Kinase experiment:

The potency of the compound towards MPS1 and 60 additional kinases belonging to kinase selectivity screening (KSS) panel is determined using either a strong anion exchanger based assay or P81 Multiscreen plate. MPS1 activity is measured using 5 nM of MPS1 recombinant protein in 50 mM HEPES pH 7.5, 2.5 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 3 μM NaVO3, 2 mM β-glycerophosphate, 0.2 mg/mL BSA, 200 μM P38-βtide substrate-peptide (KRQADEEMTGYVATRWYRAE) and 8 μM ATP with 1.5 nM 33P-γ-ATP. The assay is run in a robotized format, 10 serial 1:3 compounds dilutions (including NMS-P715, from 30 μM to 1.5 nM) are tested and IC50 determined[1].

Cell experiment:

Cells lines are seeded in 384 well-plates in the appropiate complete medium and treated with compounds (NMS-P715, etc.) dissolved in 0.1% DMSO 24 hours after seeding. The cells are incubated at 37℃ and 5% CO2 and after 72 hours the plates are processed using CellTiter-Glo assay. Inhibitory activity is evaluated comparing treated versus control data using Assay Explorer software. IC50 of proliferation is calculated using sigmoidal interpolation curve fitting. Activity Ratio is calculated as the ratio of the single cell line IC50 and the IC50 average of all the cell lines tested[1].

Animal experiment:

Mice[1]Athymic nu-nu mice, 5-6 weeks of age (20-22 g) are used in the assay. A2780 ovary carcinoma and A375 melanoma cells are transplanted s.c. into female nu-nu mice. Mice bearing a palpable tumor (100-200 mm3) are selected and randomized into control and treated groups. Treatment starts one day after randomization. NMS-P715 is typically administered by oral administration at doses of 90-100 mg/kg daily for more than seven days. Each group includs 8 animals. Tumor dimension is measured regularly by calipers during the experiments and tumor mass is calculated[1].

NMS-P715 is a selective, ATP-competitive inhibitor of MPS1, with an IC50 of 182 nM. Mps1|182 nM (IC50)|CK2|5.7 μM (IC50)|MELK|6.01 μM (IC50)|NEK6|6.02 μM (IC50)

NMS-P715 is a selective inhibitor of MPS1, with an IC50 of 182 nM. NMS-P715 is highly specific for MPS1, with no other kinases inhibited below an IC50 value of 5 μM and only 3 kinases inhibited below 10 μM (CK2, MELK, and NEK6). NMS-P715 promotes massive spindle assembly checkpoint (SAC) override with an EC50 of 65 nM. NMS-P715 (1 μM) causes mitotic acceleration in U2OS cells overexpressing YFP-α-tubulin, induces aneuploidy and inhibits the proliferation of HCT116 cells. NMS-P715 (0.5, 1 μM) affects mitotic checkpoint complex (MCC) stability and cdc20 ubiquitylation[1]. NMS-P715 (1 μM) exhibits bypass of the spindle assembly checkpoint and apoptosis in pancreatic ductal adenocarcinoma (PDAC) cell lines. NMS-P715 (0-25 μM) also selectively inhibits growth of PDAC cells[2].

NMS-P715 (10 mg/kg) exhibits an oral bioavailability of 37% and good pharmacokinetic properties in nude mice bearing subcutaneous implanted human tumor cell xenografts. NMS-P715 (90 mg/kg, p.o.) is well tolerated and cuases no signs of body weight loss or other overt toxicities in an A2780 ovary carcinoma xenograft model. NMS-P715 (100 mg/kg, p.o.) inhibits the tumor growth by appr 43% in the A375 melanoma xenograft model[1].

[1]. Colombo R, et al. Targeting the mitotic checkpoint for cancer therapy with NMS-P715, an inhibitor of MPS1 kinase. Cancer Res. 2010 Dec 15;70(24):10255-64. [2]. Slee RB, et al. Selective inhibition of pancreatic ductal adenocarcinoma cell growth by the mitotic MPS1 kinase inhibitor NMS-P715. Mol Cancer Ther. 2014 Feb;13(2):307-315.