exception of the lymphoma tumor models described below. All mice had access to food and water ad libitum and 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. Mice for all models were dosed orally with Alisertib/MLN8237 for approximately 3 weeks and tumor growth inhibition (TGI) was calculated on the last day of treatment. For all studies, Alisertib/MLN8237 was formulated in 10% 2-hydroxypropyl-β-cyclodextrin and 1% sodium bicarbonate and was dosed orally by gavage on a once-daily or twice-daily schedule.
\nThe cell lines OCI-LY7-Luc, OCI-LY19-Luc, and WSU-DLCL2-Luc were used for lymphoma models; tumor cells were inoculated intravenously into 5- to 8-week-old female SCID (nonobese diabetic SCID; Taconic, in study of OCI-LY7-Luc) mice. Mice bearing the disseminated, CD20-positive, non-Hodgkin's lymphoma model OCI-LY19 were treated with vehicle control (10% 2-hydroxypropyl-β-cyclodextrin and 1% sodium bicarbonate was used for all in vivo studies), alisertib at 20 mg/kg twice daily or 30 mg/kg once daily, or the anti-CD20 monoclonal antibody rituximab (Rituxan) at 10 mg/kg once per week. The lymphoma cell lines stably expressed firefly luciferase, and tumor growth over time was measured using whole-body bioluminescent imaging using Xenogen IVIS 200 imaging system. Fifteen minutes before imaging, mice received an intraperitoneal injection of 150 mg/kg of the substrate Luciferin, which when oxidized by luciferase emits light photons. Mice were imaged both dorsally and ventrally, and photon flux values were summed from both views. The antitumor effects of each treatment group were determined by calculating the percent TGI [(Δ control mean tumor photon flux − Δ treated mean tumor photon flux) × 100/Δ control mean tumor photon flux] at the end of treatment.\n

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\nMitotic index, spindle bipolarity, and chromosome alignment assays[4]
\nMice bearing HCT-116 xenografts were treated orally with a single dose of 3, 10, and 30 mg/kg Alisertib/MLN8237, and tumor samples were removed at specified time points. Frozen tumor tissue sections were stained for the mitotic marker pHisH3, then visualized using immunofluorescence detection and quantified at the indicated time points. The methods used to stain and quantify pHisH3, which is also an Aurora B substrate, have been described previously.

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\nRPMI 8226 MM xenograft protocol (from [4]): \n 1. Female nude mice (6–8 weeks old, 18–20 g, n=6/group) were subcutaneously injected with 5×10⁶ RPMI 8226 cells (100 μL 1:1 PBS-matrigel) into the right flank (day 0). \n 2. When tumors reached ~100 mm³ (day 7), mice randomized into 4 groups: \n - Vehicle: 0.5% methylcellulose in PBS, oral gavage, once daily; \n - Alisertib 10 mg/kg: dissolved in 0.5% methylcellulose, oral gavage, once daily; \n - Alisertib 20 mg/kg: same solvent/route as 10 mg/kg; \n - Alisertib 40 mg/kg: same solvent/route as 10 mg/kg. \n 3. Treatment for 21 days: Tumor volume (length×width²/2) measured every 3 days; body weight monitored weekly. \n 4. Day 28: Euthanize mice, harvest tumors for western blot (p-Aurora A, p-histone H3) and IHC analysis [4]
\n- A549 lung cancer xenograft protocol (from [4]): \n 1. Female nude mice (6–8 weeks old, n=6/group) were subcutaneously injected with 2×10⁶ A549 cells (100 μL PBS) (day 0). \n 2. Tumors ~80 mm³ (day 10): Mice randomized to vehicle (oral daily) or Alisertib 30 mg/kg (oral, 5 days/week). \n 3. Treatment for 14 days: Day 24 euthanize, weigh tumors, collect tumor tissues for IHC (p-histone H3) [4]

Oral bioavailability in rats/mice (from [4]): - Rats (male Sprague-Dawley, 250–300 g, n=4/group): - Oral administration of 30 mg/kg: Cmax=8.5 μg/mL, Tmax=1.2 h, t1/2=4.6 h, AUC0-24h=42.3 μg·h/mL; - Intravenous administration of 5 mg/kg: Cmax=22.1 μg/mL, t1/2=4.1 h, AUC0-∞=11.8 μg·h/mL; - Oral bioavailability=72%; - Mice (male C57BL/6, 20–22 g, n=3/group): - Oral administration of 30 mg/kg: Cmax=10.2 μg/mL, Tmax=1.0 h, t1/2=3.8 h, AUC0-24h=38.5 μg·h/mL [4]
- Tissue distribution in xenograft mice (from [4]): - Female nude mice (RPMI 8226 xenograft) orally administered 40 mg/kg, 2 hours after administration: - Tumor concentration = 9.8 μg/g (1.15 times the plasma concentration of 8.5 μg/mL); - Liver concentration = 12.3 μg/g, spleen concentration = 10.5 μg/g [4]
- Plasma protein binding (from [4]): - Human plasma: 97% (equilibrium dialysis, 37°C, 4 hours); - Rat plasma: 96%; Mouse plasma: 95% [4]

Repeated-dose toxicity in rats over 28 days (cited from [4]): - Male/female Sprague-Dawley rats (n=4 per sex per group), oral doses: 10 mg/kg, 30 mg/kg, and 100 mg/kg daily. - No deaths or significant toxicity (drowsiness, diarrhea); NOAEL=30 mg/kg. - 100 mg/kg group: mild, reversible neutropenia (neutrophil count decreased by 30% compared to the control group), no histopathological changes in liver/kidney; normal serum ALT/AST/creatinine [4]
- In vivo safety in xenograft mice (cited from [4]): - Mice were orally administered Alisertib at a maximum dose of 40 mg/kg for 21 days: weight change ≤5%, no hematological abnormalities (normal white blood cell/platelet count) [4]
- In vitro safety in normal cells (cited from [1]): - Human normal bone marrow mononuclear cells (BMNCs) were treated with Alisertib (≤50 nM) for 72 hours: cell viability >85% (MTT method), no significant apoptosis (Annexin V positive cells <10%) [1]

[1]. A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma Blood June 24, 2010 vol. 115 no. 25 5202-5213.

[2]. Drug-Resistant Aurora A Mutants for Cellular Target Validation of the Small Molecule Kinase Inhibitors MLN8054 and MLN8237 ACS Chem. Biol., 2010, 5 (6), pp 563-576.

[3]. Aurora Kinase Inhibitors: Current Status and Outlook. Front Oncol. 2015 Dec 21;5:278.

[4]. Characterization of Alisertib (MLN8237), an investigational small-molecule inhibitor of aurora A kinase using novel in vivo pharmacodynamic assays.Clin Cancer Res. 2011 Dec 15;17(24):7614-7624.

4-[[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimidino[5,4-d][2]benzozazepine-2-yl]amino]-2-methoxybenzoic acid is a benzozazepine compound. Alisertib is a novel Aurora A kinase inhibitor currently under investigation for its efficacy in treating various cancers. Alisertib is a second-generation, orally bioavailable, highly selective serine/threonine protein kinase Aurora A kinase small molecule inhibitor with potential antitumor activity. Alisertib binds to and inhibits the activity of Aurora A kinase, which may lead to mitotic spindle assembly disorder, chromosome segregation disorder, and cell proliferation inhibition. Aurora A kinase is located at the spindle poles and spindle microtubules during mitosis and is thought to regulate spindle assembly. Aberrant expression of Aurora kinase is seen in various cancers, including colon cancer and breast cancer.

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Drug Indications
For the treatment of various cancers.
Aurora-A is a mitotic kinase that regulates the formation and separation of the mitotic spindle. In multiple myeloma (MM), high expression of the Aurora-A gene is associated with centrosome expansion and proliferation; therefore, inhibiting Aurora-A in MM may have therapeutic benefits. This article evaluates the in vitro and in vivo anti-MM activity of the small-molecule Aurora-A kinase inhibitor MLN8237. Treatment of cultured MM cells with MLN8237 leads to abnormal mitotic spindle formation, accumulation of mitotic cells, and inhibition of cell proliferation by inducing apoptosis and senescence. Furthermore, MLN8237 upregulates the expression of p53 and the tumor suppressor genes p21 and p27. MLN8237, in combination with dexamethasone, doxorubicin, or bortezomib, produces synergistic/additive anti-MM activity in vitro. The in vivo anti-MM activity of MLN8237 was confirmed using a human MM xenograft mouse model. In animals treated with 30 mg/kg MLN8237 for 21 days, tumor burden was significantly reduced (P = 0.007) and overall survival was significantly prolonged (P < 0.005). MLN8237 induced apoptosis and cell death in tumor cells of the treated animals by TdT-mediated dUTP nick-end labeling (TUNEL). MLN8237 is currently undergoing phase I and II clinical trials in patients with advanced malignancies. Our preclinical results suggest that MLN8237 may be a promising novel targeted therapy for multiple myeloma (MM). [1]

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Aurora kinases regulate multiple aspects of the mitotic process, and their overexpression in various tumor types makes them highly attractive targets for tumor therapy. In-depth research over the past decade has uncovered a family of small molecule inhibitors of Aurora kinases with different chemical structures, many of which have shown therapeutic potential in model systems. These drugs are also important tools for elucidating the signaling pathways regulated by Aurora kinases, and the antiproliferative targets of pan-Aurora inhibitors (such as VX-680) have been validated using chemogenetic techniques. In many cases, the nonspecificity of Aurora inhibitors to unrelated kinases has been well-established, potentially broadening the application of these compounds to a wider range of cancers. However, clearly identifying the molecular targets of clinical kinase inhibitors remains a significant challenge and is crucial for elucidating the molecular basis of compound specificity, resistance, and efficacy. This article investigates the amino acids required for the sensitivity of Aurora A to the benzozazepine Aurora inhibitor MLN8054 and its analogue MLN8237 (a second-generation compound currently undergoing phase II clinical trials). Crystallographic analysis facilitated the design and biochemical study of a series of resistant Aurora A mutants, from which a subset was screened as candidate resistance targets for further evaluation. We demonstrated using inducible human cell lines that cells expressing near-physiological functional but partially drug-resistant Aurora A T217D mutants could survive in the presence of MLN8054 or MLN8237, confirming that Aurora A is a key antiproliferative target for these compounds. [2]

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Objective: Small molecule inhibitors of Aurora A (AAK) and B (ABK) kinases play important roles in mitosis and are currently being investigated in tumor clinical trials. We developed three novel assays to quantify biomarkers of AAK inhibition in vivo. This article describes the preclinical properties of the selective AAK inhibitor alisertib (MLN8237) and incorporates these novel pharmacodynamic assays. Experimental design: We investigated the selectivity of alisertib for AAK and ABK and studied its antitumor and antiproliferative activities in vitro and in vivo. This study used novel assays to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenograft models using immunofluorescence detection of DNA and α-tubulin, respectively. Furthermore, the effect of alicritinib on in vivo tumor cell proliferation was noninvasively measured using 18F-3'-fluoro-3'-deoxy-L-thymidine positron emission tomography (FLT-PET). Results showed that alicritinib was superior to ABK in inhibiting AAK in cells, with a selectivity exceeding 200-fold. In the HCT-116 xenograft model, alicritinib dose-dependently reduced the number of bipolar chromosomes and parallel chromosomes, a phenotype consistent with AAK inhibition. Alicritinib inhibited the proliferation of human tumor cell lines in vitro, suppressed tumor growth in a solid tumor xenograft model, and induced tumor regression in an in vivo lymphoma model. Moreover, FLT uptake decreased when alicritinib doses that caused tumor volume arrest were administered, suggesting that noninvasive imaging may be more valuable than traditional efficacy assessment methods. Conclusion: Alicritinib is a selective and potent AAK inhibitor. The novel method for measuring Aurora A pathway inhibition and its application in tumor imaging described in this article may have significant value for the clinical evaluation of small molecule inhibitors. [4]

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Mechanism of action (cited from [1,2,4]): 1. Inhibits Aurora A kinase activity, blocks centrosome maturation and spindle assembly, leading to G2/M phase cell cycle arrest; 2. Prolonged G2/M phase arrest triggers caspase-dependent apoptosis (upregulates cleavage caspase-3 and downregulates anti-apoptotic proteins); 3. Resistance in mutants: F133L/L215R/T217A mutations alter the ATP binding pocket of Aurora A, reducing the binding affinity of Alisertib [1,2,4]
-Therapeutic potential (cited from [3,4]): - Showed preclinical efficacy in hematologic malignancies (MM) and solid tumors (lung cancer, colon cancer, breast cancer) [4]; - Considered a promising Aurora A inhibitor, currently in early clinical development for relapsed/refractory cancers [3]
-Drug class (cited from [3]): Alisertib It belongs to the pyrimidine-derived Aurora kinase inhibitors, and its selectivity and oral bioavailability for Aurora A have been optimized [3]

C27H20CLFN4O4

518.923508644104

518.115

C, 62.49; H, 3.88; Cl, 6.83; F, 3.66; N, 10.80; O, 12.33

1028486-01-2

Alisertib sodium;1028486-06-7; 1208255-63-3 (sodium)

24771867

Light yellow to light pink solid powder

1.4±0.1 g/cm3

729.1±70.0 °C at 760 mmHg

394.8±35.7 °C

0.0±2.5 mmHg at 25°C

1.671

5.56

2

9

6

37

836

0

ClC1C=CC2C3C(=CN=C(NC4C=CC(C(=O)O)=C(C=4)OC)N=3)CN=C(C3C(=CC=CC=3OC)F)C=2C=1

ZLHFILGSQDJULK-UHFFFAOYSA-N

InChI=1S/C27H20ClFN4O4/c1-36-21-5-3-4-20(29)23(21)25-19-10-15(28)6-8-17(19)24-14(12-30-25)13-31-27(33-24)32-16-7-9-18(26(34)35)22(11-16)37-2/h3-11,13H,12H2,1-2H3,(H,34,35)(H,31,32,33)

4-((9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-benzo[c]pyrimido[4,5-e]azepin-2-yl)amino)-2-methoxybenzoic acid

MLN-8237; alisertib; MLN8237; MLN 8237

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: 2.08 mg/mL (4.01 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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 2: 2.08 mg/mL (4.01 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 ultrasonication.
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 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 3: ≥ 2.08 mg/mL (4.01 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:5mg/mL

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