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Purity: ≥98%
Necrosulfonamide is a potent and highly specific and potent necrosis inhibitor which blocks mixed lineage kinase domain-like protein (MLKL). Through blocking the activity of MLKL's N-terminal CC domain, necrosulfonamide prevents MLKL-mediated necrosis. It prevents necrosis from occurring after RIP3 activation. Even at a 5 μM concentration, necrosulfonamide has no impact on the apoptosis that TNF-α plus Smac mimetic induces in Panc-1 cells that do not express RIP3. The programmed necrosis (necroptosis) pathway uses the receptor-interacting serine-threonine kinase 3 (RIP3) as a key signaling molecule. This pathway is crucial for the development, tissue damage response, and antiviral immunity of many physiological and pathological conditions.
| Targets |
necrosis(MLKL)
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|---|---|
| ln Vitro |
Necrosulfonamide inhibits MLKL-mediated Necrosis by blocking its N-terminal CC domain function. Following RIP3 activation, it prevents necrosis. Even at a concentration of 5 μM , necrosulfonamide has no impact on the apoptosis induced by TNF-α plus Smac mimetic in Panc-1 cells deficient in RIP3. In human cells, necrosulfonamide effectively inhibits necrosis, but not in mouse cells. The cysteine at residue 86 in human MLKL that necrosulfonamide covalently modifies is replaced by a tryptophan residue in mouse MLKL (mixed lineage kinase domain-like protein), which accounts for necrosulfonamide's species specificity[2].
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| ln Vivo |
Necrosulfonamide (NSA) is a small molecule that targets MLKL, the final executor of necroptosis, to specifically inhibit necroptosis.
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| Enzyme Assay |
RIP1 and RIP3 were immunoprecipitated with an anti-Flag antibody. The Flag beads were incubated with 2 μCi of [32P]γ-ATP at 37°C for 1 hour with the artificial substrate MBP or purified recombinant MLKL after being washed three times with kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 0.02% BSA, 150 μM ATP, and 1 mM DTT). Then SDS-PAGE and autoradiography were applied to the reaction mixtures. We describe the discovery of a small molecule known as (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide, also known as necrosulfonamide, which specifically inhibits necrosis downstream of RIP3 activation. The mixed lineage kinase domain-like protein (MLKL) was identified as the interacting target by coimmunoprecipitation with anti-RIP3 antibodies and an affinity probe made from necrosulfonamide. The threonine 357 and serine 358 residues on MLKL were phosphorylated by RIP3 and these phosphorylation events were essential for necrosis.
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| Cell Assay |
Necrosis inhibitors induce diverse effects on MLKL phosphorylation. T/S/Z is applied to HT-29 cells for either 12 or 8 hours, with or without necrosis inhibitors. By monitoring released protease activity in the culture medium, the quantity of dead cells is calculated. The whole-cell extracts are made, and western blotting is used to analyze them. Final concentrations of 1 or 10 μM necrosulfonamide or necrostatin-1 inhibit necrosis.
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| Animal Protocol |
Male Wistar rats
1.65 mg/kg i.p. |
| References |
| Molecular Formula |
C18H15N5O6S2
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|---|---|---|
| Molecular Weight |
461.47
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| Exact Mass |
461.04637556
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| CAS # |
1360614-48-7
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| Related CAS # |
Necrosulfonamide-d4;1795144-22-7;(E/Z)-Necrosulfonamide;432531-71-0
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| Appearance |
Solid
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| SMILES |
COC1=NC=CN=C1NS(=O)(=O)C2=CC=C(C=C2)NC(=O)/C=C/C3=CC=C(S3)[N+](=O)[O-]
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| InChi Key |
FNPPHVLYVGMZMZ-XBXARRHUSA-N
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| InChi Code |
InChI=1S/C18H15N5O6S2/c1-29-18-17(19-10-11-20-18)22-31(27,28)14-6-2-12(3-7-14)21-15(24)8-4-13-5-9-16(30-13)23(25)26/h2-11H,1H3,(H,19,22)(H,21,24)/b8-4+
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| Chemical Name |
(E)-N-[4-[(3-methoxypyrazin-2-yl)sulfamoyl]phenyl]-3-(5-nitrothiophen-2-yl)prop-2-enamide
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| Synonyms |
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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: 2.5 mg/mL (5.42 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (5.42 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 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 (5.42 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Solubility in Formulation 4: 10 mg/mL (21.67 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: 6.67 mg/mL (14.45 mM) in 20% SBE-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1670 mL | 10.8349 mL | 21.6699 mL | |
| 5 mM | 0.4334 mL | 2.1670 mL | 4.3340 mL | |
| 10 mM | 0.2167 mL | 1.0835 mL | 2.1670 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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