Necrostatin-1
(Synonyms: MTH-DL-Tryptophan,Nec-1) 目录号 : GC11008
Necrostatin-1主要作用于细胞中的RIP1，Necrostatin-1是一种RIP1激酶抑制剂，IC50值为0.32 mM。
Cas No.:4311-88-0
Sample solution is provided at 25 µL, 10mM.
-
Related Biological Data
Cell death pathway and cell morphology (n = 3). (a) The cell viability treated with LDG plus inhibitors of different cell death pathways and action mechanism.
To study the specific cell death pathway induced by liposomes,LDG was administrated in combination with Ferrostatin-1,Desferrioxamine, Z-VADFMK, 3-MA, Necrostatin-1 (necrosis inhibitor, 20 μM)（GLPBIO）, and Nacetylcysteine to measure the cell viability rescuing profile using the CCK8 assay.
Nano Research, 2024: 1-17. IF: 9.8996 -
Related Biological Data
Cetuximab treatment enhances RSL3-induced ferroptosis in KRAS mutant CRC cells.E HCT116 and DLD-1 cells were treated with cetuximab (100 μg/ml) or RSL3 (1 μM) in combination with Z-VAD-FMK (10 μM), necrostatin-1 (10 μM) or 3-MA (60 μM) for 24 h,and cell viability was assessed by the CCK-8 assay.
HCT116 and DLD-1 cells were treated with necrostatin-1 (10 μM)（GLPBIO） for 24 h,and cell viability was assessed by the CCK-8 assay.
Cell Death & Disease 12.11 (2021): 1-11. PMID: 34775496 IF: 8.461 -
Related Biological Data
Cysteine depletion induces ferroptosis in K562/G01 but not K562 cells. (a) K562/G01 cells were treated with Fer-1 (2 μM), Nec-1s(10 μM), Z-VAD-FMK (10 μM), or chloroquine (25 μM) cultured in normal or cysteine depletion condition for 24 h. Cell viability was measured using the MTS kit.
K562 cells were treated with Fer-1(2μM), necrostatin-1s (10 μM)(GLPBIO), Z-VAD-FMK (10 μM), or chloroquine (25 μM) cultured in normal or cysteine depletion condition for 24 h.
Oxid Med Cell Longev 2021 (2021). PMID: 34917232 IF: 6.541 -
Related Biological Data
Elaidic acid-induced necroptosis via ERK activation. (F) SW116 cells were treated with Nec-1 (0, 75 μM) and followed with/without Elaidic acid (450 μM).
SW116 cells were treated with Nec-1 (0, 75 μM)（GLPBIO）
J Ethnopharmacol 312 (2023): 116454. PMID: 37059246 IF: 5.3999 -
Related Biological Data
Ferroptosis may be involved in iron overload-induced 661W cell death.(B) Cell viability was assessed using the CCK-8 assay after exposure to 50 μM Nec-1.
Cells were treated with 50 μM cell death inhibitor Nec-1(GLPBIO)for 4 h before the normal culture for 24 h.
Mol Nutr Food Res, 2024: 2300123. PMID: 38196088 IF: 5.2002 -
Related Biological Data
Erastin and sulfasalazine induce ferroptosis in Kasumi-1 and HL-60 cells. (C) Kasumi-1 and HL-60 cells treated with normal or 5μM erastin were cultured with Ferrostatin-1(2 μM), deferoxamine (100 μM), necrostatin-1 (10 μM), Z-VAD-FMK (10 μM) or chloroquine (25 μM) for 24 h, and cell viability was detected using the MTS assay.
Kasumi-1 and HL-60 cells treated with necrostatin-1 (GLPBIO) (10 μM) for 24 h, and cell viability was detected using the MTS assay.
Frontiers in Oncology 12 (2022): 930654. PMID: 36033479 IF: 4.7003
Quality Control & SDS
- View current batch:
- Purity: >99.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Kinase experiment [1]: | |
|
Preparation Method |
Cells were lysed, Immunoprecipitation was carried out for 16 h at 4 °C using anti-FLAG M2 agarose beads, Beads were incubated in 15 ul of the reaction buffer for 15 min at 23-25 °C in the presence of different concentrations of necrostatins (including Necrostatin-1). Kinase reaction was initiated by addition of 10 μM cold ATP and 1 μCi of [y-32P]ATP, and reactions were carried out for 30 min at 30°C. |
|
Reaction Conditions |
RIP1 kinase assay（ Necrostatin-1） was performed at 30°C for 30 min |
|
Applications |
Necrostatin-1 is a RIP1 kinase inhibitor, RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. |
| Cell experiment [2]: | |
|
Cell lines |
Cardiomyocyte progenitor cells (CMPCs) |
|
Preparation Method |
CMPCs were pretreated with vehicle or Necrostatin-1 for 30 min prior to the addition of tert-Butyl hydroperoxide in serum-free M199 medium. |
|
Reaction Conditions |
30 µM Necrostatin-1 for 30 min |
|
Applications |
Under oxidative stress conditions, CMPC mainly displayed a necrotic phenotype and by pretreatment with Necrostatin-1, we observed a 37 ± 8% reduction in necrotic cell death in CMPCs compared with vehicle. Not find differences in apoptotic-mediated cell death. Therefore, Necrostatin-1 increased the survival of CMPCs by inhibiting necrotic cell death. |
| Animal experiment [3]: | |
|
Animal models |
Eight-week-old male C57Bl/6 mice |
|
Preparation Method |
Mice undergo sham surgery or bilateral renal pedicle clamping 24 hours before intraperitoneal injection of either PBS, the highly specific RIP1 kinase inhibitor Necrostatin-1, or the inactive derivate of necrostatin-1 (Necrostatin-1i) in the presence or absence of RCM. |
|
Dosage form |
Necrostatin-1 (1.65 mg/kg body weight) was applied intraperitoneally 15 min before RCM-injection. |
|
Applications |
CIAKI Is Attenuated by Necrostatin-1, an Inhibitor of the Kinase Domain of Receptor-Interacting Protein Kinase-1. |
|
References: [1]. Degterev A, Hitomi J,et,al.Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008 May;4(5):313-21. doi: 10.1038/nchembio.83. PMID: 18408713; PMCID: PMC5434866. [2]. Feyen D, Gaetani R,et,al. Increasing short-term cardiomyocyte progenitor cell (CMPC) survival by necrostatin-1 did not further preserve cardiac function. Cardiovasc Res. 2013 Jul 1;99(1):83-91. doi: 10.1093/cvr/cvt078. Epub 2013 Apr 3. PMID: 23554461. [3]. Linkermann A, Heller JO,et,al. Nec-1The RIP1-kinase inhibitor necrostatin-1 prevents osmotic nephrosis and contrast-induced AKI in mice. J Am Soc Nephrol. 2013 Oct;24(10):1545-57. doi: 10.1681/ASN.2012121169. Epub 2013 Jul 5. Erratum in: J Am Soc Nephrol. 2014 Dec;25(12):2942. PMID: 23833261; PMCID: PMC3785275. |
|
Necrostatin-1 mainly acts on RIP1 in cells [5], Necrostatin-1 is a RIP1 kinase inhibitor with an IC50 value of 0.32 mM[1]. Necrostatin-1 can effectively inhibit RIP1 autophosphorylation, Necrostatin-1 effectively blocks RIP1-RIP3-MLKL signaling by inhibiting RIP1 phosphorylation [7]. Necrostatin-1 is also an IDO inhibitor[2].
Under oxidative stress conditions, CMPC mainly displayed a necrotic phenotype and by pretreatment with Necrostatin-1, we observed a 37 ± 8% reduction in necrotic cell death in CMPCs compared with vehicle. Not find differences in apoptotic-mediated cell death. Therefore, Necrostatin-1 increased the survival of CMPCs by inhibiting necrotic cell death [4]. The ratios of apoptotic and necrotic C2C12 cells were increased following CoCl2 treatment, typical necroptotic morphological characteristics were able to observe by TEM, whereas Necrostatin-1 exhibited a protective effect against CoCl2 induced oxidative stress. Treatment with Necrostatin-1 significantly decreased the levels of RIP1, p ERK1/2, HIF 1α, BNIP3 and ROS induced by CoCl2, and promoted C2C12 differentiation. Necrostatin-1 reversed the CoCl2 induced decrease in mitochondrial membrane potential [6].
In mice,Necrostatin-1 (Nec-1), a specific inhibitor of the receptor-interacting protein 1 (RIP1) kinase domain, prevented osmotic nephrosis and CIAKI, whereas an inactive Necrostatin-1 derivate (Nec-1i) or the pan-caspase inhibitor zVAD did not. Necrostatin-1 prevented RCM-induced dilation of peritubular capillaries, suggesting a novel role unrelated to cell death for the RIP1 kinase domain in the regulation of microvascular hemodynamics and pathophysiology of CIAKI[3].
References:
[1]: Xie T, Peng W, et,al. Structural basis of RIP1 inhibition by necrostatins. Structure. 2013 Mar 5;21(3):493-9. doi: 10.1016/j.str.2013.01.016. PMID: 23473668.
[2]: Degterev A, Huang Z, et,al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005 Jul;1(2):112-9. doi: 10.1038/nchembio711. Epub 2005 May 29. Erratum in: Nat Chem Biol. 2005 Sep;1(4):234. PMID: 16408008.
[3]: Linkermann A, Heller JO, et,al. The RIP1-kinase inhibitor necrostatin-1 prevents osmotic nephrosis and contrast-induced AKI in mice. J Am Soc Nephrol. 2013 Oct;24(10):1545-57. doi: 10.1681/ASN.2012121169. Epub 2013 Jul 5. Erratum in: J Am Soc Nephrol. 2014 Dec;25(12):2942. PMID: 23833261; PMCID: PMC3785275.
[4]: Feyen D, Gaetani R, et,al. Increasing short-term cardiomyocyte progenitor cell (CMPC) survival by necrostatin-1 did not further preserve cardiac function. Cardiovasc Res. 2013 Jul 1;99(1):83-91. doi: 10.1093/cvr/cvt078. Epub 2013 Apr 3. PMID: 23554461.
[5]: Degterev A, Hitomi J, et,al.Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008 May;4(5):313-21. doi: 10.1038/nchembio.83. PMID: 18408713; PMCID: PMC5434866.
[6]: Chen R, Xu J, et,al.Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia. Int J Mol Med. 2018 May;41(5):2565-2572. doi: 10.3892/ijmm.2018.3466. Epub 2018 Feb 6. PMID: 29436688; PMCID: PMC5846651.
[7]: Degterev A, Huang Z, et,al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005 Jul;1(2):112-9. doi: 10.1038/nchembio711. Epub 2005 May 29. Erratum in: Nat Chem Biol. 2005 Sep;1(4):234. PMID: 16408008.
Necrostatin-1主要作用于细胞[5]中的RIP1，Necrostatin-1是一种RIP1激酶抑制剂，IC50值为0.32 mM[1]。 Necrostatin-1可有效抑制RIP1自身磷酸化，Necrostatin-1通过抑制RIP1磷酸化有效阻断RIP1-RIP3-MLKL信号通路[7]。 Necrostatin-1 也是一种 IDO 抑制剂[2]。
在氧化应激条件下，CMPC 主要表现出坏死表型，通过使用 Necrostatin-1 进行预处理，我们观察到与载体相比，CMPC 中的坏死细胞死亡减少了 37 ± 8%。未发现凋亡介导的细胞死亡差异。因此，Necrostatin-1 通过抑制坏死细胞死亡来增加 CMPCs 的存活率[4]。 CoCl2 处理后 C2C12 细胞凋亡和坏死的比例增加，通过 TEM 能够观察到典型的坏死形态特征，而 Necrostatin-1 对 CoCl2 诱导的氧化应激具有保护作用。用 Necrostatin-1 处理可显着降低 CoCl2 诱导的 RIP1、p ERK1/2、HIF 1α、BNIP3 和 ROS 的水平，并促进 C2C12 分化。 Necrostatin-1逆转了CoCl2诱导的线粒体膜电位降低[6]。
在小鼠中，Necrostatin-1 (Nec-1) 是一种受体相互作用蛋白 1 (RIP1) 激酶结构域的特异性抑制剂，可预防渗透性肾病和 CIAKI，而无活性的 Necrostatin-1 衍生物 (Nec-1i) 或泛半胱天冬酶抑制剂 zVAD 则没有。 Necrostatin-1 阻止 RCM 诱导的管周毛细血管扩张，表明 RIP1 激酶结构域在调节 CIAKI 的微血管血流动力学和病理生理学方面具有与细胞死亡无关的新作用[3]。
| Cas No. | 4311-88-0 | SDF | |
| 别名 | MTH-DL-Tryptophan,Nec-1 | ||
| 化学名 | 5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylideneimidazolidin-4-one | ||
| Canonical SMILES | CN1C(=O)C(NC1=S)CC2=CNC3=CC=CC=C32 | ||
| 分子式 | C13H13N3OS | 分子量 | 259.33 |
| 溶解度 | ≥ 12.97 mg/mL in DMSO, ≥ 13.29 mg/mL in EtOH with ultrasonic | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
||
| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
 |
1 mg | 5 mg | 10 mg |
| 1 mM | 3.8561 mL | 19.2805 mL | 38.5609 mL |
| 5 mM | 0.7712 mL | 3.8561 mL | 7.7122 mL |
| 10 mM | 0.3856 mL | 1.928 mL | 3.8561 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Necrostatin-1 and necroptosis inhibition: Pathophysiology and therapeutic implications
Necrostatin-1 (Nec-1) is a RIP1-targeted inhibitor of necroptosis, a form of programmed cell death discovered and investigated in recent years. There are already many studies demonstrating the essential role of necroptosis in various diseases, including inflammatory diseases, cardiovascular diseases and neurological diseases. However, the potential of Nec-1 in diseases has not received much attention. Nec-1 is able to inhibit necroptosis signaling pathway and thus ameliorate necroptotic cell death in disease development. Recent research findings indicate that Nec-1 could be applied in several types of diseases to alleviate disease development or improve prognosis. Moreover, we predict that Nec-1 has the potential to protect against the complications of coronavirus disease 2019 (COVID-19). This review summarized the effect of Nec-1 in disease models and the underlying molecular mechanism, providing research evidence for its future application.
Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia
Necrostatin-1 (Nec-1) is a selective and potent allosteric inhibitor of necroptosis by specifically inhibiting the activity of receptor?interacting protein (RIP) 1 kinase. The aim of the present study was to determine the effect of Nec?1 on an anoxia model comprising mouse skeletal C2C12 myotubes. In the present study, a hypoxic mimetic reagent, cobalt chloride (CoCl2), was used to induce hypoxia in C2C12 myotubes. The cytotoxic effects of CoCl2?induced hypoxia were determined by a Cell Counting kit?8 assay and flow cytometry. Transmission electron microscopy (TEM) was used to characterize the morphological characteristics of dead cells at the ultrastructural level. To clarify the signaling pathways in CoCl2?mediated cell death, the expression levels of RIP1, RIP3, extracellular signal?regulated kinase (ERK)1/2, hypoxia?inducible factor (HIF)?1α and B cell lymphoma?2 adenovirus E1B 19?kDa interacting protein 3 (BNIP3) were investigated by western blotting. Oxidative stress was determined using 2',7'?dichlorofluorescin diacetate to measure intracellular reactive oxygen species (ROS) and the fluorescent dye JC?1 was used to measure mitochondrial membrane potential (Δψm). The results showed that the ratios of apoptotic and necrotic C2C12 cells were increased following CoCl2 treatment, typical necroptotic morphological characteristics were able to observe by TEM, whereas Nec?1 exhibited a protective effect against CoCl2?induced oxidative stress. Treatment with Nec?1 significantly decreased the levels of RIP1, p?ERK1/2, HIF?1α, BNIP3 and ROS induced by CoCl2, and promoted C2C12 differentiation. Nec?1 reversed the CoCl2?induced decrease in mitochondrial membrane potential. Together, these findings suggested that Nec?1 protected C2C12 myotubes under conditions of CoCl2-induced hypoxia.
Necrostatin-1 Prevents Ferroptosis in a RIPK1- and IDO-Independent Manner in Hepatocellular Carcinoma
Ferroptosis is caused by the iron-mediated accumulation of lipid peroxidation, which is distinct from apoptosis and necroptosis. Necrostatin-1 inhibits receptor-interacting serine/threonine-protein kinase 1 (RIPK1) to initiate necroptosis; it also inhibits indoleamine 2,3-dioxygenase (IDO) to regulate tumor immunity. However, few studies have examined the off-target effect of necrostatin-1 on the ferroptosis pathway. The present study examined whether necrostatin-1 could interrupt ferroptosis induced by system xc- inhibitors (sulfasalazine and erastin) and a glutathione peroxidase 4 inhibitor (RSL3) in Huh7 and SK-HEP-1 cells. Necrostatin-1 completely prevented decreases in cell viability induced by sulfasalazine and erastin; it partially blunted decreases in cell viability induced by RSL3. Necrostatin-1, ferrostatin-1, and deferoxamine repressed sulfasalazine-provoked membrane permeabilization, as detected by 7-aminoactinomycin D staining and lipid peroxidation measured using a C11-BODIPY probe. However, other RIPK1 inhibitors (necrostatin-1s and GSK2982772) and an IDO inhibitor (1-methyl-D-tryptophan) did not recover the decrease in cell viability induced by sulfasalazine. Necrostatin-1 potentiated sulfasalazine-induced expression of xCT, a catalytic subunit of system xc- in these cells. These results demonstrated that necrostatin-1 blocked ferroptosis through a mechanism independent from RIPK1 and IDO inhibition in Huh7 and SK-HEP-1 cells, indicating that its antioxidant activity should be considered when using necrostatin-1 as a RIPK1 inhibitor.
Necrostatin-1 Prevents Necroptosis in Brains after Ischemic Stroke via Inhibition of RIPK1-Mediated RIPK3/MLKL Signaling
Pharmacological studies have indirectly shown that necroptosis participates in ischemic neuronal death. However, its mechanism has yet to be elucidated in the ischemic brain. TNFα-triggered RIPK1 kinase activation could initiate RIPK3/MLKL-mediated necroptosis under inhibition of caspase-8. In the present study, we performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia in rats and used immunoblotting and immunostaining combined with pharmacological analysis to study the mechanism of necroptosis in ischemic brains. In the ipsilateral hemisphere, we found that ischemia induced the increase of (i) RIPK1 phosphorylation at the Ser166 residue (p-RIPK1), representing active RIPK1 kinase and (ii) the number of cells that were double stained with P-RIPK1 (Ser166) (p-RIPK1+) and TUNEL, a label of DNA double-strand breaks, indicating cell death. Furthermore, ischemia induced activation of downstream signaling factors of RIPK1, RIPK3 and MLKL, as well as the formation of mature interleukin-1β (IL-1β). Treatment with necrostatin-1 (Nec-1), an inhibitor of necroptosis, significantly decreased ischemia-induced increase of p-RIPK1 expression and p-RIPK1+ neurons, which showed protection from brain damage. Meanwhile, Nec-1 reduced RIPK3, MLKL and p-MLKL expression levels and mature IL-1β formation in Nec-1 treated ischemic brains. Our results clearly demonstrated that phosphorylation of RIPK1 at the Ser166 residue was involved in the pathogenesis of necroptosis in the brains after ischemic injury. Nec-1 treatment protected brains against ischemic necroptosis by reducing the activation of RIPK1 and inhibiting its downstream signaling pathways. These results provide direct in vivo evidence that phosphorylated RIPK1 (Ser 166) plays an important role in the initiation of RIPK3/MLKL-dependent necroptosis in the pathogenesis of ischemic stroke in the rodent brain.
Necrostatin-1 Attenuates Renal Ischemia and Reperfusion Injury via Meditation of HIF-1α/mir-26a/TRPC6/PARP1 Signaling
Necroptosis, oxidative stress, and inflammation are major contributors to the pathogenesis of ischemic acute kidney injury. Necrostatin-1 (Nec-1), an inhibitor of the kinase domain of receptor-interacting protein kinase-1 (RIP1), has been reported to regulate renal ischemia and reperfusion (I/R) injury; however, its underlying mechanism of action remains unclear. HK-2 cells were used to create an in vitro I/R model, in which the cells were subjected to hypoxia, followed by 2, 6, and 12 h of reoxygenation. For the in vivo study, a rat model of renal I/R was established in which samples of rat blood serum and kidney tissue were harvested after reperfusion to assess renal function and detect histological changes. Cell viability and necroptosis were analyzed using the Cell Counting Kit (CCK)-8 assay and flow cytometry, respectively. The expression levels of molecules associated with necroptosis, oxidative stress, and inflammation were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to confirm the relevant downstream signaling pathway. We found that pretreatment with Nec-1 significantly decreased hypoxia-inducible factor-1α (HIF-1α) and miR-26a expression, as well as the levels of factors associated with necroptosis (RIP1, RIP3, and Sirtuin-2), oxidative stress (malondialdehyde [MDA], NADP+/NADPH ratio), and inflammation (interleukin [IL]-1β, IL-10, and tumor necrosis factor alpha [TNF-α]) in I/R injury cells and the rat model. However, these effects could be reversed by miR-26a overexpression or TRPC6 knockdown. Mechanistic studies demonstrated that HIF-1α directly binds to the promoter region of miR-26a, and that TRPC6 is a potential target gene for miR-26a. Our findings indicate that Nec-1 can effectively protect against renal I/R injury by inhibiting necroptosis, oxidative stress, and inflammation, and may exert its effects through mediation of the HIF-1α/miR-26a/TRPC6/PARP1 signaling pathway.