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Mps1-IN-2 is a novel, ATP-competitive and potent Mps1 (monopolar spindle)/Plk1 (Polo Like Kinase 1) inhibitor with potential anticancer activity. Its IC50 and Kd values for MPS1/Plk1 are 145 nM and 12 nM, respectively, for Mps1 and 61 nM for Plk1, respectively.
| Targets |
Mps1 (Kd = 12 nM); GAK (Kd = 140 nM); CLK4 (IC50 = 15 nM)
Mps1 (IC50 = 145 nM) Plk1 (significant activity, consistent with being a ring-expanded version of the potent Plk1 inhibitor BI-2536)[1] |
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| ln Vitro |
Mps1-IN-2 is an ATP-competitive, potent, and selective Mps1 kinase inhibitor with IC50 and Kd values of 145 nM and 12 nM, respectively. Mps1-IN-2 exhibits little to no inhibition on the remaining 352 member kinases, but it demonstrates strong affinity for PLK1 and GAK, with Kds of 61 and 140 nM, respectively. Mps1-IN-2 has the ability to cause U2OS cells to bypass a checkpoint-mediated mitotic arrest[1].
Mps1-IN-2 inhibited Mps1 kinase activity with an IC50 of 145 nM when screened at 1 μM ATP. It demonstrated high selectivity against a panel of 352 kinases, with the major off-target activity being against Plk1. The compound is overall more selective than its analog Mps1-IN-1, but its significant Plk1 activity limits its use as a selective Mps1 inhibitor. It provides a unique tool to investigate the combined inhibition of Plk1 and Mps1. [1] Treatment of HCT116 colorectal cancer cells with 5-10 μM Mps1-IN-2 reduced proliferative capacity to 33% of DMSO control after 96 hours, caused severe loss of cell viability and clonal survival in colony outgrowth assays, and induced gross aneuploidy and accumulation of cells with <2c DNA content. [1] Loss of viability induced by compound treatment was associated with induction of apoptosis as evidenced by PARP cleavage beginning after 48 hours. [1] Mps1-IN-2 treatment (like Mps1-IN-1) led to a dose-dependent decrease in cyclin B protein levels in U2OS cells arrested in mitosis, indicating checkpoint override. This effect could be reversed by addition of the proteasome inhibitor MG132. [1] In U2OS cells expressing fluorescently-tagged histone H2B, Mps1-IN-2 treatment caused premature mitotic exit, chromosome mis-segregation, and aneuploidy. [1] Expression of an inhibitor-resistant Mps1 M602Q mutant restored pHistone H3 positivity to wild-type levels in the presence of up to 10 μM Mps1-IN-2, confirming on-target activity. [1] |
| Enzyme Assay |
The in vitro kinase activity of Mps1-IN-2 against Mps1 was assessed using a Lanthascreen technology-based assay. Reactions contained approximately 40 nM Mps1 kinase, 1 μM ATP (apparent Km for ATP < 1 μM), and 200 nM AF-647 E4Y substrate. The half-maximal inhibitory concentration (IC50) was determined to be 145 nM. [1]
A radioenzymatic immunoprecipitation kinase assay was performed to assess inhibitor sensitivity of wild-type versus mutant Mps1. Wild-type or M602Q mutant LAP-Mps1 was immunoprecipitated, and kinase activity in the presence of inhibitors was measured. The M602Q mutant was 19-fold less sensitive to Mps1-IN-2 compared to wild-type Mps1. [1] Kinase selectivity profiling was performed using an in vitro ATP-site competition binding assay. Approximately 400 compounds were profiled at 10 μM against a panel of 352 diverse kinases to generate a selectivity-annotated library (SAL). Mps1-IN-2 showed greater than 1000-fold selectivity for Mps1 relative to most of the kinase panel, with major exceptions being Gak and Plk1. [1] |
| Cell Assay |
To assess SAC abrogation, U2OS cells were arrested in mitosis using a combination of thymidine and nocodazole. Cells were then treated with nocodazole alone or co-administered with Mps1-IN-2 (with or without the proteasome inhibitor MG132) for 4 hours. Cells were harvested and analyzed by immunoblotting for cyclin B levels. [1]
For mitotic timing and chromosome segregation analysis, U2OS cells expressing histone H2B-GFP were treated with Mps1-IN-2 or DMSO. Cells were imaged using time-lapse fluorescence microscopy to record the time from nuclear envelope breakdown (NEBD) to anaphase initiation and to observe chromosome alignment and segregation. [1] To generate inhibitor-resistant cell lines, endogenous Mps1 in U2OS cells was replaced by simultaneous expression of an Mps1-specific shRNA plasmid and an RNAi-insensitive, LAP-tagged Mps1 M602Q allele, creating a stable cell line (UTRM10 Mps1 M602Q). These cells were used to confirm the on-target effects of Mps1-IN-2 by assessing pHistone H3 levels and aneuploidy after inhibitor treatment. [1] For proliferation assays, HCT116 cells were treated with various concentrations of Mps1-IN-2 and analyzed for cell number over a 4-day period using a fluorescent nucleic acid stain (Syto60). [1] For colony outgrowth assays, HCT116 cells were plated at low density (200 cells per dish), treated with Mps1-IN-2 or DMSO, and allowed to grow for 10 days before being fixed and stained with crystal violet to visualize and quantify colonies. [1] For cell cycle and aneuploidy analysis, cells treated with Mps1-IN-2 or DMSO for indicated times were harvested, fixed, stained with propidium iodide, and analyzed by flow cytometry to determine DNA content. [1] To assess apoptosis, cells treated with Mps1-IN-2 were harvested at various time points, and cell lysates were subjected to immunoblotting to detect cleavage of PARP. [1] |
| Animal Protocol |
The ATP site-directed kinase inhibitor Kinase Tracer 236 is fluorescently labeled, and its displacement from the kinase active site is monitored in the kinase binding assay to evaluate compound binding to TTK. In every 15 μL assay, there are five different concentrations of the test compound (Mps1-IN-2), 30 nM Kinase Tracer 236; 2 nM Eu-anti-GST Antibody; and 1% DMSO (which is the leftover from the compound dilution) in Kinase Buffer A, which also contains 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, and 0.01% Brij-35. The procedure for starting binding assays involves adding 5 μL of the test compound (from the 2-fold dilution series) to 5 μL of a kinase/antibody mixture, and then adding 5 μL of the antibody. Standard Eu-based TR-FRET settings are used to read assay plates, with excitation occurring at 340 nm and emission being observed at 615 nm (donor) and 665 nm (acceptor). A 100 µs post-excitation delay is followed by a 200 µs window for measuring emission intensities[1].
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| References | |
| Additional Infomation |
Mps1-IN-2 belongs to the piperidine class of compounds. Mps1-IN-2 is a dihydropyrimidine diazoxide ketone backbone compound and is an ATP competitive kinase inhibitor. [1] Its structural analogue, methoxy-Mps1-IN-2, is co-crystallized with the Mps1 kinase domain. Structural analysis showed that the inhibitor binds to the ATP pocket, forms a hydrogen bond with the hinge region residue G605, and forms hydrophobic interactions with the gating residue and the P ring residue. [1] The main research use of Mps1-IN-2 is as a chemical biology tool to study the function of Mps1 and spindle assembly checkpoints in cells. Its significant Plk1 activity makes it particularly suitable for studying the combined inhibitory effects of Mps1 and Plk1. [1]
The chemoinhibition of Mps1 by Mps1-IN-2 reproduced the phenotypes observed in RNAi, including defects in Mad1/Mad2 recruitment to the centromere, reduced Aurora B kinase activity, premature exit from mitosis, and induction of aneuploidy. [1] |
| Molecular Formula |
C26H36N6O3
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| Molecular Weight |
480.6024
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| Exact Mass |
480.284
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| Elemental Analysis |
C, 64.98; H, 7.55; N, 17.49; O, 9.99
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| CAS # |
1228817-38-6
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| Related CAS # |
1228817-38-6
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| PubChem CID |
44968267
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| Appearance |
Light brown to brown solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
772.7±70.0 °C at 760 mmHg
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| Flash Point |
421.1±35.7 °C
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| Vapour Pressure |
0.0±2.8 mmHg at 25°C
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| Index of Refraction |
1.633
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| LogP |
2.05
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
35
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| Complexity |
699
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O([H])C1([H])C([H])([H])C([H])([H])N(C2C([H])=C([H])C(=C(C=2[H])OC([H])([H])C([H])([H])[H])N([H])C2=NC([H])=C3C(=N2)N(C([H])([H])C([H])([H])C(N3C([H])([H])[H])=O)C2([H])C([H])([H])C([H])([H])C([H])([H])C2([H])[H])C([H])([H])C1([H])[H]
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| InChi Key |
WELBJLUKWAJOQV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C26H36N6O3/c1-3-35-23-16-19(31-13-10-20(33)11-14-31)8-9-21(23)28-26-27-17-22-25(29-26)32(18-6-4-5-7-18)15-12-24(34)30(22)2/h8-9,16-18,20,33H,3-7,10-15H2,1-2H3,(H,27,28,29)
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| Chemical Name |
9-cyclopentyl-2-[2-ethoxy-4-(4-hydroxypiperidin-1-yl)anilino]-5-methyl-7,8-dihydropyrimido[4,5-b][1,4]diazepin-6-one
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| Synonyms |
Mps1-IN-2
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~13.3 mg/mL (~27.7 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.33 mg/mL (2.77 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 13.3 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. Solubility in Formulation 2: ≥ 1.33 mg/mL (2.77 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 13.3 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. View More
Solubility in Formulation 3: ≥ 1.33 mg/mL (2.77 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0807 mL | 10.4037 mL | 20.8073 mL | |
| 5 mM | 0.4161 mL | 2.0807 mL | 4.1615 mL | |
| 10 mM | 0.2081 mL | 1.0404 mL | 2.0807 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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