MPS1 (TTK) [1][2][3];
In vitro kinase assay using recombinant human Mps1 catalytic domain (Mps1Cat): IC50 = approximately 35 nM [1];
In vitro inhibition of full-length Mps1 immunoprecipitated from mitotic HeLa cells: IC50 not explicitly stated, but 2 μM AZ3146 effectively inhibited autophosphorylation [1];
Cellular target engagement: inhibition of MPS1 autophosphorylation at T33/S37 in cells: IC50 for wild-type MPS1 = 1.48 μmol/L (MSD assay) [2];
Kinase selectivity panel: at 1 μM, AZ3146 inhibited only FAK, JNK1, JNK2, and KIT by >40% among 50 kinases tested [1];
In HCC cells, AZ3146 targets TTK to reduce cell viability (IC50 values: 7.13 μM in BEL-7404, 28.62 μM in SMMC-7721) [3].

AZ3146 inhibits human Mps1Cat in an in vitro kinase assay, with an IC50 of about 35 nM. Additionally, full-length Mps1 immunoprecipitated from human cells is potently inhibited from autophosphorylating by AZ3146 [1]. HCC cell growth can be inhibited by AZ3146, a TTK-specific kinase inhibitor. To test for cytotoxicity in vitro, SMMC-7721 and BEL-7404 cells were used. The results indicated that the IC50 values were 7.13 μM (BEL-7404) and 28.62 μM (SMMC-7721). For four days, both cells received additional treatment at IC50 concentrations. There was a noticeable reduction in cell proliferation [2]. HCT116 cells were grown for ten days at 0.8 μM (GI50) AZ3146 and for three weeks at 2 μM AZ3146. In cell viability assays, sixteen clones, designated AzR1-16, were isolated and cell lines were created. AzR3 and 4 had GI50 values of roughly 3 μM (4-fold resistance), while the remaining clones had GI50 values of roughly 9 μM (11-fold resistance). All of the clones were resistant to AZ3146-induced cell death. The parental cell line rapidly exited mitosis within 10 minutes when 2 μM AZ3146 was added, according to time-lapse microscopy analysis of mitosis [3].

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AZ3146 inhibits Mps1 kinase activity in cells: treatment with 2 μM AZ3146 for 2 h reduced the slower-migrating phosphorylated isoform of Mps1 in Phos tag gels [1]. AZ3146 (2 μM) overrides the spindle assembly checkpoint (SAC), causing cells to exit mitosis prematurely (from ~25 min to ~10 min in HeLa cells) [1]. AZ3146 inhibits kinetochore recruitment of O-Mad2 but does not displace the Mad1-C-Mad2 core complex; in HeLa cells expressing Myc-Mad2, AZ3146 reduced anti-Myc signal at kinetochores to 45±5% of control, while SM2.2 (Mad2 antibody) signal was reduced to background levels [1]. AZ3146 also inhibits chromosome alignment: after monastrol washout, 2 μM AZ3146 plus MG132 caused alignment defects in >85% of cells, and time-lapse microscopy showed unaligned chromosomes [1]. AZ3146 treatment (2 μM) reduced kinetochore localization of CENP-E to approximately 30% of control [1]. In HCT116 cells, the GI50 of AZ3146 is 0.8 μmol/L [2]. AZ3146 treatment (2 μmol/L) caused rapid mitotic exit (~10 min) in parental HCT116 cells, while AZ3146-resistant clones (e.g., AzR1 with p.S611G, AzR3 with p.I531M) maintained normal mitotic duration (~25 min) [2]. AZ3146 (2 μmol/L) abolished G1 and G2-M peaks in parental HCT116 cell cycle profiles by flow cytometry, but not in resistant clones [2]. In IP-kinase assays, the p.S611G and p.I531M MPS1 mutants showed near-wild-type kinase activity but were resistant to AZ3146 inhibition (e.g., cellular IC50 for p.S611G = 19.2 μmol/L, p.I531M = 3.4 μmol/L, double mutant >25 μmol/L for autophosphorylation) [2]. In HCC cell lines, AZ3146 inhibits cell proliferation: BEL-7404 cells treated with 7.13 μM AZ3146 for 4 days showed significant growth inhibition; SMMC-7721 cells treated with 28.62 μM AZ3146 also showed reduced viability [3].

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.

Recombinant Mps1 catalytic domain (Mps1Cat, amino acids 510-857) was incubated with AZ3146 in buffer containing 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, 0.5 μg/ml myelin basic protein (MBP), 100 μM γ[32P]ATP, and varying concentrations of AZ3146. Reactions were incubated at 30°C for 20 min, spotted onto P81 paper, washed in 0.5% phosphoric acid, and incorporated radioactivity was quantified by scintillation counting. IC50 was determined as approximately 35 nM [1].
For immunoprecipitation kinase assays, HeLa cells were arrested with nocodazole, lysed, and full-length Mps1 was immunoprecipitated. Immune complexes were washed and assayed with MBP and γ[32P]ATP in the presence or absence of 2 μM AZ3146. Reactions were stopped with SDS sample buffer, separated by SDS-PAGE, and analyzed by phosphorimaging [1].
Kinase selectivity profiling: a panel of 50 kinases was assayed by a commercial service (SelectScreen) using 1 μM AZ3146. Percent inhibition was calculated for each kinase [1].
Meso Scale Discovery (MSD) electrochemiluminescence assay: cells expressing Myc-tagged MPS1 constructs were treated with AZ3146 (0 to 32 μmol/L), and MPS1 autophosphorylation at T33/S37 was quantified using a fluorescence-based kinase assay as previously described (Naud et al., 2013). IC50 values for wild-type and mutant MPS1 were calculated from dose-response curves [2].
Recombinant full-length MPS1 proteins (wild-type and mutants) were produced, and their kinase activity in the presence of AZ3146 was measured using similar in vitro kinase assays with MBP and ATP, quantifying phosphorylation by autoradiography or scintillation counting [2].

Cell viability assays: HCT116 cells were treated with AZ3146 (0–16 μmol/L) for 4 days, and viability was measured using CellTiter-Glo Luminescent Cell Viability Assay. Colony formation was assessed after 14 days using Sulforhodamine B (SRB) colorimetric assay [2].
Flow cytometry: cells were fixed in 70% ethanol, stained with propidium iodide and RNase, and analyzed on a flow cytometer to assess cell cycle profiles after AZ3146 treatment (e.g., 1 and 2 μmol/L for 24 h) [2]. For mitotic index, cells were stained with anti-MPM2 antibodies followed by FITC-conjugated secondary antibodies [1].
Immunofluorescence: HeLa or HCT116 cells were fixed in 1% formaldehyde, permeabilized with PBS-Triton X-100, and stained with primary antibodies against MAD2, CDC20, MPS1, phospho-MPS1 (pT33/pS37), ACA (centromeres), or CENP-E, followed by fluorescent secondary antibodies and DAPI. Images were acquired on a confocal microscope [1][2].
Time-lapse microscopy: HeLa or HCT116 cells expressing Histone H2B-mCherry were treated with AZ3146 (2 μmol/L for HCT116, 2 μM for HeLa) and imaged using an inverted microscope with a motorized stage in a humidified CO2 chamber at 37°C. Time spent in mitosis was measured [1][2].
Phos tag gel electrophoresis: Mps1 from interphase or nocodazole-arrested cells treated with MG132 ± 2 μM AZ3146 for 2 h was resolved on SDS-PAGE containing 25 μM Phos tag and 50 μM MnCl2, followed by immunoblotting [1].
Cell proliferation assays (HCC): BEL-7404 and SMMC-7721 cells were seeded in 96-well plates (3×10^3 cells/well), treated with various concentrations of AZ3146 (0.1–100 μM) for 3–4 days, and cell viability was measured using CCK-8 reagent. IC50 values were calculated using Origin 8.0 software [3].
Transwell migration assay: HCC cells were transfected with TTK or siRNA, then starved overnight, and 1×10^5 cells in serum-free medium were added to upper chambers (8 μm pore size) with 10% FBS in lower chamber. After 48 h, cells on lower membrane were fixed, stained with crystal violet, and counted [3].
Soft agar colony formation: HCC cells transfected with TTK or siRNA were seeded in 24-well plates with 1% base agar and 0.5% top agar (2×10^3 cells/well). After 21 days, colonies were counted [3].
xCELLigence real-time cell analysis: After transfection with plasmids or siRNAs for 24 h, HCC cells were replated in E-plates (7.5×10^3 cells/well) and monitored every 60 min for 6 days using the RTCA-DP instrument. Cell index values were recorded [3].

[1]. Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1-C-Mad2 core complex. J Cell Biol. 2010 Jul 12;190(1):25-34.

[2]. TTK activates Akt and promotes proliferation and migration of hepatocellular carcinoma cells. Oncotarget. 2015 Oct 27;6(33):34309-20.

[3]. Naturally Occurring Mutations in the MPS1 Gene Predispose Cells to Kinase Inhibitor Drug Resistance. Cancer Res. 2015 Aug 15;75(16):3340-54.

AZ3146 is a chemical probe for studying Mps1 function in mitosis; it allowed dissection of the two-step mechanism of Mad2 recruitment, showing that Mps1 activity is required for Mad1 localization upon mitotic entry but not for maintenance, and continuously required for O-Mad2 recruitment [1]. AZ3146 has been used to generate acquired resistance models in HCT116 cells, identifying point mutations (p.S611G, p.I531M, p.M600T, p.Y568C, p.C604W) in the MPS1 kinase domain that confer cross-resistance to other MPS1 inhibitors, with structural studies revealing steric hindrance mechanisms [2]. AZ3146 also demonstrates that pre-existing drug-resistant mutations occur naturally in both cancer and normal cells, as detected by droplet digital PCR (e.g., p.S611G at 0.94% frequency in parental HCT116 cells) [2]. In hepatocellular carcinoma, AZ3146 treatment confirms TTK as a potential therapeutic target, as its inhibition reduces HCC cell growth and the compound shows specificity against TTK [3].

C24H32N6O3

452.55

452.253

1124329-14-1

56973724

Off-white to light yellow solid powder

1.3±0.1 g/cm3

621.0±65.0 °C at 760 mmHg

329.4±34.3 °C

0.0±1.8 mmHg at 25°C

1.631

2.76

1

7

6

33

669

0

YUKWVHPTFRQHMF-UHFFFAOYSA-N

InChI=1S/C24H32N6O3/c1-28-12-10-17(11-13-28)33-18-8-9-19(21(14-18)32-3)26-23-25-15-20-22(27-23)30(24(31)29(20)2)16-6-4-5-7-16/h8-9,14-17H,4-7,10-13H2,1-3H3,(H,25,26,27)

9-cyclopentyl-2-(2-methoxy-4-(1-methylpiperidin-4-yloxy)phenylamino)-7-methyl-7H-purin-8(9H)-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)