Mps1 (IC50 = 1.8 nM); ALK (IC50 = 0.26 μM); B-RAF (IC50 = 3.2 μM); ERK2 (IC50 = 3.9 μM); FAK1 (IC50 = 2.7 μM); FER (IC50 = 0.59 μM); FLT3 (IC50 = 0.08 μM); INSR (IC50 = 0.38 μM); JNK1 (IC50 = 0.11 μM); PLK4 (IC50 = 3.3 μM); STK33 (IC50 = 1.1 μM )

MPI-0479605, having an IC50 of 1.8 nM, is a strong and specific ATP-competitive inhibitor of Mps1. HCT-116 cell viability is dose-dependently lowered by MPI-0479605 (0.1–10 μM). While causing full cytokinesis, MPI-0479605 exhibits severe defects in chromosome alignment at the metaphase plate[1].

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MPI-0479605 is a potent, selective inhibitor of Mps1 kinase [1]
MPI-0479605 is a potent, ATP-competitive inhibitor of Mps1 identified by compound screening and subsequent medicinal chemistry optimization. It inhibits Mps1 with an IC50 value of 1.8 nmol/L (Fig. 1A) and is highly selective for Mps1 when tested against 120 other kinases (Supplementary Table S1). MPI-0479605 is structurally similar to reversine and MPS1-IN-1, 2 recently published inhibitors of Mps1, but differs from reversine in that it lacks activity against Aurora kinases. [1]
MPI-0479605 inhibited Mps1 function in cells as shown by the ability to override the SAC induced by the microtubule-destabilizing drug nocodazole. In nocodazole-arrested cells, MPI-0479605 triggered the time-dependent degradation of both cyclin B and securin, which normally allow for mitotic exit, as well as a decrease in BubR1 phosphorylation (Fig. 1B). As a result, cells exited mitosis and failed to undergo cytokinesis, as evidenced by an increase in the percentage of cells with more than 4N DNA content (Fig. 1C). In addition, the percentage of cells that stained positive for phosphohistone-H3 (pHH3), a marker of mitotic cells, decreased in a dose-dependent manner in response to treatment with MPI-0479605 (Supplementary Fig. S2). Consistent with inhibition of Mps1 in cells, MPI-0479605 blocked apparent autophosphorylation of Mps1 at threonine 676 in HEK293T cells overexpressing Mps1 (Fig. 1D).

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Effects of Mps1 inhibition on mitosis and cell-cycle progression [1]
The effect of MPI-0479605 on chromosome segregation was examined by imaging A549 cells during mitosis. Cells treated with MPI-0479605 exhibited severe defects in the ability to align chromosomes at the metaphase plate. A significant fraction of cells treated with MPI-0479605 entered anaphase with at least one unaligned chromosome, as was evident by the presence of lagging chromosomes during anaphase (Fig. 2A). Following completion of mitosis and reentry into interphase, cells treated with MPI-0479605, or the related compound MPI-0485812 (structure in Supplementary Fig. S3), contained micronuclei that stained positive for centromeres, but not γH2AX (Fig. 2B and data not shown), consistent with the presence of whole chromosomes, and not DNA fragments, within these micronuclei. In some cells, we observed the presence of multiple nuclei, but this was not the predominant phenotype. Despite defects in chromosome segregation, the majority of MPI-0479605–treated cells were able to complete cytokinesis, as indicated by the absence of a large population of tetraploid cells (cells with 8N DNA content). Instead, there seemed to be gain or loss of chromosomes, as shown by the divergence of DNA content from the 2N and 4N peaks, in the majority of cells (Fig. 3A). Interestingly, p53-proficient HCT-116 cells showed only a small increase in the apoptotic sub-G1 population of cells within 72 hours of compound exposure, whereas p53-deficient Colo-205 cells exhibited a strong increase in this population. This suggests that cells with wild-type p53 may be partially protected from apoptosis. To better understand whether cells are actively dividing, cells were synchronized in late G1 and treated with CSFE, which labels intracellular proteins. Upon cell division, the fluorescence intensity decreases, allowing cell division to be monitored. Labeled cells were released from the G1 block into media containing DMSO or MPI-0479605, and the number of cell divisions occurring during a 72-hour period was determined. Cells treated with MPI-0479605 were able to complete one cell division in a manner similar to the DMSO-treated cells, but thereafter, MPI-0479605–treated cells showed a significant delay and/or arrest in cell-cycle progression (Fig. 3B). DNA synthesis was monitored by the incorporation of BrdUrd. Following exposure to MPI-0479605 for 48 hours, BrdUrd incorporation was greatly suppressed, indicating that most cells were not actively cycling through S-phase (Fig. 3C). This occurred in both p53 wild-type and p53-deficient cells, suggesting that this effect is not dependent on the presence of functional p53. We conclude that inhibition of Mps1 abrogated the SAC and allowed anaphase progression in the presence of misattached chromosomes, resulting in chromosome segregation defects and aneuploidy. Subsequent to this, cell-cycle progression was retarded, presumably due to the activation of a postmitotic checkpoint or initiation of mitotic catastrophe. [1]
Recent work has shown that inactivation of the spindle checkpoint induces aneuploidy followed by activation of a p53 response and growth arrest and/or apoptosis. To determine whether aneuploidy induced by MPI-0479605 promotes a similar response, we examined the p53 signaling pathway. In p53-proficient cells, MPI-0479605 induced the expression of p53 in a time-dependent manner with maximal levels occurring by 48 hours, a time frame that is consistent with the observed inhibition of DNA synthesis (Fig. 4A). Induction of p21 protein and mRNA also occurred in a time- and dose-dependent manner, suggesting that p53 was transcriptionally active (Fig. 4A and B). Previous studies indicated that Mps1 phosphorylates p53 on threonine 18 during a postmitotic checkpoint in response to microtubule destabilization. Surprisingly, inhibition of endogenous Mps1 in the absence of microtubule-targeting drugs induced the phosphorylation of p53 on serine 15 rather than on threonine 18 (Fig. 4C and data not shown). Phosphorylation of serine 15 was prevented if cells were pretreated with CGK733, a dual inhibitor of the DNA damage kinases ATR and ataxia telangiectasia mutated (ATM; ref. 18) but not by pretreatment with the ATM-specific inhibitor KU-55933, suggesting ATR is the relevant kinase. (Fig. 4C and data not shown). Because ATR is normally activated in response to DNA damage, phosphorylation of histone H2AX, a marker for DNA damage and a direct substrate of ATR, was examined. At 48 hours posttreatment with MPI-0479605, approximately 15% of cells showed diffuse nuclear staining for γH2AX but did not display the typical focal staining observed in response to DNA damage (data not shown). By Western blotting, an increase in γH2AX was detected with the maximal signal occurring by 36 hours (Fig. 4A). Phosphorylation of H2AX in response to MPI-0479605 was inhibited with CGK733 but not KU-55933 (Fig. 4C and data not shown). Thus, both p53 and H2AX, known substrates of ATR, are phosphorylated in response to MPI-0479605 treatment.

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Inhibition of Mps1 induces a time-dependent cell death [1]
Treatment of HCT-116 cells with MPI-0479605 resulted in a significant decrease in cell viability at 48 and 72 hours (Fig. 5A). At the same time, caspase-3/7 activity was induced, with induction levels highest at 48 hours (Fig. 5B). Caspase induction was observed in both p53 wild-type and mutant cells but pretreatment of cells with the pan-caspase inhibitor z-VAD-fmk did not affect MPI-0479605–mediated cell death (Supplementary Fig. S4). To explore commitment to cell death, cells were exposed to Mps1 inhibitors for 24 hours, followed by incubation in compound-free media for up to 9 additional days. At lower doses of inhibitor, cell killing was incomplete and cell growth recovered, whereas at higher doses, cell killing was essentially complete (Fig. 5C and D). Treatment of a panel of tumor cell lines for 72 hours revealed many lines with minimal sensitivity. In contrast, treatment for 7 days led to cytotoxicity in all of the lines tested, with half-maximal growth inhibition (GI50) values ranging from 30 to 100 nmol/L in most cell lines (Supplementary Table S2). The extended time course of cell death may reflect the time required to accumulate a lethal level of chromosome segregation errors, or it may reflect the delayed cell-cycle kinetics exhibited upon Mps1 inhibition.

In HCT-116 xenografts, MPI-0479605 (30 mg/kg daily or 150 mg/kg every fourth day (Q4D), ip) suppresses tumor development by 49% and 74%. On the Colo-205 xenografts, MPI-0479605 does not exhibit inhibitory action when dosed daily; in contrast, dosing every four days results in 63% tumor growth inhibition (TGI)[1].

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MPI-0479605 displays antitumor activity in colon cancer xenograft models [1]
To ascertain the effect of Mps 1 inhibition on tumor growth, we treated mice bearing subcutaneous HCT-116 or Colo-205 human tumor cell xenografts with MPI-0479605. On the basis of pharmacokinetic analysis (Supplementary Fig. S5) and studies of maximum tolerated dose, mice were treated daily with 30 mg/kg or every 4 days with 150 mg/kg of MPI-0479605. For the HCT-116 xenografts, daily dosing at 30 mg/kg resulted in 49% TGI (P = 0.1), whereas dosing every 4 days at 150 mg/kg resulted in 74% TGI (P = 0.005) relative to vehicle-treated mice (Fig. 6A). For the Colo-205 xenografts, daily dosing did not show antitumor activity, whereas dosing every 4 days resulted in 63% TGI (P = 0.07; Supplementary Fig. S6). These animal studies provide validation for Mps1 as a cancer target, but indicate that there is associated toxicity (body weight loss and death) using these dosing regimens. The specific cause of the toxicity is unclear; however, mice treated with a single dose of MPI-0479605 exhibited significant neutropenia by day 5 (Fig. 6B). This suggests that the effects of MPI-0479605 are not limited to tumor cells.

Cell-cycle analysis [1]
To measure DNA content, fixed, permeabilized cells were stained with 7-aminoactinomycin (7-AAD). To monitor cell division, G1-synchronized cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CSFE) and released from arrest into media containing dimethyl sulfoxide (DMSO) or MPI-0479605. Cells were fixed and analyzed by flow cytometry. To measure bromodeoxyuridine (BrdUrd) incorporation, cells were labeled with BrdUrd and stained by the BrdU Flow Kit according to the manufacturer's protocol.

HCT-116 or Colo-205 cells were transplanted subcutaneously into the flanks of nude (nu+/nu+) mice, and compound treatment was initiated when tumor masses reached an average size of 100 mm3. MPI-0479605 was formulated in 5% dimethylacetamide (DMA)/12% ethanol/40% PEG-300, ispinesib was formulated in 2% cremophor/2% DMA, and 5-fluorouracil was formulated in 2% sodium bicarbonate. Tumor volume was measured with vernier calipers, and tumor growth inhibition (TGI) was calculated as follows: %TGI = 100 − 100 (change in median tumor volume of treated)/(change in median tumor volume control). [1]

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Dissolved in 5% dimethylacetamide (DMA)/12% ethanol/40% PEG-300; daily with 30 mg/kg or every 4 days with 150 mg/kg; i.p. injection

Mice bearing subcutaneous HCT-116 or Colo-205 human tumor cell xenografts.

[1]. Characterization of the cellular and antitumor effects of MPI-0479605, a small-molecule inhibitor of the mitotic kinase Mps1. Mol Cancer Ther. 2011 Dec;10(12):2267-2275.

Mps1 is a dual specificity protein kinase that is essential for the bipolar attachment of chromosomes to the mitotic spindle and for maintaining the spindle assembly checkpoint until all chromosomes are properly attached. Mps1 is expressed at high levels during mitosis and is abundantly expressed in cancer cells. Disruption of Mps1 function induces aneuploidy and cell death. We report the identification of MPI-0479605 , a potent and selective ATP competitive inhibitor of Mps1. Cells treated with MPI-0479605 undergo aberrant mitosis, resulting in aneuploidy and formation of micronuclei. In cells with wild-type p53, this promotes the induction of a postmitotic checkpoint characterized by the ATM- and RAD3-related-dependent activation of the p53-p21 pathway. In both wild-type and p53 mutant cells lines, there is a growth arrest and inhibition of DNA synthesis. Subsequently, cells undergo mitotic catastrophe and/or an apoptotic response. In xenograft models, MPI-0479605 inhibits tumor growth, suggesting that drugs targeting Mps1 may have utility as novel cancer therapeutics.[1]

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Mps1 has been proposed to play a role in Aurora B signaling, but this remains controversial. Early studies showed that Mps1 phosphorylates borealin, a chromosomal passenger protein that regulates Aurora B, suggesting that Mps1 functions upstream of Aurora B during chromosome biorientation. In addition, the Mps1 inhibitor, MPS1-IN-1 was found to inhibit both Aurora B autophosphorylation on threonine 232 and phosphorylation of its substrate histone H3 on serine 10. In contrast, 2 other newly characterized inhibitors of Mps1 failed to affect Aurora B kinase activity. Inhibition of Aurora B overrides the SAC and impairs cytokinesis, resulting in multinucleated cells. Furthermore, cells treated with Aurora B inhibitors fail to effectively engage the postmitotic checkpoint, resulting in endoreduplication and subsequent polyploidy. These phenotypes were not highly observed in response to treatment with MPI-0479605 . Cell-cycle analysis failed to show a significant increase in 8N cells after 72 hours of treatment (Fig. 3A). Also, whereas there were some multinucleated cells, the vast majority were not (Fig. 2), indicating that MPI-0479605 does not impair cytokinesis. Furthermore, in p53-proficient cells, the postmitotic checkpoint seems to be intact following treatment with MPI-0479605, as indicated by an increase in p53 and p21 and the inhibition of DNA synthesis. As a result, the majority of cells do not become polyploid. These results show that inhibition of Mps1 does not affect the ability of Aurora B to promote cytokinesis or engage the postmitotic checkpoint, suggesting Aurora B functions independently of Mps1 in this context.
Administration of MPI-0479605 to mice bearing human tumor xenografts led to partial TGI that was associated with significant toxicity. Related compounds with greater potency or superior pharmacokinetic properties (to be described elsewhere) did not show improved antitumor activity. In viability assays, MPI-0479605 inhibited the growth of normal colon cell lines (data not shown) as well as human fibroblasts immortalized with hTERT (Fig. 6C) and MPI-0479605 induced significant neutropenia in mice (Fig. 6B), indicating a lack of tumor selectivity. Recently, a novel Mps1 inhibitor was shown to have antitumor activity with no associated toxicity in 10- and 13-day xenograft studies using A2780 and A375 tumor cell lines. It will be of great interest to learn whether these responses are durable. If longer term exposure to Mps1 inhibitors as a single agent is intolerable, these compounds may still find utility when used in combination regimens, such as with tubulin-targeting agents. [1]

C22H29N7O

407.511963605881

407.243

C, 64.84; H, 7.17; N, 24.06; O, 3.93

1246529-32-7

46909588

White to gray solid powder

1.3±0.1 g/cm3

1.700

1.69

3

7

5

30

540

0

CC(C=C(N1CCOCC1)C=C2)=C2NC3=NC4=C(N=CN4)C(NC5CCCCC5)=N3

OVJBNYKNHXJGSA-UHFFFAOYSA-N

InChI=1S/C22H29N7O/c1-15-13-17(29-9-11-30-12-10-29)7-8-18(15)26-22-27-20-19(23-14-24-20)21(28-22)25-16-5-3-2-4-6-16/h7-8,13-14,16H,2-6,9-12H2,1H3,(H3,23,24,25,26,27,28)

N6-cyclohexyl-N2-(2-methyl-4-morpholinophenyl)-9H-purine-2,6-diamin

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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