Kinase experiment:

His-tagged human Mps1Cat encoding amino acids 510-857 is generated. For kinase assays, 500 ng is added to buffer (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 μg/mL BSA, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 10 mM MgCl2, and 0.5 μg/mL myelin basic protein), AZ3146, and 100 μM γ-[32P]ATP (2 μCi/assay). Reactions are incubated at 30℃ for 20 min, spotted onto P81 paper, washed in 0.5% phosphoric acid, and immersed in acetone. Phosphate incorporation is determined by scintillation counting. For immunoprecipitation kinase assays, HeLa cells are treated with nocodazole for 14 h, mitotic cells isolated, washed in PBS, and lysed for 30 min in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5% NP-40, 5 mM EDTA, 5 mM EGTA, 40 mM β-glycerophosphate, 0.2 mM PMSF, 1 mM DTT, 1 mM sodium orthovanadate, 20 mM sodium fluoride, 1 μM okadaic acid, and complete EDTA-free protease inhibitor cocktail. Full-length Mps1 is immunoprecipitated. Purified complexes are washed with lysis buffer containing 100 mM NaCl and assayed as described for the recombinant protein. To quantify 32P incorporation, reactions are stopped with SDS sample buffer and separated by SDS-PAGE followed by phosphorimaging. The plate is analyzed using a phosphorimager using AIDA software. To assess the specificity of AZ3146, a single-point screen is carried using kinase profiling service. 50 kinases are selected and assayed with 1 μM AZ3146[1].

Cell experiment:

The TTK inhibitor AZ3146 is disolved in DMSO at a concentration in 100 mM and diluted into 100 μM, 10 μM, 1 μM and 0.1 μM sequentially with DMEM containing 10% FBS before use. In vitrocytotoxicity assays are performed. HCC cells are plated into 96-well plates at the density of 3×103 per well. AZ3146 is added in the indicated concentrations the next day. The inhibitor treated cells are cultured and tested at a 24-hour intervals for 3-4 days using CCK-8[2].

AZ3146 is a potent and selective Monopolar Spindle 1 (Mps1) kinase inhibitor with IC50 value of 35 nM. AZ3146 acts by interfering with chromosome alignment and overriding the spindle assembly checkpoint.

MPS1 protein kinases play important roles in different steps in mitosis including chromosome attachment and functions at the centrosomes. Additionally, they are also involved in regulation of development and in multiple signaling pathways after mitosis.

In cellular culture, if Mps1 is inhibited by AZ3146 before mitotic entry, this inhibition abloshed the subsequent recruitment of Mad1 and Mad2 to kinetochores. However, if cells were treated with AZ3146 after mitotic entry, the Mad1-C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. , AZ3146 interferes with chromosome alignment and overrides spindle assembly checkpoint1.

Regarding the effect of AZ3146 administration in vivo, the evidence should be provided by performing the study in human or mice or other animal models.

Reference:
1. Hewitt L, Tighe A, Santaguida S, et al. Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1-C-Mad2 core complex. The Journal of cell biology.2010;190(1):25-34.