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| 25mg |
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Purity: ≥98%
MK2-IN-1 HCl is a novel, potent, non-ATP competitive and selecitve MAPKAPK2(MK2) inhibitor(IC50=0.11 uM). By screening against a large panel of 150 protein kinases at a concentration of 10 μM, MK2-IN-1 was profiled for kinase selectivity, and only CK1γ3 was significantly inhibited at levels higher than 50%. The human THP1 acute monocytic leukemia cell line secreted pro-inflammatory cytokines less frequently as a result of MK2-IN-1, which led to a dose-dependent reduction in TNFα and IL6 production. The secretion of matrixmetalloprotease (MMP)13 from the SW1353 chondrosarcoma cell line and primary human chondrocyte cultures was also inhibited by MK2-IN-1 in a dose-dependent manner. Notably, given its high level of selectivity, our data imply that MK2-IN-1 may be an excellent pharmacologic tool for specifically exploring and validating MK2 biology.
| Targets |
MK2 (IC50 = 0.11 μM)
Addition of the MK2 inhibitor MK2-IN-1 induced more alkaline phosphatase (AP)-positive colonies than the other factors in a short time , implying that MK2 may be responsible for the phosphorylation of Tfcp2l1.[2] 46C mESCs were treated with MK2-IN-1 for a period of time. The Tfcp2l1 protein level gradually increased without a change in the Tfcp2l1 transcript level within 2 h .[2] |
|---|---|
| ln Vitro |
Addition of the MK2 inhibitor MK2-IN-1 induced more alkaline phosphatase (AP)-positive colonies than the other factors in a short time , implying that MK2 may be responsible for the phosphorylation of Tfcp2l1.[2]
46C mESCs were treated with MK2-IN-1 for a period of time. The Tfcp2l1 protein level gradually increased without a change in the Tfcp2l1 transcript level within 2 h .[2] Compound 25 inhibited MK2 enzyme activity with an IC₅₀ of 0.1 µM (100 nM) in a biochemical assay performed at 100 µM ATP.[4] In a cell-based assay measuring phosphorylation of heat shock protein 27 (pHSP27), an MK2 substrate, compound 25 showed an EC₅₀ of 1.3 µM.[4] Kinase selectivity profiling against a panel of 150 protein kinases at 10 µM concentration showed that compound 25 significantly inhibited (>50%) only CK1/3, demonstrating high selectivity for MK2.[4] In LPS-stimulated human THP-1 monocytic cells, compound 25 dose-dependently inhibited the secretion of pro-inflammatory cytokines TNF-α and IL-6.[4] In IL-1β-stimulated SW1353 chondrosarcoma cells and human primary osteoarthritis-derived chondrocytes, compound 25 dose-dependently inhibited the secretion of matrix metalloproteinase-13 (MMP13).[4] Saturation-transfer-difference (STD) NMR and ¹H/¹⁵N-heteronuclear single quantum coherence (HSQC) NMR studies confirmed that the lead compound (e.g., compound 1, the progenitor of the series) binds to MK2 in a site-specific, non-ATP-competitive manner.[4] Enzymatic kinetic analysis of the initial hit (compound 1) with respect to peptide substrate (Acan peptide) revealed a noncompetitive inhibition model as the most likely mechanism.[4] |
| Enzyme Assay |
MK2 enzyme inhibition assays were performed using recombinant human MK2 kinase domain (Gln41-Thr338). The biochemical potency (IC₅₀) was determined in the presence of 100 µM ATP and a peptide substrate (Acan peptide). Activity was measured by quantifying phosphorylated product, and data were fitted to dose-response curves to derive IC₅₀ values.[4]
To determine the mode of inhibition, enzyme kinetic studies were conducted. MK2 activity was measured in the presence of saturating ATP (100 µM) and varying concentrations of peptide substrate and inhibitor (e.g., compound 1). The resulting data were globally fitted to various kinetic inhibition models (competitive, uncompetitive, noncompetitive, mixed), and the best fit was evaluated using the Akaike information criterion (AICc). The analysis identified noncompetitive inhibition as the most likely model.[4] Saturation-transfer-difference (STD) NMR was used to study binding competition with ATP. Experiments were conducted with MK2 protein, ATP, and MgCl₂. STD signals for ATP were acquired. The test compound was then added, and its binding was assessed by the appearance of new STD signals without affecting the ATP STD signals, indicating non-ATP-competitive binding.[4] ¹H/¹⁵N-heteronuclear single quantum coherence (HSQC) NMR was performed to confirm site-specific binding. Spectra of apo-MK2 were compared with spectra of MK2 in complex with the inhibitor (e.g., compound 1). Chemical shift perturbations specific to the inhibitor confirmed site-specific binding distinct from the ATP-binding site.[4] |
| Cell Assay |
Cell-based potency (EC₅₀) was determined by measuring the inhibition of MK2-dependent phosphorylation of heat shock protein 27 (HSP27) in cells. Cells were treated with the compound, lysed, and the levels of phosphorylated HSP27 (pHSP27) were quantified, typically using an immunoassay.[4]
Cytotoxicity (CC₅₀) was assessed by treating cells with various concentrations of the compound for a defined period (likely 24-72 hours) and measuring cell viability/proliferation using standard assays (e.g., ATP-based or dye-based viability assays).[4] For functional assays in THP-1 cells, cells were stimulated with lipopolysaccharide (LPS) in the presence or absence of increasing concentrations of compound 25. After incubation, culture supernatants were collected, and the secretion of TNF-α and IL-6 was measured by ELISA or similar immunoassays.[4] For functional assays in SW1353 chondrosarcoma cells and primary human chondrocytes, cells were stimulated with interleukin-1β (IL-1β) in the presence or absence of compound 25. After incubation, culture supernatants were collected, and the secretion of MMP13 was measured by ELISA or similar immunoassays.[4] |
| Animal Protocol |
An oral pharmacokinetic (PK) study was conducted in rats. Compound 25 was administered as a single oral dose (10 mg/kg). Blood samples were collected over time, and plasma concentrations were measured to determine pharmacokinetic parameters such as area under the curve (AUC).[4]
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| ADME/Pharmacokinetics |
In a rat oral pharmacokinetic study, a single dose of 10 mg/kg of compound 25 resulted in an area under the curve (AUC) of 2300 nM·h. [4] Compound 25 did not show significant inhibitory effects on a range of major human cytochrome P450 (CYP) enzymes (e.g., CYP1A2, 2C9, 2C19, 2D6, 3A4) at concentrations up to 30 µM. [4]
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| Toxicity/Toxicokinetics |
In cell-based assays, compound 25 showed cytotoxicity (CC₅₀) >20 µM, indicating a significant difference (>10-fold) between its cytotoxicity and its cytotoxicity. [4]
This study did not describe organ-specific toxicity, drug interactions, plasma protein binding, or lethal dose. [4] |
| References | |
| Additional Infomation |
Compound 25 (chemical structure: N-methyl-5-(4-chlorophenyl)-N-(4-(piperazin-1-yl)phenyl)furan-2-carboxamide) is a representative optimized lead compound among a series of non-ATP competitive MK2 inhibitors based on the furan-2-carboxamide skeleton. [4]
This series of compounds was discovered through high-throughput screening using an automated ligand identification system (ALIS), an affinity selection-mass spectrometry platform. [4] The non-ATP competitive binding mode is a key feature of this series of compounds, which have potential advantages in selectivity and efficacy in a high cellular ATP environment compared to ATP competitive kinase inhibitors. [4] The biological principle of targeting MK2 is based on its downstream regulation of pro-inflammatory cytokines (e.g., TNF-α, IL-6) and matrix degrading enzymes (e.g., T cells, T cell receptors... MMP13) downstream of p38 MAPK, suggesting its association with inflammatory diseases such as rheumatoid arthritis and osteoarthritis. [4] Compound 25 is considered an excellent pharmacological tool compound for the specific study of MK2 biology due to its high efficiency, selectivity and good in vitro pharmacokinetic properties. [4] |
| Molecular Formula |
C27H26CL2N4O2
|
|---|---|
| Molecular Weight |
509.43
|
| Exact Mass |
508.143
|
| CAS # |
1314118-94-9
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| Related CAS # |
MK2-IN-1;1314118-92-7
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| PubChem CID |
70681199
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| Appearance |
Light yellow to pink solid
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| LogP |
6.447
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
6
|
| Heavy Atom Count |
35
|
| Complexity |
645
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
ClC1C=CC(=CC=1)C1=CC=C(C(N(CC2C=CC=CN=2)C2C=CC(=CC=2)N2CCNCC2)=O)O1.Cl
|
| InChi Key |
AZDOSXSDARWKGX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H25ClN4O2.ClH/c28-21-6-4-20(5-7-21)25-12-13-26(34-25)27(33)32(19-22-3-1-2-14-30-22)24-10-8-23(9-11-24)31-17-15-29-16-18-31;/h1-14,29H,15-19H2;1H
|
| Chemical Name |
5-(4-chlorophenyl)-N-(4-piperazin-1-ylphenyl)-N-(pyridin-2-ylmethyl)furan-2-carboxamide;hydrochloride
|
| Synonyms |
MK2-IN-1; MK2-IN 1; MK2 IN-1; MK25; MK-25; MK 25; MK2 Inhibitor IV; MK2 Inhibitor-IV
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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: ~100 mg/mL (~196.3 mM)
Ethanol: ~2 mg/mL (~3.9 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (3.28 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 16.7 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: ≥ 1.67 mg/mL (3.28 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 16.7 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: ≥ 1.67 mg/mL (3.28 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 | 1.9630 mL | 9.8149 mL | 19.6298 mL | |
| 5 mM | 0.3926 mL | 1.9630 mL | 3.9260 mL | |
| 10 mM | 0.1963 mL | 0.9815 mL | 1.9630 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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