Necrosis; MLKL/mixed lineage kinase domain-like protein
Necrosulfonamide (NSA) selectively targets the Mixed Lineage Kinase Domain-like Protein (MLKL); no IC50, Ki, or EC50 values for this target were described in the literature. [1]

Necrosulfonamide (NSA) specifically targets the Mixed Lineage Kinase Domain-like Protein (MLKL), which acts downstream of receptor-interacting serine-threonine kinase 3 (RIP3) in the necroptosis pathway; no IC50, Ki, or EC50 values for this target were described in the literature. [2]

Necrosulfonamide (NSA) exerts its in vivo effects by targeting MLKL-mediated necroptosis; no IC50, Ki, or EC50 values for this target were described in the literature. [4]

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]。

---

1. 通过对20万种化合物的高通量筛选及后续构效关系（SAR）研究，发现NSA是一种强效坏死性凋亡抑制剂。细胞坏死性凋亡由肿瘤坏死因子-α（TNF-α）、Smac模拟物与z-VAD-fmk（简称T/S/Z）联合诱导产生；采用正向化学遗传学方法，利用基于NSA构建的化学探针进一步证实，NSA通过选择性结合MLKL阻断坏死小体（necroposome）的形成。[1]

2. NSA在RIP3激活下游特异性阻断细胞坏死。两种实验方法证实MLKL是NSA的作用靶点：（1）基于NSA构建亲和探针，与细胞裂解液孵育后捕获相互作用蛋白，鉴定发现MLKL是主要结合蛋白；（2）采用抗RIP3抗体进行共免疫沉淀（co-IP），结果显示MLKL可与RIP3共沉淀，证实二者存在物理相互作用。此外，研究发现RIP3可将MLKL的苏氨酸357（T357）和丝氨酸358（S358）位点磷酸化，这两个磷酸化位点对细胞坏死的启动至关重要；用NSA处理细胞或敲低MLKL表达，均可在RIP3形成胞质离散斑点的阶段阻断坏死，表明NSA在RIP3聚集后、MLKL介导膜破坏前中断坏死性凋亡级联反应。[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。

---

在氯化铝（AlCl₃）诱导的阿尔茨海默病（AD）大鼠模型中（AlCl₃以17 mg/kg/天的剂量口服给药6周），以1.65 mg/kg/天的剂量腹腔注射NSA连续6周，表现出显著的改善作用：[4]

- 行为学改善：NSA提升大鼠的空间学习与记忆能力，具体表现为Morris水迷宫测试中逃避潜伏期缩短、穿越原平台位置次数增加，以及Y迷宫测试中自发交替行为率升高。

- 生化指标调节：NSA降低海马区异常升高的促炎因子TNF-α、β淀粉样前体蛋白切割酶1（BACE1）、β淀粉样肽、糖原合成酶激酶-3β（GSK-3β）、磷酸化tau蛋白及乙酰胆碱酯酶（AChE）水平，同时恢复海马区降低的乙酰胆碱（ACh）水平。

- 作用机制：NSA的治疗效果与其抑制坏死性凋亡关键执行分子MLKL的磷酸化有关，进而阻断AD模型大鼠海马区MLKL介导的坏死性凋亡。

- 组织病理学支持：海马组织切片检查显示，NSA处理组大鼠的神经元损伤减轻、β淀粉样肽沉积减少，与生化检测结果一致。[4]

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。

---

1. 基于亲和探针的NSA靶点鉴定实验：[2]

通过对NSA化学结构进行修饰，引入反应性基团（如光交联剂）和纯化标签（如生物素），构建NSA亲和探针。将经坏死性凋亡诱导（T/S/Z或其他坏死性刺激）的细胞制备成裂解液，与NSA亲和探针孵育；随后利用链霉亲和素包被磁珠（结合生物素标签）或针对纯化标签的抗体捕获探针-蛋白复合物。捕获的蛋白经洗脱后，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，再经质谱（MS）或Western blot鉴定，最终确认MLKL是NSA的主要相互作用蛋白。

2. 检测RIP3与MLKL相互作用的共免疫沉淀（co-IP）实验：[2]

用坏死性刺激（如T/S/Z）处理细胞以激活RIP3，随后用含蛋白酶和磷酸酶抑制剂的裂解缓冲液提取总蛋白。将蛋白裂解液与抗RIP3抗体在4°C下孵育过夜，形成抗体-RIP3复合物；加入蛋白A/G琼脂糖珠继续孵育数小时，使抗体-RIP3复合物沉淀。用洗涤缓冲液去除非特异性结合蛋白后，用Laemmli样品缓冲液洗脱沉淀的复合物并加热变性；通过Western blot实验，用抗MLKL抗体检测洗脱液中是否存在MLKL，以此证实RIP3与MLKL的物理相互作用。[2]

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。

---

1. 细胞坏死性凋亡诱导及NSA抑制实验：[2]

将培养细胞（如HeLa或HT29细胞）接种于多孔板，贴壁过夜后，向培养基中加入TNF-α、Smac模拟物与z-VAD-fmk（T/S/Z）的混合物诱导坏死性凋亡。加入诱导剂后，用不同浓度的NSA（或溶剂对照）处理细胞，孵育特定时间（如6~24小时）；通过检测培养基中乳酸脱氢酶（LDH，膜损伤标志物）的释放量，或显微镜观察细胞形态（如肿胀、膜破裂），评估NSA对细胞坏死性凋亡的抑制效果。

2. MLKL敲低及坏死性凋亡验证实验：[2]

用靶向MLKL的小干扰RNA（siRNA）（或非靶向siRNA作为对照），通过转染试剂转染细胞；转染48~72小时后，确认MLKL高效敲低，再用T/S/Z诱导坏死性凋亡。通过LDH释放检测或形态学分析评估坏死性凋亡程度，同时用抗MLKL抗体进行Western blot，验证MLKL的敲低效率。

3. 检测MLKL磷酸化的Western blot实验：[2]

将经坏死性刺激（加或不加NSA）处理的细胞用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解；采用BCA法测定蛋白浓度，取等量蛋白经SDS-PAGE分离后，转移至聚偏二氟乙烯（PVDF）膜上，用脱脂牛奶封闭。将膜与抗磷酸化MLKL（p-MLKL，特异性识别T357/S358位点）、总MLKL或RIP3的一抗在4°C下孵育过夜；洗涤后加入辣根过氧化物酶（HRP）标记的二抗，最后用增强化学发光（ECL）检测系统显示蛋白条带，证实NSA可抑制MLKL磷酸化或阻断其下游功能。

4. 检测RIP3斑点的免疫荧光染色实验：[2]

将生长在盖玻片上的细胞用坏死性刺激和NSA（或溶剂）处理后，用多聚甲醛固定、Triton X-100透化，再用牛血清白蛋白（BSA）封闭；加入抗RIP3一抗在4°C下孵育过夜，随后加入荧光素标记的二抗；用DAPI染色细胞核后，将盖玻片封片，通过荧光显微镜观察RIP3的定位。实验显示，NSA处理或MLKL敲低后，细胞内会形成离散的RIP3斑点，但不会发生后续的坏死性凋亡。[2]

Male Wistar rats
1.65 mg/kg
i.p.

---

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]

---

Alzheimer’s disease (AD) rat model construction and NSA treatment protocol: [4]

1. Animal selection and grouping: Male rats (specific strain not specified) were randomly divided into three groups: normal control group, AD model group, and NSA-treated AD group.

2. AD model induction: Rats in the AD model and NSA-treated groups were administered AlCl₃ via oral gavage at a dose of 17 mg/kg/day for 6 consecutive weeks to induce AD-like neuropathologies.

3. NSA administration: Concurrently with AlCl₃ treatment, rats in the NSA-treated group received intraperitoneal injections of NSA at a dose of 1.65 mg/kg/day for 6 weeks. The normal control group and AD model group received intraperitoneal injections of vehicle (e.g., saline or DMSO) at the same frequency.

4. Behavioral testing: After 6 weeks of treatment, spatial learning and memory were evaluated using the Morris Water Maze and Y-Maze:
- Morris Water Maze: Rats were trained to find a hidden platform in a water tank, with escape latency (time to find the platform) recorded daily. A probe trial was conducted 24 hours after the last training session to measure the number of crossings over the former platform position.
- Y-Maze: Rats were placed in one arm of a Y-shaped maze, and the spontaneous alternation rate (percentage of consecutive entries into different arms) was recorded over a specified period.

5. Tissue collection and analysis: After behavioral testing, rats were euthanized, and the hippocampus was dissected. Hippocampal tissues were used for:
- Biochemical analysis: Protein extraction for Western blotting to detect TNF-α, BACE1, β-amyloid, GSK-3β, p-tau, AChE, ACh, and p-MLKL levels.
- Histopathological analysis: Tissue fixation, paraffin embedding, sectioning, and staining (e.g., HE staining for neuronal morphology or immunohistochemistry for β-amyloid deposition) to assess hippocampal damage. [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.

---

\nThrough 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]

---

\n\nThe 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]

---

\n\nProgrammed 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]

---

\n\nAlzheimer'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]

---

1. Background of NSA discovery: NSA was identified through high-throughput screening of 200,000 compounds and subsequent SAR optimization, aiming to find inhibitors of necroptosis—a programmed necrotic cell death pathway involved in various pathological conditions. [1]

2. Role in necroptosis pathway: NSA is a key tool compound for studying necroptosis, as it specifically targets MLKL (a downstream effector of RIP3) and blocks necroptosis at a late stage (after RIP3 activation and aggregation). This discovery validated MLKL as a critical mediator of RIP3-dependent necrosis, advancing the understanding of the necroptosis signaling cascade. [2]

3. Therapeutic potential in neurodegenerative diseases: In a rat AD model, NSA demonstrated therapeutic efficacy by targeting MLKL-mediated necroptosis, reducing AD-related neuropathologies (e.g., β-amyloid accumulation, tau hyperphosphorylation) and improving cognitive function. This suggests that NSA or MLKL inhibitors may represent promising therapeutic strategies for AD and other neurodegenerative diseases associated with 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

193

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)

配方 1 中的溶解度: 2.5 mg/mL (5.42 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: 2.5 mg/mL (5.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: 2.5 mg/mL (5.42 mM) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

配方 4 中的溶解度: 10 mg/mL (21.67 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 5 中的溶解度: 6.67 mg/mL (14.45 mM) in 20% SBE-β-CD in Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

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));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。