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Purity: ≥98%
MD2-IN-1, a chalcone derivative, is a novel potent and specific inhibitor of Myeloid differentiation protein 2 (MD2) with potential anti-inflammatory activity. It inhibits MD2 with a KD of 189 μM for the recombinant human MD2 (rhMD2). Acute lung injury (ALI) is a life-threatening acute inflammatory disease with limited options available for therapy. Myeloid differentiation protein 2, a co-receptor of TLR4, is absolutely required for TLR4 sense LPS, and represents an attractive target for treating severe inflammatory diseases.MD2-IN-1 contains the moiety of (E)-4-phenylbut-3-en-2-one, which is considered to be the core structure of current MD2 inhibitors. The anti-inflammatory activities of MD2-IN-1 was evaluated in MPMs. MD2-IN-1 binds to the hydrophobic pocket of MD2 via hydrogen bonds with Arg(90) and Tyr(102) residues. MD2-IN-1 also blocked the LPS-induced activation of TLR4/MD2 -downstream pro-inflammatory MAPKs/NF-κB signaling pathways. In a rat model with ALI induced by intracheal LPS instillation, administration with MD2-IN-1 exhibited significant protective effect against ALI, accompanied by the inhibition of TLR4/MD2 complex formation in lung tissues. Taken together, the results of this study suggest the specific MD2 inhibitor from chalcone derivatives is a potential candidate for treating acute inflammatory diseases.
| Targets |
TLR4's coreceptor is myeloid differentiation protein 2 (MD2). Of all the derivatives, compound 20 (MD2-IN-1) had the most potent inhibitory effect on the production of TNF-α and IL-6 produced by LPS. While pretreatment with MD2-IN-1 suppressed the increase in TLR4/MD2 complexes to vehicle levels, LPS alone significantly enhanced the quantity of TLR4/MD2 complexes relative to vehicle. SPR study revealed that xanthohumol bound to MD2 with a KD value of 460 μM, whereas MD2-IN-1 bound to rhMD2 protein in a dose-dependent manner with a KD value of 189 μM. The binding of FITC-LPS to MD2 in the cell surface membrane was dose-dependently decreased by pretreatment with several dosages of MD2-IN-1, with an average fluorescence intensity inhibition of 65% at 10 μM. Additionally, pretreatment with MD2-IN-1 dose-dependently prevents MPM-induced MAPK phosphorylation caused by LPS [1].
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| ln Vitro |
TLR4's coreceptor is myeloid differentiation protein 2 (MD2). Of all the derivatives, compound 20 (MD2-IN-1) had the most potent inhibitory effect on the production of TNF-α and IL-6 produced by LPS. While pretreatment with MD2-IN-1 suppressed the increase in TLR4/MD2 complexes to vehicle levels, LPS alone significantly enhanced the quantity of TLR4/MD2 complexes relative to vehicle. SPR study revealed that xanthohumol bound to MD2 with a KD value of 460 μM, whereas MD2-IN-1 bound to rhMD2 protein in a dose-dependent manner with a KD value of 189 μM. The binding of FITC-LPS to MD2 in the cell surface membrane was dose-dependently decreased by pretreatment with several dosages of MD2-IN-1, with an average fluorescence intensity inhibition of 65% at 10 μM. Additionally, pretreatment with MD2-IN-1 dose-dependently prevents MPM-induced MAPK phosphorylation caused by LPS [1].
Compound 20 dose-dependently inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) production in mouse peritoneal macrophages (MPMs), with IC₅₀ values of 4.27 μM and 2.09 μM, respectively. Its inhibitory activity was stronger than the positive control xanthohumol at equivalent concentrations. [1] Compound 20 (at 2.5, 5, 10 μM) dose-dependently inhibited LPS-induced phosphorylation of ERK, JNK, and p38 MAPKs, and prevented LPS-induced IκB-α degradation in MPMs. [1] Compound 20 (10 μM) effectively inhibited LPS-induced nuclear translocation of NF-κB p65 and its DNA-binding activity in MPMs. [1] Compound 20 (10 μM) significantly inhibited LPS-induced upregulation of TNF-α, IL-6, IL-1β, and COX-2 mRNA transcripts in MPMs. [1] Compound 20 (at 0.1, 1, 10 μM) dose-dependently reduced the binding of FITC-labeled LPS to MD2 on the surface of RAW264.7 macrophages, with ~65% inhibition at 10 μM. [1] Compound 20 (10 μM) pretreatment significantly reduced the LPS-induced formation of the TLR4/MD2 complex in MPMs, as shown by immunoprecipitation. [1] Compound 20 showed no obvious cytotoxicity in macrophages at the tested concentrations (up to 10 μM). [1] |
| ln Vivo |
The LPS-induced rise in protein concentration in BALF was considerably attenuated by administering MD2-IN-1. A treatment of MD2-IN-1 reduced LPS-induced pulmonary edema, and the lung wet/dry weight ratio in the LPS treatment group was considerably greater than in the control group. Furthermore, areas of inflammatory infiltrates, bleeding, interstitial edema, thickening of the alveolar wall, and loss of lung tissue are all visible histological changes brought on by LPS. In the group receiving MD2-IN-1 treatment, these histological alterations improved [1].
In a rat model of acute lung injury (ALI) induced by intratracheal instillation of LPS (5 mg/kg), intragastric administration of compound 20 (20 mg/kg/day for one week prior to LPS challenge) significantly attenuated lung injury. [1] Treatment with compound 20 reduced the LPS-induced increase in total protein concentration in bronchoalveolar lavage fluid (BALF) and decreased the lung wet/dry weight ratio, indicating alleviation of pulmonary edema. [1] Compound 20 treatment ameliorated LPS-induced histopathological changes in lung tissue, including inflammatory infiltration, hemorrhage, interstitial edema, and alveolar wall thickening. [1] Compound 20 administration reduced the LPS-induced influx of total cells and neutrophils into BALF, decreased macrophage infiltration (CD68 staining), and lowered myeloperoxidase (MPO) activity in lung tissue. [1] Compound 20 treatment significantly inhibited the LPS-induced upregulation of TNF-α, IL-6, and IL-1β mRNA expression in lung tissue. [1] Immunoprecipitation assay of lung tissues confirmed that compound 20 treatment inhibited the LPS-induced formation of the TLR4/MD2 complex in vivo. [1] |
| Enzyme Assay |
The direct interaction between compound 20 and recombinant human MD2 (rhMD2) protein was assessed by surface plasmon resonance (SPR). rhMD2 was immobilized on an HTE sensor chip activated with nickel sulfate. Compound 20 samples at concentrations of 6.25, 12.5, 25, 50, and 100 μM were prepared in running buffer and injected simultaneously at a flow rate of 30 μL/min for a 120-second association phase, followed by a 120-second dissociation phase. Binding kinetics were analyzed using a 1:1 Langmuir binding model to calculate the KD value. [1]
Fluorescence spectroscopy was used to study competitive binding to the MD2 hydrophobic pocket. rhMD2 protein and a fluorescent probe (bis-ANS) were mixed in buffer. After fluorescence stabilization, compound 20 (5-30 μM) was added, and the decrease in relative fluorescence units was measured, indicating displacement of the probe. [1] A competitive ELISA was performed to assess the effect of compound 20 on LPS binding to MD2. An anti-rhMD2 antibody was coated onto a plate, followed by incubation with rhMD2 protein. Biotin-labeled LPS was then added with or without compound 20 (0.1 and 1.0 μM). Binding was detected by measuring absorbance. [1] Similar competitive ELISAs were performed using mutant MD2 proteins (R90A, Y102A, R90A/Y102A) to confirm the critical binding residues. [1] |
| Cell Assay |
For cytokine inhibition assays, mouse peritoneal macrophages (MPMs) were pretreated with various concentrations of compound 20 for 2 hours, then stimulated with LPS (0.5 μg/mL) for 22 hours. The levels of TNF-α and IL-6 in the culture medium were quantified by ELISA. [1]
For Western blot analysis of signaling proteins, MPMs were pretreated with compound 20 for 30 minutes, then stimulated with LPS (0.5 μg/mL) for 20 minutes. Cell lysates were prepared, proteins were separated by gel electrophoresis, transferred to membranes, and probed with specific antibodies against phospho- and total forms of MAPKs (ERK, JNK, p38) and IκB-α. [1] For NF-κB nuclear translocation assay, MPMs were pretreated with compound 20 (10 μM) for 30 minutes, stimulated with LPS for 1 hour, then fixed and stained with a Cy3-labeled antibody against NF-κB p65 and DAPI for nuclei. Fluorescence microscopy was used for visualization. [1] Electrophoretic mobility shift assay (EMSA) was used to assess NF-κB DNA-binding activity. Nuclear extracts from MPMs pretreated with compound 20 and stimulated with LPS were incubated with a biotin-labeled NF-κB probe. DNA/protein complexes were separated on a non-denaturing gel, transferred to a membrane, and detected. [1] For quantitative PCR (qPCR), MPMs were pretreated with compound 20 (10 μM) for 30 minutes, stimulated with LPS for 6 hours, and total RNA was extracted. cDNA was synthesized, and mRNA levels of TNF-α, IL-6, IL-1β, and COX-2 were measured using SYBR Green and normalized to β-actin. [1] For flow cytometry analysis of LPS binding, RAW264.7 macrophages were starved, then incubated with FITC-labeled LPS in the presence or absence of compound 20 (0.1-10 μM) for 30 minutes. Cells were fixed and analyzed by flow cytometry to measure fluorescence intensity. [1] For immunoprecipitation of the TLR4/MD2 complex, MPMs were treated with LPS in the presence or absence of compound 20 (10 μM) for 5 minutes. Cell lysates were incubated with an anti-MD2 antibody overnight, followed by protein A+G agarose. Precipitated complexes were analyzed by Western blot for TLR4. [1] |
| Animal Protocol |
An acute lung injury (ALI) model was induced in male Sprague-Dawley rats. Rats were randomly divided into control, LPS, and compound 20 + LPS groups. [1]
The compound 20 + LPS group received intragastric administration of compound 20 at a dose of 20 mg/kg body weight per day, dissolved in vehicle (0.9% saline), continuously for one week prior to LPS challenge. [1] Under anesthesia, ALI was induced by intratracheal instillation of LPS (E. coli 055:B5, 5 mg/kg, dissolved in 50 μL of 0.9% saline). Control rats received intratracheal saline. [1] Six hours after LPS induction, rats were euthanized. Bronchoalveolar lavage fluid (BALF) was collected, and lung tissues were harvested for analysis of wet/dry weight ratio, histopathology, immunohistochemistry, mRNA expression, and protein complex formation. [1] |
| References | |
| Additional Infomation |
Compound 20 is a chalcone derivative containing (E)-4-phenylbut-3-en-2-one structural units, which are considered the core structure of many MD2 inhibitors. Its chemical structure comprises a 3,4,5-trimethoxyphenyl (ring A) and a 4-hydroxyphenyl (ring B). [1] The synthesis of Compound 20 and its analogues involves a Claisen-Schmidt condensation reaction between substituted acetophenone and an aromatic aldehyde, followed by purification and characterization by nuclear magnetic resonance and mass spectrometry. [1] Molecular docking simulations showed that Compound 20 binds to the hydrophobic pocket of MD2 and forms hydrogen bonds with Arg⁹⁰ and Tyr¹⁰² residues. Mutation studies confirmed this result, showing that mutant MD2 (R90A, Y102A) lost its binding ability and inhibitory activity. [1] The main mechanism of action is that compound 20, as a specific MD2 inhibitor, competitively blocks the binding of LPS to MD2, thereby preventing the formation of the TLR4/MD2/LPS complex, the activation of subsequent downstream signaling pathways (MAPKs/NF-κB), and the production of pro-inflammatory cytokines. [1] This study shows that compound 20 has great potential as a candidate drug for the treatment of acute inflammatory diseases such as acute lung injury (ALI) and sepsis. [1]
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| Molecular Formula |
C20H22O6
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| Molecular Weight |
358.39
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| Exact Mass |
358.141
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| CAS # |
111797-22-9
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| Related CAS # |
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| PubChem CID |
5724738
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.5
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
26
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| Complexity |
451
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1C(/C=C/C(=O)C2=CC(OC)=C(C=C2)OC)=CC(=C(OC)C=1OC)OC
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| InChi Key |
ZKYRYELHPFTZTI-SOFGYWHQSA-N
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| InChi Code |
InChI=1S/C20H22O6/c1-22-16-9-7-14(12-17(16)23-2)15(21)8-6-13-10-18(24-3)20(26-5)19(11-13)25-4/h6-12H,1-5H3/b8-6+
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| Chemical Name |
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3.25 mg/mL (9.07 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 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: 3.25 mg/mL (9.07 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 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: ≥ 3.25 mg/mL (9.07 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 | 2.7903 mL | 13.9513 mL | 27.9026 mL | |
| 5 mM | 0.5581 mL | 2.7903 mL | 5.5805 mL | |
| 10 mM | 0.2790 mL | 1.3951 mL | 2.7903 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.
Inhibition of TNF-α and IL-6 production as indicated by chalcone derivatives.Sci Rep.2016 Apr 27;6:25130. |
Compound 20 (MD2-IN-1) is a specific inhibitor of MD2. |
Compound20(MD2-IN-1)binds with MD2 via interacting with Arg90and Tyr102. |
Effects of compound20(MD2-IN-1)on the LPS-induced activation of MAPK and NF-κB and expression of inflammatory genes.Sci Rep.2016 Apr 27;6:25130. |
Proposed model of signaling pathway involved in compound 20(MD2-IN-1) preventing LPS-induced TLR4 signaling pathway activation and acute lung injury.Sci Rep.2016 Apr 27;6:25130. |
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