Monopolar spindle 1 (MPS1) (IC50 = 14 nM)

An in vitro kinase assay designed to measure the inhibition of MPS1 enzymatic activity led to the identification of three top-scoring compounds: Mps-BAY1, a triazolopyridine, and Mps-BAY2a and Mps-BAY2b, two imidazopyrazines (Supplementary Figure 1). Both these classes of compounds contain H-bond donor/acceptor nitrogen atoms, which are common among molecules that bind to the ATP pocket -and associated hinge region- of protein kinases. Mps-BAY1 Mps-BAY2a and Mps-BAY2b inhibited human MPS1 with an IC50 ranging between 1 and 10 nM (Supplementary Table 1). When used at a high concentration (10 μM), Mps-BAY1, Mps-BAY2a and Mps-BAY2b exhibited a restricted inhibitory effect on a panel of 220 human kinases compared with the broad-spectrum kinase inhibitors reversine and anthra[1,9-cd]pyrazole-6(2H)-one (SP600125) (Supplementary Table 2).10, 15 Of note, Mps-BAY1, Mps-BAY2a and Mps-BAY2b failed to inhibit several kinases that are known for their role in mitosis. Mps-BAY1, Mps-BAY2a and Mps-BAY2b inhibited the activation of the SAC with an IC50 of 130 nM, 95 nM and 670 nM, respectively, as monitored in an assay in which the disappearance of histone 3 (H3) phosphorylation (a post-translational modification occurring in prophase/metaphase) was assessed in HeLa cells responding to 300 nM nocodazole (data not shown). Thus, Mps-BAY1, Mps-BAY2a and Mps-BAY2b are efficiently taken up by cultured cells and can reach their molecular target. In line with this notion, all these MPS1 inhibitors reduced the proliferation of the vast majority of primary and transformed human and rat cells tested, and exerted even higher antiproliferative effects on mouse cells (Supplementary Table 3). Mps-BAY2a caused heterogeneous antiproliferative responses within a collection of human colon carcinoma cell lines, with sensitivities (IC50) ranging from 160 nM to >10 μM (Supplementary Table 4). Noteworthy, neither CIN nor microsatellite instability (MIN) was clearly associated with the resistance/sensitivity of human colorectal cancer cell lines to Mps-BAY2a (Supplementary Table 4). Mps-BAY1, Mps-BAY2a and Mps-BAY2b had a major impact on the cell cycle progression and survival of human colorectal carcinoma HCT 116 (Figure 1 and Supplementary Figure 2) and human cervical carcinoma HeLa cells (Supplementary Figures 3 and 4), both of which are particularly sensitive to these compounds (Supplementary Table 3). Thus, Mps-BAY1, Mps-BAY2a and Mps-BAY2b induced a dose- and time-dependent perturbation of the cell cycle, manifesting with an increase in the frequency of cells exhibiting a hyperploid DNA content (>4n) (Figures 1a–c and Supplementary Figures 2 and 3), as well as with a progressive accumulation of dying cells (i.e., cells that had lost their mitochondrial transmembrane potential, Δψm) and cell corpses (with ruptured plasma membranes) (Figure 1d and Supplementary Figure 4). These findings identify Mps-BAY1, Mps-BAY2a and Mps-BAY2b as new MPS1 inhibitors with potent antiproliferative and cytotoxic effects[1].

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Characteristics of cell cycle perturbations as induced by Mps-BAY1 and Mps-BAY2a [1]
We then investigated the precise impact of MPS1 inhibitors on cell cycle progression. Upon the administration of Mps-BAY1, Mps-BAY2a or Mps-BAY2b, the fraction of HCT 116 cells that incorporated the DNA precursor 5-ethynyl-2′-deoxyuridine (EdU, which is only taken up in the S phase of the cell cycle) decreased over time, although such an inhibition was more pronounced with SP600125 (Figure 2a). Of note, a significant fraction of the cells still replicated their DNA even after 48 h of exposure to MPS1 inhibitors. We then performed an in-depth cytofluorometric and (fluorescence) microscopic analysis of the levels of cyclin E and B1, two markers that accumulate in the G1 and G2 phase of the cell cycle, respectively. In response to Mps-BAY1, Mps-BAY2a and Mps-BAY2b (standard dose: 1 μM, 1 μM and 3 μM, respectively), the frequency of cyclin B1+ HCT 116 cells diminished, although these effects were less consistent than those mediated by SP600125 (Figures 2b and c).

Finally, researchers evaluated the therapeutic potential of Mps-BAY2b plus paclitaxel, in vivo, on HeLa-Matu cervical carcinomas growing in immunodeficient mice. Researchers used Mps-BAY2b because it displayed a higher in vivo stability than Mps-BAY1 and Mps-BAY2a (Supplementary Table 5). Twenty-four hours after the administration of paclitaxel, HeLa-Matu cell-derived xenografts displayed higher levels of phosphorylated H3 than untreated tumors, as determined by immunohistochemistry. A short (1 h) exposure of tumor-bearing, paclitaxel-treated mice to Mps-BAY2b resulted in the decrease of H3 phosphorylation (Figure 8a). This finding indicates that Mps-BAY2b is efficiently distributed in vivo, reaches xenotransplanted tumors and penetrates cancer cells to inhibit MPS1. In this xenograft model, the combination of Mps-BAY2b and paclitaxel induced higher levels of apoptosis and a higher incidence of giant mononuclear cells (nuclear diameter >25 μm) than either agent employed as a standalone intervention (Figure 8b). Moreover, the coadministration of paclitaxel and Mps-BAY2b exerted superior antineoplastic effects compared with the administration of vehicle and either paclitaxel or Mps-BAY2b alone (Figure 8c). Altogether, these data underscore the possibility to advantageously combine MPS1 inhibitors with MT-targeting agents [1].

Cytofluorometric studies [1]
For the simultaneous quantification of plasma membrane integrity and Δψm, cells were collected and stained with 1 μg/ml propidium iodide and 40 nM 3,3′-dihexyloxacarbocyanine iodide (DiOC6) for 30 min at 37 °C. For the assessment of cell cycle distribution, cells were collected, stained with 50 μg/ml PI and analyzed by cytofluorometry as previously described. For EdU incorporation assays, cells were incubated with 10 μM EdU for 30 min at 37 °C, fixed, permeabilized and stained with the fluorescent dye azide and PI, according to the manufacturer's instructions. For the simultaneous measurement of DNA content and cyclin B1 levels, fixed cells were costained with 10 μM 4′,6-diamidino-2-phenylindole and mouse antiserum specific for cyclin B1 as previously reported. Cytofluorometric acquisitions were performed on a FACSCalibur cytofluorometer equipped with a 70-μm nozzle or a Gallios cytofluorometer.

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Immunofluorescence and videomicroscopy[1]
Immunofluorescence microscopy was performed according to conventional procedures.58 Images were captured using a Zeiss Axio Observer.Z1 microscope equipped with the ApoTome system. For videomicroscopy, HCT 116 cells stably expressing a H2B-GFP chimera were grown in black/clear 96-well imaging plates under standard conditions and subjected to pulsed observations (every 13 min for up to 72 h) with a BD pathway 855 automated live-cell microscope. Images were analyzed with the open-source software ImageJ. Cell fate profiles are illustrated as previously described.

For the quantification of circulating MPS1 inhibitors, Mps-Bay1, Mps-BAY2a and Mps-BAY2b were administered to female athymic nu/nu mice p.o. in a solubilized form (n=2 mice per compound and time point). Serum samples were prepared 1, 7 and 24 h after administration and precipitated with ice-cold 1 : 5 (v:v) acetonitrile/water. Supernatants were analyzed for Mps-BAY1, Mps-BAY2a and Mps-BAY2b content via liquid chromatography–tandem mass spectroscopy. For tumor xenograft studies, 50-day-old female athymic nu/nu mice with an average body weight of 20–22 g were used after an acclimation period of 14 days. Human HeLa-Matu cervical carcinoma cells derived from exponentially growing cultures were resuspended in 1 : 1 (v:v) FBS-free growth medium/Matrigel (BD Biosciences) to a final concentration of 1.5 × 107 cells/ml. Thereafter, 1.5 × 106 cells were subcutaneously implanted into the inguinal region. Tumor area (monitored with a common caliper and approximated to the product of the longest diameter by its perpendicular) and body weight were determined twice a week. When tumors reached an area of approximately 21 mm2, animals were randomized into the following groups (eight mice per group): control, receiving 3 : 1 (v:v) polyethylene glycol/water (vehicle) once a week p.o.; Mps-BAY2b, receiving 30 mg/kg Mps-BAY2b in 3 : 1 polyethylene glycol/water once a week p.o.; paclitaxel, receiving 10 mg/kg paclitaxel in 1 : 1 : 18 (v:v:v) cremophor/ethanol/PBS once a week i.v.; and Mps-BAY2b plus paclitaxel, receiving 30 mg/kg Mps-BAY2b in 3 : 1 polyethylene glycol/water p.o. plus 10 mg/kg paclitaxel in 1 : 1 : 18 cremophor/ethanol/PBS once a week i.v. When tumor area exceeded 150 mm2, animals were euthanized according to the German Animal Welfare Guidelines. For immunohistochemical studies, when tumors reached a size of 40–50 mm2, animals were randomized into the following groups (three mice per group): control, receiving 3 : 1 polyethylene glycol/water (vehicle) once p.o.; Mps-BAY2b, receiving 30 mg/kg Mps-BAY2b in 3 : 1 polyethylene glycol/water once p.o.; and paclitaxel, receiving 30 mg/kg paclitaxel in 1 : 1 : 18 cremophor/ethanol/PBS once i.p. For hematoxylin and eosin staining, when tumors reached a size of 50–80 mm2, animals were randomized into the following groups (four mice per group): control, receiving 3 : 1 polyethylene glycol/water (vehicle) twice daily for 2 days p.o.; Mps-BAY2b, receiving 30 mg/kg Mps-BAY2b in 3 : 1 polyethylene glycol/water twice daily for 2 days p.o.; paclitaxel, receiving 8 mg/kg paclitaxel in 1 : 1 : 18 cremophor/ethanol/PBS once i.v.; and Mps-BAY2b plus paclitaxel, receiving 30 mg/kg Mps-BAY2b in 3 : 1 polyethylene glycol/water twice daily for 2 days p.o. plus 10 mg/kg paclitaxel in 1 : 1 : 18 cremophor/ethanol/PBS i.v. once. Seventy-two hours after the first treatment, tumors were recovered, fixed with 4% (w/v) PFA for 4 h and embedded into paraffin. Ten-micrometer-thick tissue sections were then stained with hematoxylin and eosin according to standard protocols and analyzed as previously described.[1]

[1]. Jemaa, et al. "Characterization of novel MPS1 inhibitors with preclinical anticancer activity." Cell Death & Differentiation 20.11 (2013): 1532-1545.

Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A synergistic interaction between paclitaxel and MPS1 inhibitors could also be demonstrated in vivo, as the combination of these agents efficiently reduced the growth of tumor xenografts and exerted superior antineoplastic effects compared with either compound employed alone. Altogether, these results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals. [1]

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Here, we reported the identification and functional characterization of three novel and potent MPS1 inhibitors, the triazolopyridine Mps-BAY1 and the imidazopyrazines Mps-BAY2a and Mps-BAY2b. All these agents were capable of abrogating the functionality of the SAC, as demonstrated by the incapacity of cells exposed to MPS1 inhibitors to sustain a mitotic arrest upon exposure to MT poisons. Even in the absence of SAC activators, both classes of MPS1 inhibitors markedly increased the rate of chromosome misalignments resulting from erroneous MT–KT attachments and promoted a premature anaphase entry (i.e., before the formation of a correct equatorial metaphase plate). These results are in line with previous findings obtained with other MPS1-specific inhibitors, upon MPS1 depletion1 or following the conditional knockout of TTK,11 confirming the central implication of this mitotic kinase in SAC function and chromosome congression. [1]

C20H23N5O

349.429523706436

349.19

C, 68.74; H, 6.63; N, 20.04; O, 4.58

1263420-68-3

1263420-68-3;

67974488

Typically exists as solid at room temperature

3.8

2

4

6

26

487

0

O=C(C1C=CC(=CC=1)C1=CN=C2C(=NC=CN12)NCC(C)C)NC1CC1

QTOFVYOMOKYCEQ-UHFFFAOYSA-N

InChI=1S/C20H23N5O/c1-13(2)11-22-18-19-23-12-17(25(19)10-9-21-18)14-3-5-15(6-4-14)20(26)24-16-7-8-16/h3-6,9-10,12-13,16H,7-8,11H2,1-2H3,(H,21,22)(H,24,26)

N-cyclopropyl-4-[8-(2-methylpropylamino)imidazo[1,2-a]pyrazin-3-yl]benzamide

Mps-BAY2b; 1263420-68-3; Mps-BAY 2b; CHEMBL3410060; N-cyclopropyl-4-[8-(2-methylpropylamino)imidazo[1,2-a]pyrazin-3-yl]benzamide; N-cyclopropyl-4-{8-[(2-methylpropyl)amino]imidazo[1,2-a]pyrazin-3-yl}benzamide; N-Cyclopropyl-4-(8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl)benzamide; SCHEMBL9938616;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)