Necrosis; MLKL/mixed lineage kinase domain-like protein

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].

Necrosulfonamide (NSA) is a small molecule that targets MLKL, the final executor of necroptosis, to specifically inhibit necroptosis.

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.

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.

Male Wistar rats
1.65 mg/kg
i.p.

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Rats were randomly allocated into four groups (8 rats/group). Group 1 (Control group) comprised normal vehicle-treated rats. Group 2 (AlCl3 group; AD group) comprised rats that were treated with AlCl3, dissolved in distilled water, orally at a dose of 17 mg/kg daily for 6 consecutive weeks, and represented the AD group. Group 3 (AlCl3 + necrosulfonamide (NSA) group) comprised rats that were treated with AlCl3, as in group 2, concomitantly with necrosulfonamide (NSA), dissolved in dimethyl sulfoxide, intraperitoneally at a dose of 1.65 mg/kg daily for 6 weeks. Group 4 (necrosulfonamide (NSA) group) comprised normal rats that were treated with NSA dissolved in dimethyl sulfoxide at a dose of 1.65 mg/kg/day intraperitoneally for 6 weeks. The dose of NSA was selected based on a pilot experiment conducted prior to the main study. In this preliminary study, the dose efficacy was evaluated based on histological examination of the hippocampus for amyloid plaque deposits and neuronal degeneration, learning and memory evaluation by Morris water maze and Y-maze tests, and analysis of hippocampal p-MLKL, p-tau, and β-amyloid levels, in AlCl3 + NSA-treated rats compared to AlCl3-treated rats.[4]

[1]. Med Chem Commun. 2014, 5, 333.

[2]. Cell . 2012 Jan 20;148(1-2):213-27.

[3]. Mol Cell . 2014 Apr 10;54(1):133-146.

[4]. ACS Chem Neurosci . 2020 Oct 21;11(20):3386-3397.

Necrosulfonamide is a sulfonamide that is a 3-methoxypyrazin-2-yl derivative of (E)-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide. Necrosulfonamide specifically blocks necrosis downstream of the activation of RIP3 (the receptor-interacting serine-threonine kinase 3), a key signalling molecule in the programmed necrosis (necroptosis) pathway. It has a role as a necroptosis inhibitor and a neuroprotective agent. It is a sulfonamide, a member of pyrazines and a member of thiophenes.

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Through high-throughput screening of 200 000 compounds and subsequent structure–activity relationship (SAR) studies we identified necrosulfonamide (NSA) as a potent small molecule inhibitor for necroptosis, induced by a combination of TNF-a, Smac mimetic, and z-VAD-fmk (T/S/Z). Applying a forward chemical genetic approach, we utilized an NSA based chemical probe to further reveal that NSA selectively targeted the Mixed Lineage Kinase Domain-like Protein (MLKL) to block the necrosome formation.[1]

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The receptor-interacting serine-threonine kinase 3 (RIP3) is a key signaling molecule in the programmed necrosis (necroptosis) pathway. This pathway plays important roles in a variety of physiological and pathological conditions, including development, tissue damage response, and antiviral immunity. Here, we report the identification of a small molecule called (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide--hereafter referred to as necrosulfonamide--that specifically blocks necrosis downstream of RIP3 activation. An affinity probe derived from necrosulfonamide and coimmunoprecipitation using anti-RIP3 antibodies both identified the mixed lineage kinase domain-like protein (MLKL) as the interacting target. MLKL was phosphorylated by RIP3 at the threonine 357 and serine 358 residues, and these phosphorylation events were critical for necrosis. Treating cells with necrosulfonamide or knocking down MLKL expression arrested necrosis at a specific step at which RIP3 formed discrete punctae in cells. These findings implicate MLKL as a key mediator of necrosis signaling downstream of the kinase RIP3.[2]

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Programmed necrotic cell death induced by the tumor necrosis factor alpha (TNF-α) family of cytokines is dependent on a kinase cascade consisting of receptor-interacting kinases RIP1 and RIP3. How these kinase activities cause cells to die by necrosis is not known. The mixed lineage kinase domain-like protein MLKL is a functional RIP3 substrate that binds to RIP3 through its kinase-like domain but lacks kinase activity of its own. RIP3 phosphorylates MLKL at the T357 and S358 sites. Reported here is the development of a monoclonal antibody that specifically recognizes phosphorylated MLKL in cells dying of this pathway and in human liver biopsy samples from patients suffering from drug-induced liver injury. The phosphorylated MLKL forms an oligomer that binds to phosphatidylinositol lipids and cardiolipin. This property allows MLKL to move from the cytosol to the plasma and intracellular membranes, where it directly disrupts membrane integrity, resulting in necrotic death.[3]

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Alzheimer's disease (AD) is a progressively debilitating neurodegenerative disorder that has no effective remedy, so far, with available therapeutic modalities being only symptomatic and of modest efficacy. Necroptosis is a form of controlled cell death with a recently emerging link to the pathogenesis of several neurodegenerative diseases. This study investigated the role of necroptosis in the pathogenesis of AD and evaluated the potential beneficial effect of the necroptosis inhibitor, necrosulfonamide (NSA), in a rat model of AD. AD was induced by oral administration of AlCl3 (17 mg/kg/day) for 6 consecutive weeks. Administration of NSA (1.65 mg/kg/day) intraperitoneally for 6 weeks significantly amended AlCl3-induced spatial learning and memory deficits, as demonstrated by enhanced rat performance in Morris water and Y-mazes. NSA alleviated the abnormally high hippocampal expression of tumor necrosis factor-alpha (TNF-α), β-site amyloid precursor protein cleaving enzyme 1 (BACE1), β-amyloid, glycogen synthase kinase-3β (GSK-3β), phosphorylated tau protein, and acetylcholinesterase with concordant replenishment of acetylcholine. The amendments of AD perturbations achieved by NSA correlated with its inhibitory effect on the phosphorylation of the key necroptotic executioner, mixed lineage kinase domain-like protein (MLKL). Histopathological alterations supported the biochemical findings. In conclusion, NSA treatment represents a promising anti-Alzheimer's approach, mitigating AD neuropathologies via targeting MLKL-dependent necroptosis.[4]

C18H15N5O6S2

461.47

461.046

1360614-48-7

Necrosulfonamide-d4;1795144-22-7;(E/Z)-Necrosulfonamide;432531-71-0

1566236

Light yellow to yellow solid

1.6±0.1 g/cm3

257 °C(dec.)

1.695

4.08

2

10

7

31

760

0

S(C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(/C(/[H])=C(\[H])/C1=C([H])C([H])=C([N+](=O)[O-])S1)=O)(N([H])C1C(=NC([H])=C([H])N=1)OC([H])([H])[H])(=O)=O

FNPPHVLYVGMZMZ-XBXARRHUSA-N

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+

(E)-N-[4-[(3-methoxypyrazin-2-yl)sulfamoyl]phenyl]-3-(5-nitrothiophen-2-yl)prop-2-enamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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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.
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 corn oil and mix evenly.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)