Necrostatin-1

IC50: Necrostatin-1 (Nec-1), (R)-5-([7-chloro-1H-indol-3-yl]methyl)-3-methylimidazolidine-2,4-dione (Nec-1a) (Figure 1A) (Degterev et al., 2008), exhibited an inhibitory constant (IC50) of 0.32 mM for RIP1 [1].

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. Necrostatin-1, identified as a small-molecule inhibitor of necroptosis, is also a selective allosteric inhibitor of the death domain receptor–associated adaptor kinase RIP1.

In vitro: Previous study indicated that necrostatin-1 was a selective allosteric inhibitor of the death domain receptor–associated adaptor kinase RIP1 in vitro. In this study, RIP1 was found to be the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, two other necrostatins, necrostatin-3 and necrostatin-5, were also shown to target the RIP1 kinase step in the necroptosis pathway, but through different mechanism compared with that of necrostatin-1. The findings established necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis [2].

In vivo: A previous study was designed to investigate the protective effects and mechanisms of Nec-1 in concanavalin A-induced hepatitis in mice. It was found that in Nec-1-treated mice the amelioration in liver functions and histopathological changes and the suppression of inflammatory cytokine production were observed. Western blotting analyses showed that the expression of TNF-α, IFN-γ, IL2, IL6, and RIP1 was significantly reduced in the Nec-1-treated mice, which was further confirmed by immunofluorescence and immunohistochemistry. In addition, autophagosome formation was significantly reduced by Nec-1 treatment. These results indicated that Nec-1 could prevent concanavalin A -induced liver injury via RIP1-related and autophagy-related pathways [3].

Clinical trial: Up to now, Necroptosis is still in the preclinical development stage.

References:
[1] Xie T, Peng W, Liu Y, Yan C, Maki J, Degterev A, Yuan J, Shi Y.  Structural basis of RIP1 inhibition by necrostatins. Structure. 2013;21(3):493-9.
[2] Degterev A, Hitomi J, Germscheid M, Ch'en IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, Hedrick SM, Gerber SA, Lugovskoy A, Yuan J.  Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008;4(5):313-21.
[3] Yingqun Zhou, Weiqi Dai, Chunlei Lin, Fan Wang, Lei He, Miao Shen, Ping Chen, Chenfen Wang, Jie Lu, Ling Xu, Xuanfu Xu, and Chuanyong Guo.  Protective Effects of Necrostatin-1 against Concanavalin A-Induced Acute Hepatic Injury in Mice. Mediators of Inflammation. http://dx.doi.org/10.1155/2013/706156


Necrostatin-1 treatment inhibits osteocyte necroptosis and trabecular deterioration in ovariectomized rats.[Pubmed:27703177]

Sci Rep. 2016 Oct 5;6:33803.

Estrogen (E2) deficiency has been associated with accelerated osteocyte apoptosis. Our previous study showed necroptosis accelerated the loss of osteocytes in E2 deficiency-induced osteoporosis in rats in addition to apoptosis, but the mechanism involved remains. Necroptosis is a caspase-independent form of programmed cell death. In the necroptosis pathway, receptor interaction proteins 1 and 3 (RIP1/3) play vital roles. Necrostatin-1 (Nec-1) has been confirmed to be a specific inhibitor of necroptosis. However, the effect of Nec-1 on postmenopausal osteoporosis remains ambiguous. The aim of this study was to investigate the effect of Nec-1 on osteocytes in ovariectomized (OVX) rats. We found that an increased number of necroptotic osteocytes was related to the production of tumor necrosis factor-alpha (TNF-alpha) in OVX rats. Treatment with Nec-1 significantly decreased RIP1 and RIP3 expression in OVX rats and inhibited osteocyte necroptosis induced by TNF-alpha in vitro. Both E2 and Nec-1 treatment markedly ameliorated trabecular bone deterioration. Nec-1 also significantly elevated the levels of bone formation markers and decreased bone resorption markers. These data suggest that the role of Nec-1 on alleviating bone loss might be associated with Nec-1 restraining TNF-alpha-induced osteocyte necroptosis in rats with E2 deficiency-induced osteoporosis. This process may represent a novel therapeutic strategy for the treatment of postmenopausal osteoporosis.

Induction of Apoptosis in TNF-Treated L929 Cells in the Presence of Necrostatin-1.[Pubmed:27739412]

Int J Mol Sci. 2016 Oct 7;17(10). pii: ijms17101678.

It has been shown that necroptosis-caspase-independent programmed necrotic cell death-can be induced by treatment with tumor necrosis factor (TNF) in the L929 murine fibrosarcoma cell line, even in the absence of a caspase inhibitor. Although it was reported that Necrostatin-1-a specific inhibitor of necroptosis-inhibited TNF-induced necroptosis in L929 cells, it has not been elucidated whether the cells eventually die by apoptosis in the presence of Necrostatin-1. In this paper, induction of apoptosis was demonstrated in TNF-treated L929 cells in the presence of Necrostatin-1. Co-treatment with cycloheximide expedited apoptosis induction in Necrostatin-1/TNF-treated L929 cells: typical apoptotic morphological changes, including membrane blebbing and nuclear fragmentation, induction of caspase-3 activity, proteolytic activation of caspases-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase (PARP) (a well-known substrate of caspase-3) were observed. Moreover, co-treatment with Z-VAD-fmk (a pan-caspase inhibitor) inhibited apoptosis by completely inhibiting caspases, resulting in a shift from apoptosis to necroptosis. In contrast, co-treatment with Z-Asp-CH2-DCB (a caspase inhibitor preferential to caspase-3) inhibited apoptosis without expediting necroptosis. These results indicate that apoptosis can be induced in TNF-treated L929 cells when the cells are protected from necroptosis, and support the notion that partial activation of caspase-8 in the presence of a caspase inhibitor preferential to caspase-3 suppresses both apoptosis and necroptosis.

Effects of necrostatin-1, an inhibitor of necroptosis, and its inactive analogue Nec-1i on basal cardiovascular function.[Pubmed:27875900]

Physiol Res. 2016 Nov 23;65(5):861-865.

Inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIP1) by Necrostatin-1 (Nec-1) alleviates cardiac injury due to prevention of necroptotic cell death. Its inactive analogue Necrostatin-1i (Nec-1i), lacking RIP1 activity, serves as a suitable control. It is unknown if these agents influence the heart function in the absence of damaging stimuli. For this purpose, we measured intraarterial blood pressure (systolic - sBP and diastolic - dBP) and ECG parameters after a bolus administration of Nec-1 and Nec-1i in rats during 30 min. Nec-1, unlike Nec-1i, increased sBP and dBP, as well as heart rate reaching the peak at 20 min. The P wave duration tended to be decreased and the duration of the PR interval was shortened by Nec-1 indicating faster conduction of the impulses through atria to the ventricles. The drugs did not influence the QTc interval duration and no episode of ventricular arrhythmia was observed. In summary, Nec-1 temporarily modulates blood pressure and electrical function of the healthy heart. These effects of Nec-1 are likely due to its off-target action or RIP1 has an important role in the regulation of cardiovascular function independently of its action on the necroptotic pathway.

MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells.[Pubmed:28250973]

Cell Death Discov. 2017 Feb 27;3:17013.

Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson's disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by Necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3'-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson's disease.

Rip1 (receptor-interacting protein kinase 1) mediates necroptosis and contributes to renal ischemia/reperfusion injury.[Pubmed:22237751]

Kidney Int. 2012 Apr;81(8):751-61.

Loss of kidney function in renal ischemia/reperfusion injury is due to programmed cell death, but the contribution of necroptosis, a newly discovered form of programmed necrosis, has not been evaluated. Here, we identified the presence of death receptor-mediated but caspase-independent cell death in murine tubular cells and characterized it as necroptosis by the addition of Necrostatin-1, a highly specific receptor-interacting protein kinase 1 inhibitor. The detection of receptor-interacting protein kinase 1 and 3 in whole-kidney lysates and freshly isolated murine proximal tubules led us to investigate the contribution of necroptosis in a mouse model of renal ischemia/reperfusion injury. Treatment with Necrostatin-1 reduced organ damage and renal failure, even when administered after reperfusion, resulting in a significant survival benefit in a model of lethal renal ischemia/reperfusion injury. Unexpectedly, specific blockade of apoptosis by zVAD, a pan-caspase inhibitor, did not prevent the organ damage or the increase in urea and creatinine in vivo in renal ischemia/reperfusion injury. Thus, necroptosis is present and has functional relevance in the pathophysiological course of ischemic kidney injury and shows the predominance of necroptosis over apoptosis in this setting. Necrostatin-1 may have therapeutic potential to prevent and treat renal ischemia/reperfusion injury.

Identification of RIP1 kinase as a specific cellular target of necrostatins.[Pubmed:18408713]

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

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.

Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.[Pubmed:16408008]

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

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.

Necrostatin-1 (Nec-1) is a potent necroptosis inhibitor with an EC50 of 490 nM in Jurkat cells. Necrostatin-1 inhibits RIP1 kinase (EC50=182 nM). Necrostatin-1 is also an IDO inhibitor.

Necrostatin-1,4311-88-0,Natural Products,TNF-α, buy Necrostatin-1 , Necrostatin-1 supplier , purchase Necrostatin-1 , Necrostatin-1 cost , Necrostatin-1 manufacturer , order Necrostatin-1 , high purity Necrostatin-1