Danusertib (PHA-739358) is a small-molecule 3-aminopyrazole derivative with potential antineoplastic activity. Danusertib binds to and inhibits the Aurora kinases, which may result in cell growth arrest and apoptosis in tumor cells in which Aurora kinases are overexpressed.

FGFR1:47 nM, Abl:25 nM, TrkA:31 nM, Aurora A:13 nM, RET:31 nM

Danusertib significantly inhibits the proliferation of K562 cells and suppresses tumor growth over a 10-day treatment period. Administering 25 mg/kg of Danusertib (b.d.i.v.) to HL-60 xenograft rats resulted in a 75% inhibition of tumor growth, with complete regression observed in one animal.

In cellular assays, treatment with Danusertib in wild-type and p53-deficient mouse embryonic fibroblasts (MEFs) resulted in the arrest of wild-type cells in mitosis (4N) for up to 48 hours, whereas p53-deficient cells did not exhibit arrest at the 4N DNA stage and proceeded through mitosis, leading to DNA content >8N. Danusertib treatment was associated with increased levels of p53 protein and an upregulation of p21 protein, which is known to be transcriptionally regulated by p53. Additionally, Danusertib inhibited the activity of other kinases, such as FGFR1, Abl, Ret, and Trka, with IC50 values of 47 nM, 25 nM, 31 nM, and 31 nM, respectively.

Biochemical kinase Assays: The Km values for ATP and the specific substrate are initially determined, and each assay is then run at optimized ATP (2Km) and substrate (5Km) concentrations. This setting enabled direct comparison of IC50 values of Danusertib across the applied kinase selectivity screening panel for the evaluation of the selectivity profile.

For short-term expansion assays, 1 × 103 CD34+ cells are plated in triplicates in 96-well plates containing 100 μL of serum-free medium per well supplemented with human stem-cell factor (100 ng/mL), human Flt-3 Ligand (100 ng/mL), human thrombopoietin (50 ng/mL), human interleukin-3 and -6 (IL-3 and IL-6, respectively, both 20 ng/mL), and granulocyte colony-stimulating factor (20 ng/mL) along with Danusertib at the indicated concentrations. After 5 days, an additional 100 μL of cytokine and Danusertib containing medium are added. Cell numbers within each individual well are estimated on days 3, 6, and 9 or on days 3, 6, and 12 for healthy donor samples. (Only for Reference)