Necrostatin-1 is a specific RIP1 inhibitor and inhibits TNF-α-induced necroptosis (EC50: 490 nM in Jurkat cells).

Necrostatin-1 efficiently inhibited kinase activity of overexpressed protein in a dose-dependent manner. Similar to the results with overexpressed protein, Necrostatin-1 efficiently suppressed endogenous RIP1 kinase activity [1]. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells [2].

Necrostatin-1 (Nec-1) prevented osmotic nephrosis and contrast-induced AKI (CIAKI), whereas an inactive Nec-1 derivate (Nec-1i) or the pan-caspase inhibitor zVAD did not. In addition, Nec-1 prevented RCM-induced dilation of peritubular capillaries [3].

The assay was performed essentially as described. 293T cells were transfected with pcDNA3-FLAG-RIP1 vector, vectors encoding RIP1 mutant proteins or pcDNA3-RIP2-Myc and pcDNA3-FLAG-RIP3 vectors using standard Ca3(PO4)2 precipitation procedure. Culture medium was replaced 6 h after the transfection and cells were lysed 48 h later in the TL buffer consisting of 1% Triton X-100, 150 mM NaCI, 20 mM HEPES, pH 7.3, 5 mM EDTA, 5 mM NaF, 0.2 mM NaVO3 and complete protease inhibitor cocktail. Immunoprecipitation was carried out for 16 h at 4 °C using anti-FLAG M2 agarose beads, followed by three washes with TL buffer and two washes with 20 mM HEPES, pH 7.3. Beads were incubated in 15 μl of the reaction buffer containing 20 mM HEPES, pH 7.3, 10 mM MnCl2 and 10 mM MgCl2 for 15 min at 23–25 °C in the presence of different concentrations of necrostatins. For these assays, compound stocks (in DMSO) were diluted to appropriate concentrations in DMSO before the addition to the reactions to maintain final concentration of DMSO for all samples at 3%. Kinase reaction was initiated by addition of 10 μM cold ATP and 1 mCi of [γ-32P] ATP, and reactions were carried out for 30 min at 30 °C. Reactions were stopped by boiling in SDS-PAGE sample buffer and subjected to 8% SDS-PAGE. RIP1 band was visualized by analysis in a Storm 8200 Phosphorimager. Similar protocol was used for endogenous RIP1 kinase reactions, except mouse monoclonal RIP1 antibody and protein magnetic beads or rabbit RIP1 antibody-coupled agarose beads were used. For recombinant baculovirally expressed RIP1, protein was expressed in Sf9 cells according to manufacturer's instructions and purified using glutathione-sepharose beads. Protein was eluted in 50 mM Tris-HCl, pH 8.0 supplemented with 10 mM reduced glutathione, and eluted protein was used in the kinase reactions, supplemented with 5 × kinase reaction buffer (100 mM HEPES, pH 7.3, 50 mM MnCl2, 50 mM MgCl2, 50 μM cold ATP and 5 μCi of [γ-32P]ATP) [1].

Determination of EC50 was performed in FADD-deficient Jurkat cells treated with human TNFα as previously described. Briefly, cells were seeded into 96-well plates and treated with a range of necrostatin concentrations (30 nM to 100 μM, 11 dose points) in the presence and absence of 10 ng ml–1 human TNFα for 24 h. For these and all other cellular assays, compound stocks (in DMSO) were diluted to appropriate concentrations in DMSO before addition to the cells to maintain final concentration of DMSO for all samples at 0.5%. Cell viability was determined using CellTiter-Glo luminescent cell viability assay. Ratio of luminescence in compound and TNF-treated wells to compound-treated, TNF-untreated wells was calculated (viability, %) [1].

24 hours after reperfusion, mice received intravenous application of 200 μl PBS or RCM via the tail vein. A single dose of zVAD (10 mg/kg body weight) or Nec-1 (1.65 mg/kg body weight) was applied intraperitoneally 15 min. before RCM-injection. To test the activity of zVAD, we applied zVAD from the same byculture to anti-Fas-treated Jurkat cells to assure its quality before mice were treated with this compound. Mice were harvested another 24 hours after RCM-application (48 hours after reperfusion). Blood samples were obtained from retroorbital bleeding and serum levels of urea and creatinine 5 were determined according to clinical standards in the central laboratory of the University Hospital Schleswig-Holstein, Campus Kiel, Germany, employing an enzymatic ultraviolettest for urea and an enzymatic peroxidase-dependent test for creatinine according to the manufacturer's instructions. Kidneys were conserved for histology. In addition to the demonstrated experiments, we compared the PBS group to mice that only received IRI without 200 μl of PBS and detected no changes in serum concentrations of urea and creatinine or histologically [3].

4311-88-0

C13H13N3OS

259.33

Necrostatin 1;Nec-1;Necrostatin-1

[1]Linkermann A, 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. [2]Wang S, Li F, Qiao R, et al. Arginine-Rich Manganese Silicate Nanobubbles as a Ferroptosis-Inducing Agent for Tumor-Targeted Theranostics[J]. ACS nano. 2018 Dec 26;12(12):12380-12392. [3]Yao X, Ma S, Peng S, et al. Zwitterionic Polymer Coating of Sulfur Dioxide‐Releasing Nanosystem Augments Tumor Accumulation and Treatment Efficacy[J]. Advanced Healthcare Materials. 2020, 9(5): 1901582. [4]Degterev A, et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008 May;4(5):313-21. [5]hang C, Liu Z, Zhang Y, et al. Z“Iron free” zinc oxide nanoparticles with ion-leaking properties disrupt intracellular ROS and iron homeostasis to induce ferroptosis[J]. Cell Death & Disease. 2020, 11(3): 1-15. [6]Zhang Y, Fan B Y, Pang Y L, et al. Neuroprotective effect of deferoxamine on erastin-induced ferroptosis in primary cortical neurons[J]. Neural regeneration research. 2020, 15(8): 1539. [7]Huang C, et al. Shikonin kills glioma cells through necroptosis mediated by RIP-1. PLoS One. 2013 Jun 28;8(6):e66326. [8]Yan B, Ai Y, Sun Q, et al. Membrane Damage during Ferroptosis Is Caused by Oxidation of Phospholipids Catalyzed by the Oxidoreductases POR and CYB5R1[J]. Molecular Cell. 2020 [9]Wu H, Cheng X, Huang F, et al. Aprepitant Sensitizes Acute Myeloid Leukemia Cells to the Cytotoxic Effects of Cytosine Arabinoside in vitro and in vivo[J]. Drug Design. Development and Therapy. 2020, 14: 2413.

[1]Yan B, Ai Y, Sun Q, et al. Membrane Damage during Ferroptosis Is Caused by Oxidation of Phospholipids Catalyzed by the Oxidoreductases POR and CYB5R1. Molecular Cell. 2020 [2]Yang K H, Tang J Y, Chen Y N, et al. Nepenthes Extract Induces Selective Killing, Necrosis, and Apoptosis in Oral Cancer Cells. Journal of Personalized Medicine. 2021, 11(9): 871. [3]Wu X, Lu Y, Qin X. Combination of Compound Kushen Injection and cisplatin shows synergistic antitumor activity in p53-R273H/P309S mutant colorectal cancer cells through inducing apoptosis. Journal of Ethnopharmacology. 2021: 114690. [4]D’Onofrio N, Martino E, Balestrieri A, et al. Diet‐derived ergothioneine induces necroptosis in colorectal cancer cells by activating the SIRT3/MLKL pathway. FEBS letters. 2022 [5]Du S, Zeng F, Sun H, et al. Prognostic and therapeutic significance of a novel ferroptosis related signature in colorectal cancer patients. Bioengineered. 2022, 13(2): 2498-2512. [6]Wu H, Cheng X, Huang F, et al. Aprepitant Sensitizes Acute Myeloid Leukemia Cells to the Cytotoxic Effects of Cytosine Arabinoside in vitro and in vivo. Development and Therapy. 2020, 14: 2413 [7]Su G, Yang W, Wang S, et al. SIRT1-autophagy axis inhibits excess iron-induced ferroptosis of foam cells and subsequently increases IL-1Β and IL-18. Biochemical and Biophysical Research Communications. 2021, 561: 33-39. [8]Zhang Y, Fan B Y, Pang Y L, et al. Neuroprotective effect of deferoxamine on erastininduced ferroptosis in primary cortical neurons. Neural Regeneration Research. 2020, 15(8): 1539 [9]Wang S, Li F, Qiao R, et al. Arginine-Rich Manganese Silicate Nanobubbles as a Ferroptosis-Inducing Agent for Tumor-Targeted Theranostics. ACS nano. 2018 Dec 26;12(12):12380-12392. [10]Yang W, Liu S, Li Y, et al. Pyridoxine induces monocyte-macrophages death as specific treatment of acute myeloid leukemia. Cancer Letters. 2020

DMSO:40 mg/mL (154.24 mM)
Powder: -20°C for 3 years
In solvent: -80°C for 2 years