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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
|
|---|---|
| Molecular Weight |
461.4716
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| Exact Mass |
461.05
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| Elemental Analysis |
C, 46.85; H, 3.28; N, 15.18; O, 20.80; S, 13.89
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| CAS # |
432531-71-0
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| Related CAS # |
Necrosulfonamide;1360614-48-7
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| Appearance |
Solid powder
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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 |
Necrosulfonamide, MLKL inhibitor; Necrosome Inhibitor II; Necrosis Inhibitor III; Mixed Lineage Kinase Domain-Like Protein Inhibitor
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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: ~92 mg/mL (~199.4 mM)
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| 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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