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MD2-TLR4-IN-1 (compound 22m) is an inhibitor of myeloid differentiation protein 2/toll-like receptor 4 (MD2-TLR4) complex, inhibiting lipopolysaccharides (LPS)-induced expression of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in macrophages with IC50 of 0.89 μM and 0.53 μM, respectively.
| Targets |
MD2-TLR4-IN-1 targets myeloid differentiation protein 2 (MD2)-Toll-like receptor 4 (TLR4) complex (IC50 = 0.15 μM for inhibiting LPS-MD2 binding; EC50 = 0.32 μM for suppressing LPS-induced NF-κB activation in RAW264.7 cells) [1]
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|---|---|
| ln Vitro |
- Inhibition of LPS-MD2 binding: MD2-TLR4-IN-1 dose-dependently blocked the interaction between LPS and recombinant human MD2, with IC50 = 0.15 μM, as determined by fluorescence polarization assay. It bound directly to MD2 (KD = 0.09 μM) without interacting with TLR4 alone [1]
- Suppression of TLR4 downstream signaling: The compound inhibited LPS (100 ng/mL)-induced activation of NF-κB and MAPK pathways in RAW264.7 macrophages. At 1 μM, phosphorylation of IκBα (p-IκBα) and p65 (p-p65) was reduced by 78% and 82% respectively, and phosphorylation of ERK1/2, JNK, and p38 was inhibited by 70%, 68%, and 65% [1] - Reduction of pro-inflammatory cytokines: MD2-TLR4-IN-1 (0.1-2 μM) dose-dependently decreased LPS-induced production of TNF-α, IL-6, and IL-1β in RAW264.7 cells and primary mouse bone marrow-derived macrophages (BMDMs). At 1 μM, TNF-α, IL-6, and IL-1β levels were reduced by 75%, 72%, and 68% (RAW264.7) and 78%, 70%, and 65% (BMDMs) [1] - High target selectivity: MD2-TLR4-IN-1 (10 μM) showed no significant inhibition against other TLR family members (TLR2, TLR3, TLR5, TLR7) or cytokine receptors (TNF-R1, IL-6R). It did not affect LPS-induced TLR2 signaling in HEK293T-TLR2 cells [1] - Minimal cytotoxicity: At concentrations up to 20 μM, MD2-TLR4-IN-1 exhibited no obvious cytotoxicity to RAW264.7 cells, BMDMs, or normal human bronchial epithelial cells (BEAS-2B), with cell viability > 90% after 24 hours of treatment [1] |
| ln Vivo |
- Amelioration of LPS-induced acute lung injury (ALI) in mice: C57BL/6 mice were intraperitoneally administered MD2-TLR4-IN-1 (10 mg/kg, 20 mg/kg) 1 hour before LPS (5 mg/kg, intratracheal instillation) challenge. At 24 hours post-LPS, the lung wet/dry weight ratio (a marker of pulmonary edema) was reduced by 42% (10 mg/kg) and 60% (20 mg/kg) compared to vehicle control [1]
- Reduction of lung inflammation: Treated mice (20 mg/kg) showed a 65% decrease in total inflammatory cell infiltration (neutrophils and macrophages) in bronchoalveolar lavage fluid (BALF). BALF levels of TNF-α, IL-6, and IL-1β were reduced by 68%, 62%, and 58% respectively [1] - Improvement of lung histopathology: Lung tissue sections from treated mice (20 mg/kg) showed reduced alveolar wall thickening, interstitial edema, and inflammatory cell infiltration. The lung injury score was decreased from 8.2 (control) to 3.5 [1] - Inhibition of TLR4 signaling in vivo: Lung tissues from treated mice (20 mg/kg) showed reduced p-p65 and p-IκBα levels (by 70% and 65% respectively) and downregulated mRNA expression of TNF-α, IL-6, and IL-1β (by 62%, 58%, and 55% respectively) [1] |
| Enzyme Assay |
- MD2 binding assay: Recombinant human MD2 protein was mixed with fluorescently labeled LPS analog and MD2-TLR4-IN-1 at gradient concentrations (0.01-1 μM) in binding buffer (pH 7.4). Fluorescence polarization was measured to assess the inhibition of LPS-MD2 interaction, and IC50 was calculated. Surface plasmon resonance (SPR) assay was used to determine direct binding affinity (KD = 0.09 μM) between MD2-TLR4-IN-1 and MD2 [1]
- TLR4 signaling pathway inhibition assay: HEK293T cells co-transfected with TLR4, MD2, CD14, and NF-κB-luciferase reporter plasmids were pre-treated with MD2-TLR4-IN-1 (0.05-2 μM) for 1 hour, then stimulated with LPS (100 ng/mL) for 18 hours. Luciferase activity was measured to evaluate NF-κB activation inhibition, and EC50 was calculated [1] |
| Cell Assay |
- Macrophage cytokine production assay: RAW264.7 cells or primary BMDMs were seeded into 96-well plates (5×10⁴ cells/well) and pre-treated with MD2-TLR4-IN-1 (0.1-2 μM) for 1 hour, then stimulated with LPS (100 ng/mL) for 24 hours. TNF-α, IL-6, and IL-1β levels in cell supernatants were detected by ELISA [1]
- Western blot for TLR4 downstream signaling: RAW264.7 cells were seeded into 6-well plates (5×10⁵ cells/well) and pre-treated with MD2-TLR4-IN-1 (0.5-2 μM) for 1 hour, then stimulated with LPS (100 ng/mL) for 30 minutes. Cells were lysed, and p-p65, p-IκBα, p-ERK1/2, p-JNK, p-p38, and GAPDH proteins were detected by western blot. Band intensities were quantified by densitometry [1] - TLR selectivity assay: HEK293T cells transfected with TLR2, TLR3, TLR5, or TLR7 reporter plasmids were pre-treated with MD2-TLR4-IN-1 (10 μM) for 1 hour, then stimulated with corresponding ligands (Pam3CSK4 for TLR2, poly(I:C) for TLR3, flagellin for TLR5, imiquimod for TLR7). Reporter gene activity was measured to evaluate selectivity [1] - Cell viability assay: RAW264.7 cells, BMDMs, and BEAS-2B cells were seeded into 96-well plates (5×10³ cells/well) and treated with MD2-TLR4-IN-1 (0.1-20 μM) for 24 hours. Cell viability was measured by tetrazolium salt-based assay [1] |
| Animal Protocol |
- LPS-induced ALI mouse model: 6-8 week-old male C57BL/6 mice were randomly divided into sham group, LPS control group, and MD2-TLR4-IN-1 treatment groups (10 mg/kg, 20 mg/kg, n=8 per group). The compound was dissolved in a mixture of DMSO, PEG400, and sterile water (volume ratio 1:3:6) to prepare injectable solution [1]
- Drug administration and LPS challenge: MD2-TLR4-IN-1 was administered intraperitoneally at the indicated doses 1 hour before LPS (5 mg/kg) was intratracheally instilled to induce ALI. The sham group received normal saline, and the LPS control group received equal volume of vehicle [1] - Sample collection and analysis: At 24 hours post-LPS challenge, mice were sacrificed. BALF was collected to count inflammatory cells and detect cytokine levels. Lungs were excised to measure wet/dry weight ratio, prepare tissue sections for histopathological scoring, and extract proteins for western blot analysis [1] |
| References | |
| Additional Infomation |
Chemical classification: MD2-TLR4-IN-1 is a small molecule MD2-TLR4 antagonist belonging to the 3-(indole-5-yl)-indazole derivative class [1] - Mechanism of action: This compound binds directly to the LPS binding pocket of MD2, preventing LPS from interacting with MD2 and forming an active MD2-TLR4 complex. This blocks the downstream TLR4-mediated NF-κB and MAPK signaling pathways, thereby inhibiting the production of pro-inflammatory cytokines and alleviating the inflammatory response [1] - Target background: MD2 is an extracellular adaptor protein that can bind to LPS and form a complex with TLR4 to initiate an innate immune response. Abnormal activation of the MD2-TLR4 pathway by LPS is an important factor in the pathogenesis of acute lung injury, sepsis and other inflammatory diseases [1]. - Therapeutic potential: MD2-TLR4-IN-1 is a potent, selective and highly active MD2-TLR4 antagonist that has shown good efficacy in improving LPS-induced acute lung injury. It is expected to be used to treat inflammatory diseases mediated by MD2-TLR4 activation, such as acute respiratory distress syndrome (ARDS) and sepsis[1].
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| Molecular Formula |
C22H14CL2N4O
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|---|---|
| Molecular Weight |
421.2788
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| Exact Mass |
420.054
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| CAS # |
2249801-12-3
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| PubChem CID |
138454798
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| Appearance |
White to off-white solid powder
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| LogP |
5.4
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
29
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| Complexity |
597
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C([H])=C([H])C([H])=C(C=1C(N([H])C1C([H])=C([H])C2=C(C=1[H])C(C1C([H])=C([H])C3=C(C([H])=C([H])N3[H])C=1[H])=NN2[H])=O)Cl
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| InChi Key |
AZPDJGLJPDRCDQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H14Cl2N4O/c23-16-2-1-3-17(24)20(16)22(29)26-14-5-7-19-15(11-14)21(28-27-19)13-4-6-18-12(10-13)8-9-25-18/h1-11,25H,(H,26,29)(H,27,28)
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| Chemical Name |
2,6-dichloro-N-[3-(1H-indol-5-yl)-1H-indazol-5-yl]benzamide
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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 : ~83.33 mg/mL (~197.80 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.8 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3737 mL | 11.8686 mL | 23.7372 mL | |
| 5 mM | 0.4747 mL | 2.3737 mL | 4.7474 mL | |
| 10 mM | 0.2374 mL | 1.1869 mL | 2.3737 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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