Kinase experiment:

The inhibitory activity of putative kinase inhibitors and the potency of selected compounds are determined using a trans-phosphorylation assay. Specific peptide or protein substrates are trans-phosphorylated by their specific serine-threonine or tyrosine kinase, in the presence of ATP traced with 33P-γ-ATP, at optimized buffer and cofactors conditions. At the end of the phosphorylation reaction, more than 98% unlabeled ATP and radioactive ATP is captured by adding an excess of the ion exchange dowex resin; the resin then settles down to the bottom of the reaction plate by gravity. Supernatant, containing the phosphorylated substrate, is subsequently withdrawn and transferred into a counting plate, followed by evaluation by b-counting. Inhibitory potency evaluation for all the tested kinases is performed at 25℃ using a 60 min end-point assay where the concentrations of ATP and substrates are kept equal to 2 × αKm and saturated (>5 × αKm), respectively.

Cell experiment:

Cells are seeded into 96- or 384-well plates at densities ranging from 10,000 to 30,000/cm2 for adherent and 100,000/mL for nonadherent cells in appropriate medium supplemented with 10% fetal calf serum. After 24 hours, cells are treated in duplicate with serial dilutions of NMS-P937, and 72 hours later, the viable cell number is assessed by the CellTiter-Glo Assay. IC50 values are calculated with a sigmoidal fitting algorithm. Experiments are carried out independently at least twice.

Animal experiment:

For carcinoma xenograft studies, 5- to 6-week-old female Hsd, athymic nu/nu mice (average weight, 20-22 g), are used. HCT116, HT29, Colo205 colorectal, and A2780 ovarian human carcinoma cell lines are inoculated subcutaneously. Mice bearing a palpable tumor (100-200 mm3) are treated with vehicle or NMS-P937 following doses and schedules starting from the day after randomization. Tumor dimensions are measured regularly with Vernier calipers, and tumor growth inhibition (TGI) is calculated. Toxicity is evaluated on the basis of body weight reduction. For leukemia studies, 5- to 6-week-old female severe combined immunodeficient mice (SCID; average weight, 20-22 g) are used. The AmL cell line HL-60 (5×106 cells) is injected subcutaneously and treatments initiated when tumor size reaches 200 to 250 mm3. Tumor dimensions and TGI are assessed. For disseminated models, 5×106 AmL primary cells (AmL-PS) are injected intravenously and treatments start after 2 days. Mice are monitored daily for clinical signs of disease, and the median survival time is determined for each group.

NMS-1286937 (NMS-P937) is a potent and selective inhibitor of Polo-like kinase 1 (PLK1) with IC50 value of 2 nM [1].

PLK1 is a serine/threonine protein kinase and plays an important role in the cell cycle control machinery. PLK1 is involved in mitotic entry, bipolar mitotic spindle formation, centrosome duplication, transition from metaphase to anaphase, maintenance of genomic stability and cytokinesis [1].

NMS-1286937 is an orally bioavailable PLK1 inhibitor. In cell lines established from solid tumors, leukemias and lymphomas, NMS-P937 inhibited tumor cell proliferation. In A2780 cells, NMS-P937 caused a G2-M cell-cycle block. In the mitotic phase, NMS-P937 induced apoptotic death with misaligned chromosomes and an aberrant number of spindles poles [2]. In human osteosarcoma (OS) cell lines, NMS-P937 inhibited migration and clonogenic ability of cell lines. In ABCB1-overexpressing, Doxorubicin (DX)-resistant cell lines, combination of NMS-P937 and DX reverted DX-resistance by inhibiting ABCB1 transport activity [3].

In Harlan nu/nu mice, NMS-P937 exhibited a good pharmacokinetic profile with low clearance, high AUC and Cmax, and acceptable oral bioavailability. In mice xenografted with human HCT116 colon adenocarcinoma cells, NMS-P937 (45 mg/kg) inhibited tumor growth by 83% and reduced body weight by 16% [1].

References:
[1]. Beria I, Bossi RT, Brasca MG, et al. NMS-P937, a 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivative as potent and selective Polo-like kinase 1 inhibitor. Bioorg Med Chem Lett, 2011, 21(10): 2969-2974.
[2]. Valsasina B, Beria I, Alli C, et al. NMS-P937, an orally available, specific small-molecule polo-like kinase 1 inhibitor with antitumor activity in solid and hematologic malignancies. Mol Cancer Ther, 2012, 11(4): 1006-1016.
[3]. Sero V, Tavanti E, Vella S, et al. Targeting polo-like kinase 1 by NMS-P937 in osteosarcoma cell lines inhibits tumor cell growth and partially overcomes drug resistance. Invest New Drugs, 2014, 32(6): 1167-1180.