Necrosis; MLKL/mixed lineage kinase domain-like protein

Necrosulfonamide 通过阻断 N 末端 CC 结构域功能来抑制 MLKL 介导的坏死。 RIP3 激活后，可防止坏死。即使浓度为 5 μM，necrosulfonamide 对 TNF-α 加 Smac 模拟物在 RIP3 缺陷的 Panc-1 细胞中诱导的细胞凋亡也没有影响。在人类细胞中，necrosulfonamide 可有效抑制坏死，但在小鼠细胞中则不然。 necrosulfonamide 共价修饰的人 MLKL 残基 86 处的半胱氨酸被小鼠 MLKL（混合谱系激酶结构域样蛋白）中的色氨酸残基取代，这解释了 necrosulfonamide 的物种特异性[2]。

Necrosulfonamide (NSA) 是一种小分子，靶向坏死性凋亡的最终执行者 MLKL，特异性抑制坏死性凋亡。

使用抗 Flag 抗体对 RIP1 和 RIP3 进行免疫沉淀。 Flag 珠子用激酶缓冲液（50 mM HEPES，pH 7.5，10 mM MgCl2）洗涤 3 次后，与人工底物 MBP 或纯化的重组 MLKL 一起在 37°C 下与 2 μCi 的 [32P]γ-ATP 一起孵育 1 小时。 、50 mM NaCl、0.02% BSA、150 μM ATP 和 1 mM DTT）。然后对反应混合物进行SDS-PAGE和放射自显影。我们描述了一种称为 (E)-N-(4-(N-(3-甲氧基吡嗪-2-基)氨磺酰基)苯基)-3-(5-硝基噻吩-2-基)丙烯酰胺的小分子的发现，还称为坏死磺酰胺，它特异性抑制 RIP3 激活下游的坏死。通过与抗 RIP3 抗体和由 necrosulfonamide 制成的亲和探针进行共免疫沉淀，混合谱系激酶结构域样蛋白 (MLKL) 被鉴定为相互作用靶标。 MLKL 上的苏氨酸 357 和丝氨酸 358 残基被 RIP3 磷酸化，这些磷酸化事件对于坏死至关重要。

坏死抑制剂对 MLKL 磷酸化产生多种影响。 T/S/Z 适用于 HT-29 细胞 12 或 8 小时，有或没有坏死抑制剂。通过监测培养基中释放的蛋白酶活性，计算死亡细胞的数量。制备全细胞提取物，并使用蛋白质印迹对其进行分析。 1 或 10 μM necrosulfonamide 或 necrostatin-1 的最终浓度可抑制坏死。

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

461.4716

461.046

C, 46.85; H, 3.28; N, 15.18; O, 20.80; S, 13.89

432531-71-0

Necrosulfonamide;1360614-48-7

1566236

Light yellow to yellow solid powder

1.6±0.1 g/cm3

1.695

4.08

195.95

2

10

7

31

760

0

COC1=NC=CN=C1NS(=O)(=O)C2=CC=C(C=C2)NC(=O)C=CC3=CC=C(S3)[N+](=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

Necrosulfonamide, MLKL inhibitor; Necrosome Inhibitor II; Necrosis Inhibitor III; Necrosulfonamide; 1360614-48-7; 432531-71-0; (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophen-2-yl)acrylamide; CHEBI:63770; (E/Z)-Necrosulfonamide; (2E)-N-{4-[(3-methoxypyrazin-2-yl)sulfamoyl]phenyl}-3-(5-nitrothiophen-2-yl)prop-2-enamide; (E)-N-[4-[(3-methoxypyrazin-2-yl)sulfamoyl]phenyl]-3-(5-nitrothiophen-2-yl)prop-2-enamide; Mixed Lineage Kinase Domain-Like Protein Inhibitor

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)

DMSO: ~92 mg/mL (~199.4 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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