Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury

An Addendum to this article was published on 15 February 2013

A Corrigendum to this article was published on 01 September 2005

This article has been updated


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.

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Figure 1: Identification of Nec-1 as a necroptosis inhibitor.
Figure 2: Efficient inhibition of all manifestations of necroptosis by Nec-1.
Figure 3: Specificity of Nec-1.
Figure 4: Roles of oxidative stress and autophagy in necroptosis.
Figure 5: Nec-1 inhibits RIP kinase–induced necroptosis.
Figure 6: Inhibition of in vivo ischemic injury by Nec-1.

Change history

  • 31 January 2013

    In this Article1, we described a small-molecule inhibitor of necroptosis, termed Necrostatin-1 (Nec-1). Since the original publication, additional data regarding the properties of Nec-1 have been reported, including off-target activity and metabolic stability in mice, that are important in designing in vitro and, especially, in vivo experiments with Nec-1. Teng et al.2 reported an optimized derivative of Nec-1, termed 7-Cl-O-Nec-1 (66 in ref. 2), that was used in ref. 1 to demonstrate the protection in an ischemic brain injury model. This molecule showed higher activity in inhibiting necroptosis in Jurkat cells than Nec-1 (EC50 = 210 nM versus EC50 = 490 nM), no nonspecific cytotoxicity at high concentrations (~100 μM) and reasonable pharmacokinetic characteristics following intravenous administration in mice. Degterev et al.3 subsequently reported that Nec-1 shows limited metabolic stability, which is substantially improved with 7-Cl-O-Nec-1. Takahashi et al.4 also reported that Nec-1 showed paradoxical toxicity at lower, but not higher, doses in a mouse model of systemic inflammatory stress syndrome (SIRS). No such toxicity was observed with 7-Cl-O-Nec-1. Thus, for in-cell and in vivo experiments, we recommend the use of 7-Cl-O-Nec-1. Muller et al.5 reported that Nec-1, also known by its chemical name of methylthiohydantoin-tryptophan, is a micromolar inhibitor of indolamine 2,3-deoxygenase (IDO) with EC50 = 11.4 μM in a cell-based assay. Thus, given the ~20-fold higher activity of Nec-1 in a necroptotic assay, the use of lower concentrations of this molecule could be helpful in distinguishing between inhibition of necroptosis and IDO-related processes. Another known inhibitor of IDO, 1-methyl-DL-tryptophan, lacks activity against necroptosis as reported by both Degterev et al.3 and Takahashi et al.4 Notably, both reports show that optimized 7-Cl-O-Nec-1 lacks activity against IDO. Overall, potential nonspecific toxicity, inhibition of IDO and limited stability of Nec-1 should be taken into account when the molecule is used in vivo, whereas 7-Cl-O-Nec-1 lacks these liabilities and thus represents a superior choice for in vivo studies.

  • 01 September 2005

    In the legend to Supplementary Figure 1 online, the second sentence in panel d should read "FADD-deficient Jurkat cells were treated with indicated concentrations (on log scale, in M) of Nec-1 (1) and Nec-1i (2) for 24 h."


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This work was supported in part by grants from the US National Institute of General Medicine (R01 GM64703) and National Institute on Aging (R37 AG012859) to J.Y., the National Institute of Neurological Disorders and Stroke (R01 NS37141-08) to M.M. and J.Y., and funding from the Harvard Center for Neurodegeneration and Repair to G.D.C. A.D. is a recipient of a National Institute on Aging Mentored Research Scientist Career Development Award and an American Health Assistance Foundation Pilot Award. We thank X. Teng for help in preparing compounds for animal testing; M. Lipinski and R. Olea-Sanchez for critical reading of the manuscript; C. Ayata for helpful suggestions with MCAO experiments; and G. Nunez, T. Jacks, J. Blenis and T. Yoshimori for providing RIP constructs, pSRP vector and mutant Jurkat cells, and anti-LC3 antibody, respectively.

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Correspondence to Junying Yuan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Nec-1(1) specifically and efficiently inhibits apoptosis. (PDF 123 kb)

Supplementary Fig. 2

Nec-1 inhibits multiple necroptosis-associated morphological changes. (PDF 132 kb)

Supplementary Fig. 3

Additive effects of 7-Cl-Nec-1(3) and zVAD.fmk on inhibition of ischemia-induced neuronal death in vivo. (PDF 12 kb)

Supplementary Methods (PDF 50 kb)

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Degterev, A., Huang, Z., Boyce, M. et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 1, 112–119 (2005).

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