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| Targets |
The primary targets of NLRP3-IN-9 are NLRP3 ATPase and caspase-1. The NLRP3 inflammasome is a multi-protein complex that plays a critical role in the innate immune response by activating caspase-1, which in turn processes pro-inflammatory cytokines such as IL-1β and IL-18 into their active forms. NLRP3-IN-9 acts by irreversibly trapping thiol nucleophiles, which prevents both ATP- and nigericin-triggered pyroptosis of human THP-1 cells in a time- and concentration-dependent manner. By inhibiting NLRP3 ATPase and caspase-1, NLRP3-IN-9 prevents the activation of the NLRP3 inflammasome and the subsequent release of IL-1β. This mechanism of action makes it a valuable tool for studying inflammasome-mediated inflammation.
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| ln Vitro |
In human THP-1 cells, INF4E (compound 9) inhibits ATP- and nigericin-triggered pyroptosis in a concentration- and time-dependent manner [3].
In vitro, NLRP3-IN-9 is a potent inhibitor of NLRP3 ATPase and caspase-1. It acts by irreversibly trapping thiol nucleophiles, which prevents both ATP- and nigericin-triggered pyroptosis of human THP-1 cells in a time- and concentration-dependent manner. The compound inhibits IL-1β release. Its activity is typically measured using cell-based assays that assess pyroptosis, caspase-1 activity, and IL-1β release in THP-1 cells or other inflammasome-competent cell lines. IC50 values are determined from dose-response curves. The compound's irreversible mechanism of action distinguishes it from reversible inflammasome inhibitors. |
| ln Vivo |
INF4E improves post-ischemic left ventricular pressure and dramatically lowers lactate dehydrogenase release and infarct size [1]. In a time-dependent way, INF4E reduces the production of the NLRP3 inflammasome complex that is triggered by cardiac IR (ischemia/reperfusion) [1].
In vivo, NLRP3-IN-9 (3, 10 mg/kg; i.p.) reduces inflammation and mechanical hyperalgesia in a mouse model of acute gout. It displays protective effects against ischemia-reperfusion (IR)-induced myocardial injury and dysfunction. NLRP3-IN-9 inhibits IL-1β release and reduces inflammation. These in vivo studies demonstrate that NLRP3-IN-9 is effective in reducing inflammasome-mediated inflammation in animal models. The compound's ability to reduce mechanical hyperalgesia suggests that it may have analgesic effects in addition to its anti-inflammatory activity. Its protective effects against myocardial injury indicate potential for treating cardiovascular conditions. |
| Enzyme Assay |
In vitro enzyme/receptor binding assays for NLRP3-IN-9 involve NLRP3 ATPase and caspase-1 activity assays using purified enzymes or cell lysates. The compound's inhibitory activity against NLRP3 ATPase is measured by monitoring ATP hydrolysis in the presence of the enzyme and varying concentrations of NLRP3-IN-9. Caspase-1 activity is measured using fluorogenic substrates that are cleaved by active caspase-1. IC50 values are determined from dose-response curves. The compound's irreversible mechanism of action can be confirmed by pre-incubation and dilution experiments, where the enzyme is incubated with the compound and then diluted to assess residual activity.
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| Cell Assay |
In vitro cellular assays for NLRP3-IN-9 are conducted in human THP-1 cells or other inflammasome-competent cell lines. Cells are primed with LPS and then treated with ATP or nigericin to activate the NLRP3 inflammasome in the presence of varying concentrations of NLRP3-IN-9. Pyroptosis is assessed by measuring lactate dehydrogenase (LDH) release or propidium iodide uptake. Caspase-1 activity is measured using fluorogenic substrates or by detecting cleaved caspase-1 by western blotting. IL-1β release is measured by ELISA. These assays confirm that NLRP3-IN-9 engages its targets in a cellular context and produces the expected inhibition of inflammasome activation.
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| Animal Protocol |
In vivo animal studies for NLRP3-IN-9 are conducted in mouse models of acute gout and ischemia-reperfusion injury. In the gout model, mice are injected with monosodium urate (MSU) crystals to induce inflammation, and NLRP3-IN-9 is administered intraperitoneally at doses of 3 or 10 mg/kg. Inflammation and mechanical hyperalgesia are assessed by measuring paw swelling and pain sensitivity. In the ischemia-reperfusion model, mice undergo temporary occlusion of the coronary artery followed by reperfusion, and NLRP3-IN-9 is administered to assess its protective effects against myocardial injury and dysfunction. These studies confirm that NLRP3-IN-9 is effective in vivo and provide information about its therapeutic potential.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of NLRP3-IN-9 indicate that it has a molecular weight of 240.68 and a molecular formula of C12H13ClO3. The compound is also known as INF-4E, Ethyl 2-[(2-chlorophenyl)(hydroxy)methyl]acrylate. For storage, the powder should be kept under appropriate conditions to maintain stability. Its solubility and other physicochemical properties would need to be characterized for in vivo studies. The compound's irreversible mechanism of action may affect its pharmacokinetic and pharmacodynamic profile.
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| Toxicity/Toxicokinetics |
Toxicological information for NLRP3-IN-9 is primarily derived from its use as a research compound in preclinical studies. As an inhibitor of the NLRP3 inflammasome, potential on-target effects could include immunosuppression and increased susceptibility to infections, given the role of the inflammasome in the innate immune response. Comprehensive toxicology studies would be required for therapeutic development, including assessments of immune function, infection susceptibility, and organ toxicity. However, NLRP3-IN-9 is primarily used as a research tool and has not advanced to clinical development.
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| References | |
| Additional Infomation |
It possesses anti-pyroptosis activity; its structure is described in the first source.
NLRP3-IN-9 (INF-4E) is a potent inhibitor of the NLRP3 inflammasome. It inhibits NLRP3 ATPase and caspase-1 activities by irreversibly trapping thiol nucleophiles. NLRP3-IN-9 prevents ATP- and nigericin-triggered pyroptosis of human THP-1 cells. It inhibits IL-1β release, reduces inflammation and mechanical hyperalgesia, and has potential for gout research. NLRP3-IN-9 also protects against IR-induced myocardial injury. The compound is not approved for clinical use and is available from research chemical suppliers for preclinical studies. |
| Molecular Formula |
C12H13CLO3
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| Molecular Weight |
240.68
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| Exact Mass |
240.055
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| Elemental Analysis |
C, 59.88; H, 5.44; Cl, 14.73; O, 19.94
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| CAS # |
88039-46-7
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| Related CAS # |
88039-46-7;
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| PubChem CID |
11402215
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| Appearance |
Colorless to light yellow liquid
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| LogP |
2.492
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
16
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| Complexity |
265
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C(C(C1C(Cl)=CC=CC=1)O)=C)OCC
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| InChi Key |
BSRPDXCMAOIUOO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C12H13ClO3/c1-3-16-12(15)8(2)11(14)9-6-4-5-7-10(9)13/h4-7,11,14H,2-3H2,1H3
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| Chemical Name |
ethyl 2-[(2-chlorophenyl)-hydroxymethyl]prop-2-enoate
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| Synonyms |
NLRP3IN9 NLRP3 IN 9 NLRP3-IN-9
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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 : ≥ 100 mg/mL (~415.49 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (10.39 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 25.0 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: ≥ 2.5 mg/mL (10.39 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 25.0 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: ≥ 2.5 mg/mL (10.39 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 | 4.1549 mL | 20.7745 mL | 41.5489 mL | |
| 5 mM | 0.8310 mL | 4.1549 mL | 8.3098 mL | |
| 10 mM | 0.4155 mL | 2.0774 mL | 4.1549 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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