RIP1 kinase (EC50 = 182 nM)

Necrostatin-1 (1-100 μM) 可防止内源性和过度表达的 RIP1 自身磷酸化。已发现 Necrostatin-1 的抗坏死活性主要由 RIP1 介导。 [1] Necrostatin-1 可有效防止多种刺激诱导的一系列细胞类型的坏死性细胞死亡。 necrostatin-1（之前被鉴定为小分子坏死性凋亡抑制剂）的 EC50 为 490 nM，可阻断 Jurkat 细胞中 RIP 激酶和 TNF-α 诱导的坏死性凋亡。 [2]

Necrostatin-1 （nec1）是受体相互作用蛋白激酶1 （RIPK1）的特异性小分子抑制剂，可特异性抑制RIPK1的磷酸化。RIPK1通过与受体相互作用丝氨酸/苏氨酸蛋白激酶3（RIPK3）相互作用调节炎症和细胞死亡。我们假设Nec-1可能在骨关节炎（OA）患者中具有抗炎作用，因为OA的病理生理涉及炎症相关信号通路的激活和细胞凋亡。在这项研究中，我们探讨了Nec-1对白细胞介素(IL)-1β-诱导的小鼠软骨细胞炎症和不稳定的内侧半月板（DMM）小鼠模型的影响。用Nec-1抑制RIPK1在体内和体外均能显著抑制分解代谢，但不抑制软骨下骨的变化。Nec-1可抑制IL-1β诱导的基质金属蛋白酶（MMP）和ADAMTs5型金属肽酶（ADAMTs5）表达升高。然而，加入高迁移率组盒1 （HMGB1）部分消除了这一作用，表明HMGB1和Nec-1在保护原代软骨细胞中发挥了重要作用。此外，Nec-1降低toll样受体4 （TLR4）和基质细胞衍生因子-1 （SDF-1）的表达，减弱TLR4与HMGB1的相互作用。Western blot结果显示，Nec-1显著抑制il -1β-诱导的NF-κB转录活性，但对MAPK通路无明显抑制作用。在体内采用显微计算机断层扫描、免疫组织化学染色和Safranin O/Fast Green染色评估OA软骨的破坏程度。结果表明，NEC-1能显著降低OA软骨的破坏程度。因此，Nec-1可能是治疗OA的新候选药物[3]。

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为了研究坏死性上睑垂在缺血性脑损伤中的作用，我们测定了Nec-1对小鼠大脑中动脉闭塞（MCAO）缺血性损伤的影响。脑室内给予Nec-1显著（P < 0.05）减少MCAO后梗死面积，提示坏死下垂可能参与这种形式的病理性死亡（图6a）。为了进行更详细的分析，我们切换到7-Cl-Nec-1，它在体外显示出更大的活性。7-Cl-Nec-1也提供了显著（P < 0.05）的剂量依赖性梗死面积减少和MCAO后神经学评分的成比例改善（图6b） [2]。

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Necrostatin-1 (Nec-1) 是受体相互作用蛋白激酶 1 (RIPK1) 的特异性小分子抑制剂，可阻止 RIPK1 的磷酸化。

体外激酶测定[1]
试验基本上按所述进行。采用标准的Ca3(PO4)2沉淀法，用pcDNA3-FLAG-RIP1载体、编码RIP1突变蛋白的载体或pcDNA3-RIP2-Myc和pcDNA3-FLAG-RIP3载体转染293T细胞。转染后6 h更换培养基，48 h后将细胞溶解在TL缓冲液中，该缓冲液由1% Triton X-100、150 mM NaCI、20 mM HEPES、pH 7.3、5 mM EDTA、5 mM NaF、0.2 mM NaVO3 （ortho）和完整的蛋白酶抑制剂混合物组成。用anti-FLAG M2琼脂糖珠在4℃下免疫沉淀16 h，然后用TL缓冲液洗涤3次，用20 mM HEPES洗涤2次，pH 7.3。在含有20 mM HEPES、pH 7.3、10 mM MnCl2和10 mM MgCl2的15 μl反应缓冲液中，在23-25℃下，不同浓度的坏死性他汀类药物存在下，培养15分钟。在这些试验中，在加入反应之前，将复合原液（在DMSO中）在DMSO中稀释到适当的浓度，以保持所有样品的DMSO最终浓度为3%。加入10 μM的冷ATP和1 mCi的[γ-32P] ATP引发激酶反应，在30℃下反应30 min。在SDS-PAGE样品缓冲液中煮沸停止反应，并进行8%的SDS-PAGE。在Storm 8200磷成像仪上分析显示了RIP1波段。除了小鼠单克隆RIP1抗体和蛋白磁珠或兔RIP1抗体偶联琼脂糖珠外，内源性RIP1激酶反应采用类似的方案。对于重组杆状病毒表达的RIP1，根据制造商的说明书在Sf9细胞中表达蛋白，并使用谷胱甘肽-sepharose bead纯化。蛋白在50 mM Tris-HCl中洗脱，pH 8.0，添加10 mM还原性谷胱甘肽，洗脱后的蛋白用于激酶反应，添加5 ×激酶反应缓冲液（100 mM HEPES, pH 7.3, 50 mM MnCl2, 50 mM MgCl2, 50 μM冷ATP和5 μCi的[γ-32P]ATP）。

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RIP1 的磷酸化需要其激酶活性。 RIP1 激酶测定按照方法中的概述进行，在 [γ-32P]ATP 存在下在 30°C 下进行 30 分钟，使用 FLAG 标记的野生型 (WT) 或 RIP1 激酶失活点突变体 (K45M) 的表达构建体或带有 FLAG 标记的野生型 (WT) 或 K45M。 SDS-PAGE 后，对样品进行放射自显影以鉴定 RIP1 带。该放射自显影图以及所有其他放射自显影图显示了放射性谱带基于比率的相对强度。为了确保激酶反应中存在等量的蛋白质，使用抗 RIP1 抗体对珠子样本进行蛋白质印迹分析。

细胞活力测定[3]
采用CCK-8法（Boster）评估细胞增殖和活力。简单地说，原代软骨细胞以每孔10000个细胞的密度在96孔板中接种。第二天，每天加入含有DMSO（载体）或等体积Necrostatin-1 (Nec-1) (30 μmol/L)的培养基。24、48、72 h后更换100 μL含10% CCK-8溶液的培养基，37℃暗箱孵育1 h。用ELX800酶标仪在450 nm处测定吸光度。

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细胞凋亡分析[3]
采用Annexin V-FITC/碘化丙啶（PI）试剂盒检测IL-1β (5 ng/mL)加或不加Necrostatin-1 (Nec-1) (30 μmol/L)对软骨细胞凋亡的影响。处理后收集软骨细胞，在冰冷的PBS中洗涤三次，并在结合缓冲液中重悬。然后，在缓冲液中加入5 μL PI和5 μL Annexin V，在4℃的黑暗条件下放置15 min。采用FACS Calibur流式细胞仪对早期和晚期凋亡细胞进行分析。

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细胞EC50测定坏死性他汀类药物[1]
如前所述，用人TNFα处理fadd缺陷Jurkat细胞，测定EC50。简单地说，将细胞接种到96孔板中，在10 ng ml-1人TNFα存在和不存在的情况下，用一系列necrostatin浓度（30 nM至100 μM， 11个剂量点）处理24小时。对于这些和所有其他细胞实验，在加入细胞之前，将化合物原液（DMSO）稀释到DMSO的适当浓度，以保持所有样品的DMSO最终浓度为0.5%。采用CellTiter-Glo荧光细胞活力测定法测定细胞活力。计算化合物和tnf处理井与化合物处理井和未处理井的发光比（活力，%），并在GraphPad Prizm中通过非线性回归计算EC50。

For drug administration, we dissolved 7-Cl-Necrostatin-1 (Nec-1) or other derivatives in 4% methyl-β-cyclodextrin (Sigma) solution in PBS and administered it through intracerebroventricular administration at the time points indicated. Typically, we performed two 2-μl injections of 4 mM stock solution (unless otherwise indicated). For preocclusion delivery, we performed injections 5 min before the onset of 2-h MCAO occlusion and immediately after the cessation of the occlusion, at the time of the reperfusion. For postocclusion delivery, we performed injections at the time of reperfusion after 2 h of MCAO as well as 2 h after the onset of reperfusion. In the case of infusion, we infused 20 μl of compound over a 30-min time period. In the case of injection 6 h after occlusion, we injected a single 4-μl dose. In the case of zVAD.fmk administration, we added it to the Necrostatin-1 (Nec-1) formulation and administered a total dose of 160 ng.[2]

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Twelve-week-old male C57BL/6 mice were housed in a light- and temperature-controlled room and fed a standard diet. The body weight of animals was presented in the Supplementary Table I. The destabilised medial meniscus (DMM) surgical model of OA was produced on the right knee according to a previously published protocol (Glasson et al., 2007). Forty mice were divided into four groups (n = 10 per group): (1) sham group: sham-operated mice administered 15 μL vehicle (5% DMSO, 45% PEG300, and ddH2O), (2) the sham + Necrostatin-1 (Nec-1) group: sham-operated mice treated with 15 μL Necrostatin-1 (Nec-1) (0.0468 mg/Kg), (3) the DMM group: DMM surgery mice administered 15 μL vehicle, and (4) the DMM + Necrostatin-1 (Nec-1) group: DMM surgery mice administered 15 μL Nec-1. The Necrostatin-1 (Nec-1) solution or vehicle was injected intra-articularly twice weekly for 8 weeks before sacrifice.[3]

[1].Nat Chem Biol . 2008 May;4(5):313-21.

[2]. Nat Chem Biol . 2005 Jul;1(2):112-9.

[3]. Front Pharmacol . 2018 Nov 27;9:1378.

5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylidene-4-imidazolidinone is an organonitrogen compound and an organooxygen compound. It is functionally related to an alpha-amino acid.

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Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.[1]

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The mechanism of apoptosis has been extensively characterized over the past decade, but little is known about alternative forms of regulated cell death. Although stimulation of the Fas/TNFR receptor family triggers a canonical 'extrinsic' apoptosis pathway, we demonstrated that in the absence of intracellular apoptotic signaling it is capable of activating a common nonapoptotic death pathway, which we term necroptosis. We showed that necroptosis is characterized by necrotic cell death morphology and activation of autophagy. We identified a specific and potent small-molecule inhibitor of necroptosis, necrostatin-1, which blocks a critical step in necroptosis. We demonstrated that necroptosis contributes to delayed mouse ischemic brain injury in vivo through a mechanism distinct from that of apoptosis and offers a new therapeutic target for stroke with an extended window for neuroprotection. Our study identifies a previously undescribed basic cell-death pathway with potentially broad relevance to human pathologies.[2]

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Necrostatin-1 (Nec-1) is a specific small molecule inhibitor of receptor-interacting protein kinase 1 (RIPK1) that specifically inhibits phosphorylation of RIPK1. RIPK1 regulates inflammation and cell death by interacting with receptor-interacting serine/threonine protein kinases 3(RIPK3). We hypothesized that Nec-1 may have anti-inflammatory efficacy in patients with osteoarthritis (OA), as the pathophysiology of OA involves the activation of inflammation-related signaling pathways and apoptosis. In this study, we explored the effects of Nec-1 on interleukin (IL)-1β-induced inflammation in mouse chondrocytes and the destabilised medial meniscus (DMM) mouse model. Inhibiting RIPK1 with Nec-1 dramatically suppressed catabolism both in vivo and in vitro, but did not inhibit changes in subchondral bone. Nec-1 abolished the in vitro increases in matrix metalloproteinase (MMP) and ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTs5) expression induced by IL-1β. However, adding high-mobility group box 1 (HMGB1) partially abrogated this effect, indicating the essential role of HMGB1 and Nec-1 in the protection of primary chondrocytes. Furthermore, Nec-1 decreased the expression of Toll-like receptor 4 (TLR4) and stromal cell-derived factor-1 (SDF-1), and attenuated the interaction between TLR4 and HMGB1. Western blot results suggested that Nec-1 significantly suppressed IL-1β-induced NF-κB transcriptional activity, but not MAPK pathway. Micro-computed tomography, immunohistochemical staining, and Safranin O/Fast Green staining were used in vivo to assess the degree of destruction of OA cartilage. The results show that NEC-1 can significantly reduce the degree of destruction of OA cartilage. Therefore, Nec-1 may be a novel therapeutic candidate to treat OA.[3]

C13H13N3OS

259.33

259.077

C, 60.21; H, 5.05; N, 16.20; O, 6.17; S, 12.36

4311-88-0

Necrostatin-1 (inactive control);64419-92-7

2828334

Light yellow solid powder

1.4±0.1 g/cm3

441.9±37.0 °C at 760 mmHg

151ºC

221.1±26.5 °C

0.0±1.1 mmHg at 25°C

1.738

1.26

80.22

2

2

2

18

373

0

S=C1N(C([H])([H])[H])C(C([H])(C([H])([H])C2=C([H])N([H])C3=C([H])C([H])=C([H])C([H])=C23)N1[H])=O

TXUWMXQFNYDOEZ-UHFFFAOYSA-N

InChI=1S/C13H13N3OS/c1-16-12(17)11(15-13(16)18)6-8-7-14-10-5-3-2-4-9(8)10/h2-5,7,11,14H,6H2,1H3,(H,15,18)

5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylideneimidazolidin-4-one

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 (9.64 mM) (饱和度未知) 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), 澄清溶液。
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。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (9.64 mM) (饱和度未知) 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), 澄清溶液。
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。

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配方 3 中的溶解度: 5% DMSO+45% PEG 300+ddH2O: 10mg/mL

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配方 4 中的溶解度: 12.5 mg/mL (48.20 mM) in 0.5% CMC-Na/saline water (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。

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配方 5 中的溶解度: 1.67 mg/mL (6.44 mM) in 10% (50% EtOH 50% Cremophor EL) 90% 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。

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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));
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;
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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。