Aurora A (Ki = 0.6 nM); Aurora B (Ki = 18 nM); Aurora C (Ki = 4.6 nM)

When BaF3 cells transfected with ABL or FLT-3 (mutant and wild-type) kinases are exposed to tozasertib, the cells show G2/M arrest, endoreduplication, and apoptosis in addition to comparable cytotoxicity (IC50 of about 300 nM). suppressor phenotype akin to AUR B. Time-dependently, tozasertib inhibits the growth of CAL-62. The number and size of colonies was dramatically decreased by about 70% for 8305C and by 90% for CAL-62, 8505C, and BHT-101 after 14 days of tozasertib treatment. Different ATC cell lines were treated with tozasertib, which decreased growth with an IC50 ranging from 25 to 150 nM. Tozasertib dramatically reduces many cell lines' capacity to establish colonies in soft agar. Analyses of Caspase-3 activity revealed that Tozasertib caused apoptosis in many cell types. After being exposed to tozasertib for 12 hours, CAL-62 cells accumulated cells with a DNA content of less than 4N. Time-lapse imaging revealed that Tozasertib-treated CAL-62 cells exit metaphase without proliferating. Moreover, the administration of tozasertib results in the elimination of histone H3 phosphorylation [2]. In patient-derived samples, tozasertib significantly inhibits BCR-Abl with the T315I mutation [3].

VX-680 gives rise to a marked decrease in tumor size in a human AML (HL-60) xenograft model. In mude mice treateed with VX-680 at 75 mg/kg, twice a day intraperitoneally (b.i.d. i.p.) for 13 days, mean tumor volumes are reduced by 98%. Tumor growth decrease is dose dependent and significant at a dose of 12.5 mg/kg b.i.d. VX-680 is well tolerated, with a small decrease in body weight observed only at the highest dose. VX-680 also triggers tumor regresson in pancreatic and colon xenograft models. VX-680 also displays potent antitumor activity when infused i.v. in mude rats bearing established HCT116 tumors. A higher dose of VX-680 (2 mg/kg/h) improves efficacy with a 56% decrease in mean tumor volume.

Protein Expression and Purification of ABL2 Kinase Domain[2]
For expression of the protein, baculovirus obtained from the Sf9 cell culture was used to infect Trichoplusia ni (Hi5) cells grown in suspension to a density of 2 × 106 cells/mL. At 48 h postinfection the cells were harvested by centrifugation and cell pellets were stored at −80 °C. Cells were resuspended in a buffer consisting of 5 mM imidazole, 500 mM NaCl, 50 mM Hepes, pH 7.4, 5% glycerol, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), supplemented with complete protease inhibitor mixture, and lysed by sonication. The lysate was centrifuged at 45000g for 1 h at 4 °C. The supernatant was filtered and loaded onto nickel-chelating resin. After being washed, the protein was eluted with the above buffer plus 50−300 mM imidazole and the eluates were combined. The hexahistidine tag was removed by overnight treatment with TEV protease at 4 °C. The digested ABL2 was concentrated to 2.5 mL volume and loaded onto a Superdex75 gel filtration column (HiLoad 16/60, GE Healthcare) equilibrated in 10 mM Hepes, pH 8.0, 300 mM NaCl, and 0.5 mM TCEP.[2]
The protein identity was verified by electrospray ionization time-of-flight mass spectrometry. Prior to removal of the hexahistidine tag, the observed mass was 33 414 Da compared to a calculated mass of 33 502; it is likely that the difference in mass was due to removal of the N-terminal methionine followed by acetylation. After removal of the hexahistidine tag the observed mass was 30 980 Da, matching exactly the calculated mass.

---

Crystallization and Data Collection[2]
The ABL2:imatinib complex (PDB code 3GVU, Table ​Table2)2) was crystallized at 4 °C in 200 nL drops from a 1:1 ratio of ABL2:imatinib (4 mg/mL protein containing 1 mM imatinib) and reservoir solution (20% PEG3350, 0.1 M citrate, pH 5.5). The crystals were then cryoprotected in reservoir solution with 20% (v/v) PEG300 and flash-frozen in liquid nitrogen. X-ray diffraction data was collected at 100 K on beamline X10SA at the Swiss Light Source (SLS).

Western blot[3]
The cells were lysed in RIPA buffer (50 mM Tris–HCl (pH 7.4), 1% NP-40, 0.5% sodium deoxycholate, 150 mM sodium chloride, 1 mM EDTA, 1× Protease Inhibitor Cocktail Set III), sonicated, and then centrifuged at 15 000 g for 20 min. Protein concentrations were determined by the Bradford assay. Aliquots of 50 μg cell protein extracts were electrophoresed on a 12.5% polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were then washed with TBST (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.05% Tween-20), saturated with 5% low-fat milk in TBST and then incubated at 4 °C overnight with antibodies against Aurora-A (1:500), Aurora-B (1:500), Aurora-C (1:500), or actin (1:1000) in TBS-T. After washing, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies against mouse or rabbit IgG (1:20 000) in TBST and developed using the chemiluminescence Super Signal kit. Aurora kinases and actin immunoreactive bands were quantified by scanning densitometry, using Molecular Analyst PC software for the Bio-Rad model 670 scanning densitometer. The different Aurora kinases/actin ratios were calculated and the values obtained for Tozasertib (VX680; MK0457)-treated cells were normalized against those found in control cells and reported as a fold of variation.

---

Proliferation assay[3]
The CAL-62 cells were cultured in the absence (dimethyl sulfoxide, DMSO) or the presence of 500 nM Tozasertib (VX680; MK0457) for different periods of time (1–5 days). The dose-dependent effects of Tozasertib (VX680; MK0457) on cell proliferation were evaluated by treating the different ATC cells for 4 days with different concentrations of the Aurora inhibitor (5–500 nM). The cells were pulse labeled with 30 mM BrdU for 2 h before the end of the incubation time. The BrdU incorporation was analyzed by means of a colorimetric immunoassay using the cell proliferation ELISA kit (Roche Applied Science), according to the manufacturer's instructions. The results from Tozasertib (VX680; MK0457)-treated cells were compared with those observed in control cells and expressed as a fold of variation versus control.

---

FACS analysis[3]
The CAL-62 cells were cultured in the absence (DMSO) or the presence of 250 nM Tozasertib (VX680; MK0457) for 6, 12, or 72 h. In some experiments, the cells were treated with 30 mM BrdU 20 min before harvesting. The cells were collected in PBS by scraping with a rubber policeman and fixed in ice-cold ethanol. The cell samples were analyzed for DNA content (propidium iodide) and/or BrdU content (FITC) as previously described (30) using an EPICS Elite Flow cytometer.

---

Colony formation in soft agar[3]
Petri dishes of 3.5 cm in diameter were first prepared by adding 3 ml complete media with 0.4% soft agar. Adherent ATC cell cultures were trypsinized and centrifuged to obtain a single-cell suspension of 150 000 viable cells/ml. The suspension was mixed with complete medium containing 0.4% soft agar at a ratio 1:2 and then divided into two aliquots, one of which was supplemented with Tozasertib (VX680; MK0457) 250 nM and the other with the vehicle (DMSO). These suspensions were plated onto the Petri dishes, 1 ml/dish, and incubated at 37 °C and 5% CO2. Treated and non-treated plates were photographed, few hours after plating to exclude the presence of cell aggregates (data not shown) and again after 2 weeks. The size of the colonies was measured by MetaVue software and those larger than 50 μm in diameter were scored.

---

Caspase-3 assay[3]
The different ATC cells were cultured in the absence (DMSO) or presence of 250 nM Tozasertib (VX680; MK0457) for 72 h. Following treatment, the cells were rinsed with PBS and collected by scraping in PBS. The cells were then used to evaluate caspase-3 activity using the caspase-3/CPP32 fluorimetric assay kit.

---

Time-lapse analysis[3]
The CAL-62 cells were cultured in the absence (DMSO) or presence of 250 nM Tozasertib (VX680; MK0457) and observed for 24 h under a microscope (Leica DM-IRBE) equipped with an incubation chamber at 37 °C and 5% CO2. The cell pictures were performed every 5 min using the MetaVue software.

---

Immunofluorescence (IF)[3]
The CAL-62 cells cultured on glass cover slips were treated with 250 nM Tozasertib (VX680; MK0457) or vehicle (DMSO) for 6 h. The cells were fixed in cold methanol for 2 min, washed, and preincubated with 3% BSA in PBS for 1 h at room temperature. After three washes with PBS, the cells were incubated with the antibodies anti-TACC3 (1:100), anti-Aurora-A (1:200), anti-Aurora-B (1:200), anti-Aurora-C (1:200), anti-P-histone H3 (1:1000), anti-γ-tubulin (1:200), or anti-β-tubulin (1:200) for 2 h at room temperature. After washing, the cover slips were incubated with TRITC-conjugated anti-goat or anti-rabbit (1:100) and FITC-conjugated anti-mouse (1:100) antibodies for 1 h at room temperature and then mounted in Vectashield containing 1 μg/ml DAPI. The cover slips were observed with a microscope Leica DMRXA.

The Aurora kinases are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumors, and lead to aneuploidy and tumorigenesis. Here we report the discovery of a highly potent and selective small-molecule inhibitor of Aurora kinases, VX-680, that blocks cell-cycle progression and induces apoptosis in a diverse range of human tumor types. This compound causes profound inhibition of tumor growth in a variety of in vivo xenograft models, leading to regression of leukemia, colon and pancreatic tumors at well-tolerated doses. Our data indicate that Aurora kinase inhibition provides a new approach for the treatment of multiple human malignancies.[1]

---

Dissolved in 50% PEG300 in 50 mM phosphate buffer; 50, 75 mg/kg; i.p. injection

Female athymic NCr-nu mice bearing HL-60 leukemia cells

Metabolism / Metabolites
Tozasertib has known human metabolites that include Unii-1S9W4WJ8WL and Unii-wuu5ros9AF.

[1]. VX-680, a potent and selective smallmolecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med. 2004; 10:262-7.

[2]. Crystal structures of ABL-related gene (ABL2) in complex with imatinib, tozasertib (VX-680), and a type I inhibitor of the triazole carbothioamide class.J Med Chem. 2011 Apr 14;54(7):2359-67. Epub 2011 Mar 18.

[3]. Effects of the Aurora kinase inhibitor VX-680 on anaplastic thyroid cancer-derived cell lines. Endocr Relat Cancer. 2008 Jun;15(2):559-68.

N-[4-[[4-(4-methyl-1-piperazinyl)-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyrimidinyl]thio]phenyl]cyclopropanecarboxamide is a N-arylpiperazine.
See also: Tozasertib Lactate (annotation moved to).

---

ABL2 (also known as ARG (ABL related gene)) is closely related to the well-studied Abelson kinase cABL. ABL2 is involved in human neoplastic diseases and is deregulated in solid tumors. Oncogenic gene translocations occur in acute leukemia. So far no structural information for ABL2 has been reported. To elucidate structural determinants for inhibitor interaction, we determined the cocrystal structure of ABL2 with the oncology drug imatinib. Interestingly, imatinib not only interacted with the ATP binding site of the inactive kinase but was also bound to the regulatory myristate binding site. This structure may therefore serve as a tool for the development of allosteric ABL inhibitors. In addition, we determined the structures of ABL2 in complex with VX-680 and with an ATP-mimetic type I inhibitor, which revealed an interesting position of the DFG motif intermediate between active and inactive conformations, that may also serve as a template for future inhibitor design.[2]

---

Anaplastic thyroid cancers (ATC) are aggressive tumors, which exhibit cell cycle misregulations leading to uncontrolled cellular proliferation and genomic instability. They fail to respond to chemotherapeutic agents and radiation therapy, and most patients die within a few months of diagnosis. In the present study, we evaluated the in vitro effects on ATC cells of VX-680, an inhibitor of the Aurora serine/threonine kinases involved in the regulation of multiple aspects of chromosome segregation and cytokinesis. The effects of VX-680 on proliferation, apoptosis, soft agar colony formation, cell cycle, and ploidy were tested on the ATC-derived cell lines CAL-62, 8305C, 8505C, and BHT-101. Treatment of the different ATC cells with VX-680 inhibited proliferation in a time- and dose-dependent manner, with the IC50 between 25 and 150 nM. The VX-680 significantly impaired the ability of the different cell lines to form colonies in soft agar. Analysis of caspase-3 activity showed that VX-680 induced apoptosis in the different cell lines. CAL-62 cells exposed for 12 h to VX-680 showed an accumulation of cells with > or =4N DNA content. Time-lapse analysis demonstrated that VX-680-treated CAL-62 cells exit metaphase without dividing. Moreover, histone H3 phosphorylation was abrogated following VX-680 treatment. In conclusion, our data demonstrated that VX-680 is effective in reducing cell growth of different ATC-derived cell lines and warrant further investigation to exploit its potential therapeutic value for ATC treatment.[3]

C23H28N8OS

464.59

464.21

C, 59.46; H, 6.07; N, 24.12; O, 3.44; S, 6.90

639089-54-6

5494449

White to off-white solid powder

1.4±0.1 g/cm3

1.708

1.18

3

8

7

33

650

0

GCIKSSRWRFVXBI-UHFFFAOYSA-N

InChI=1S/C23H28N8OS/c1-15-13-20(29-28-15)25-19-14-21(31-11-9-30(2)10-12-31)27-23(26-19)33-18-7-5-17(6-8-18)24-22(32)16-3-4-16/h5-8,13-14,16H,3-4,9-12H2,1-2H3,(H,24,32)(H2,25,26,27,28,29)

(N-[4({4-(4-methylpiperazin-1-yl)-6-[(3-methyl-1H-pyrazol-5 -yl)amino]pyrimidin-2-yl}thio)phenyl]cyclopropanecarboxamide)

Tozasertib, MK-0457; VX-680; MK 0457; MK-0457; VX680; VX 680; MK0457; VE 465; VE465; VE-465

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (4.48 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (4.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (4.48 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.

---

Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol:30mg/mL

---

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