Onvansertib  (Synonyms: NMS-1286937; NMS-P937)

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 Supernatant, containing the phosphorylated substrate, is subsequently withdrawn and transferred into a counting plate, followed by evaluation by b-counting.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°C using a 60 min end-point assay where the concentrations of ATP and substrates are kept equal to 2 × αK_m and saturated (>5 × αK_m), respectively.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Beria I, 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 May 15;21(10):2969-74.  [Content Brief]

  [2]. Valsasina B, 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 Apr;11(4):1006-16.  [Content Brief]

  [3]. Casolaro A, et al. The Polo-Like Kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia. PLoS One. 2013;8(3):e58424.  [Content Brief]