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)

Tozasertib (VX-680) 是三种Aurora激酶的强效抑制剂，其表观抑制常数（Ki（app））分别为Aurora-A、Aurora-B和Aurora-C的0.6、18及4.6 nM。VX-680导致细胞聚集在4N DNA含量处，并强力抑制了多种肿瘤细胞类型的增殖，IC50值在15至113 nM之间[1]。不同的甲状腺癌细胞（ATC细胞）经VX-680处理后，其增殖在时间和剂量上表现出依赖性抑制，IC50值在25至150 nM之间。VX-680显著阻碍了不同细胞系在软琼脂中形成菌落的能力。通过对半胱天冬酶-3活性的分析表明，VX-680在不同细胞系中诱导了凋亡[2]。

在裸鼠体内，用Tozasertib以每日两次，每次75 mg/kg的剂量通过腹腔注射(b.i.d. i.p.)治疗13天后，与对照组相比，平均肿瘤体积减少了98%。在十只动物中有四只的最终肿瘤体积比治疗前的初始体积还要小。肿瘤生长的减少与剂量成正比，且在12.5 mg/kg b.i.d.剂量下显著。Tozasertib具有良好的耐受性，仅在最高剂量下观察到体重轻微下降（75 mg/kg b.i.d.时，体重下降5%）。Tozasertib还在胰腺和结肠异种移植模型中诱导肿瘤退缩。在一个确立的人类胰腺(MIA PaCa-2)异种移植模型中，用Tozasertib以每日两次，每次50 mg/kg通过腹腔注射(b.i.d i.p.)治疗，使得十个肿瘤中有七个出现退缩，并且与治疗前的初始肿瘤体积相比，平均肿瘤体积减少了22% [1]。

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].