Tozasertib (MK-0457) is a pan-Aurora kinase inhibitor (Kis: 0.6/18/4.6 nM for Aurora A/Aurora B/Aurora C). It shows selectivity against more than 190 different kinases.

Aurora B:18 nM (Ki), Aurora A:0.6 nM (Ki), Aurora C:4.6 nM (Ki)

Recombinant Aurora-1 (62-344), Aurora-2 (1-403) and Aurora-3 (1-309) were expressed as N-terminal, His6-tagged fusion proteins using a baculovirus expression system. The proteins were purified by affinity chromatography using Ni-NTA agarose, followed by size exclusion using a Superdex 200 26/60 column. Inhibition of kinase activity was assessed using a standard enzyme-coupled system or a radiometric, phosphocellulose-peptide capture assay as previously described [1].

Logarithmically growing MCF-7 cells were incubated with either VX-680 or DMSO for 48 h. Single-cell suspensions were fixed in 70% ethanol for 15 min, incubated with RNase (1 mg/ml) at 37 °C for 30 min, labeled with 400 μl propidium iodide (50 μg/ml) for at least 15 min at room temperature. Cell-cycle profiles were determined by flow cytometric analysis [1].

For the HL-60 study, female athymic NCr-nu mice were inoculated subcutaneously with 10^7 HL-60(TB) leukemia cells into the right axillary area. Treatment was administered i.p. b.i.d. after tumors reached 150–200 mm^3. VX-680 was prepared in a vehicle of 50% PEG 300 in 50 mM phosphate buffer. Cisplatin, formulated in saline, was administered i.p. q.4.d. for a total of three injections, at a dose of 5.4 mg/kg. For the MIA PaCa-2 studies, female MF1 nude mice were inoculated with 10^7 MIA PaCa-2 cells into the dorsal flank. Treatment was administered i.p. b.i.d. after tumors reached 175 mm^3. VX-680 was prepared in a vehicle of 50% PEG 300 in 50 mM phosphate buffer. 5-fluorouracil, formulated in saline, was administered i.v. q.4.d. at a dose of 50 mg/kg. For the HCT116 study, female Hsd RH rnu/nu rats were inoculated with 10^7 HCT116 cells into the right flank. Treatment was administered once the tumors reached 700–950mm^3. VX-680 was administered continuously through an indwelling femoral catheter, followed by a saline infusion for 4 d before repeating the dose cycle. For all studies, tumor volume was determined by caliper measurements three times a week [1].

VX 680, MK-0457

DMSO: 122.5 mg/mL (263.67 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 2 mg/mL (4.3 mM), Sonication is recommended.