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实验参考方法

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.

产品描述

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.

Chemical Properties

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	For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.
Shipping Condition	Evaluation sample solution : ship with blue ice All other available size: ship with RT , or blue ice upon request

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Research Update

Necrostatin-1 and necroptosis inhibition: Pathophysiology and therapeutic implications

Pharmacol Res2021 Jan;163:105297.PMID: 33181319DOI: 10.1016/j.phrs.2020.105297

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

Int J Mol Med2018 May;41(5):2565-2572.PMID: 29436688DOI: 10.3892/ijmm.2018.3466

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 Attenuates Renal Ischemia and Reperfusion Injury via Meditation of HIF-1α/mir-26a/TRPC6/PARP1 Signaling

Mol Ther Nucleic Acids2019 Sep 6;17:701-713.PMID: 31422287DOI: 10.1016/j.omtn.2019.06.025

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.

Necrostatin-1 Prevents Necroptosis in Brains after Ischemic Stroke via Inhibition of RIPK1-Mediated RIPK3/MLKL Signaling

Aging Dis2019 Aug 1;10(4):807-817.PMID: 31440386DOI: 10.14336/AD.2018.0728

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 Prevents Ferroptosis in a RIPK1- and IDO-Independent Manner in Hepatocellular Carcinoma

Antioxidants (Basel)2021 Aug 25;10(9):1347.PMID: 34572979DOI: 10.3390/antiox10091347

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.

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