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

Abstract

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."

References

  1. 1

    Degterev, A., Boyce, M. & Yuan, J. A decade of caspases. Oncogene 22, 8543–8567 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Wallach, D. et al. Tumor necrosis factor receptor and Fas signaling mechanisms. Annu. Rev. Immunol. 17, 331–367 (1999).

    CAS  Article  Google Scholar 

  3. 3

    Vercammen, D. et al. Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways. J. Exp. Med. 188, 919–930 (1998).

    CAS  Article  Google Scholar 

  4. 4

    Matsumura, H. et al. Necrotic death pathway in Fas receptor signaling. J. Cell Biol. 151, 1247–1256 (2000).

    CAS  Article  Google Scholar 

  5. 5

    Schulze-Osthoff, K., Krammer, P.H. & Droge, W. Divergent signalling via APO-1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death. EMBO J. 13, 4587–4596 (1994).

    CAS  Article  Google Scholar 

  6. 6

    Holler, N. et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat. Immunol. 1, 489–495 (2000).

    CAS  Article  Google Scholar 

  7. 7

    Chan, F.K. et al. A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. J. Biol. Chem. 278, 51613–51621 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Kawahara, A., Ohsawa, Y., Matsumura, H., Uchiyama, Y. & Nagata, S. Caspase-independent cell killing by Fas-associated protein with death domain. J. Cell Biol. 143, 1353–1360 (1998).

    CAS  Article  Google Scholar 

  9. 9

    Khwaja, A. & Tatton, L. Resistance to the cytotoxic effects of tumor necrosis factor alpha can be overcome by inhibition of a FADD/caspase-dependent signaling pathway. J. Biol. Chem. 274, 36817–36823 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Lin, Y. et al. Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation. J. Biol. Chem. 279, 10822–10828 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Ruemmele, F.M., Dionne, S., Levy, E. & Seidman, E.G. TNFα-induced IEC-6 cell apoptosis requires activation of ICE caspases whereas complete inhibition of the caspase cascade leads to necrotic cell death. Biochem. Biophys. Res. Commun. 260, 159–166 (1999).

    CAS  Article  Google Scholar 

  12. 12

    Wilson, C.A. & Browning, J.L. Death of HT29 adenocarcinoma cells induced by TNF family receptor activation is caspase-independent and displays features of both apoptosis and necrosis. Cell Death Differ. 9, 1321–1333 (2002).

    CAS  Article  Google Scholar 

  13. 13

    Nieminen, A.L. Apoptosis and necrosis in health and disease: role of mitochondria. Int. Rev. Cytol. 224, 29–55 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Lo, E.H., Dalkara, T. & Moskowitz, M.A. Mechanisms, challenges and opportunities in stroke. Nat. Rev. Neurosci. 4, 399–415 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Li, M. & Beg, A.A. Induction of necrotic-like cell death by tumor necrosis factor α and caspase inhibitors: novel mechanism for killing virus-infected cells. J. Virol. 74, 7470–7477 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Kain, S.R. & Ma, J.T. Early detection of apoptosis with annexin V-enhanced green fluorescent protein. Methods Enzymol. 302, 38–43 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Fiers, W., Beyaert, R., Declercq, W. & Vandenabeele, P. More than one way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene 18, 7719–7730 (1999).

    CAS  Article  Google Scholar 

  18. 18

    Klionsky, D.J. & Emr, S.D. Autophagy as a regulated pathway of cellular degradation. Science 290, 1717–1721 (2000).

    CAS  Article  Google Scholar 

  19. 19

    Gozuacik, D. & Kimchi, A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23, 2891–2906 (2004).

    CAS  Article  Google Scholar 

  20. 20

    Kabeya, Y. et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–5728 (2000).

    CAS  Article  Google Scholar 

  21. 21

    Kabeya, Y. et al. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J. Cell Sci. 117, 2805–2812 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Kuma, A. et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Yu, L. et al. Regulation of an ATG7-beclin-1 program of autophagic cell death by caspase-8. Science 304, 1500–1502 (2004).

    CAS  Article  Google Scholar 

  24. 24

    Gwag, B.J., Lobner, D., Koh, J.Y., Wie, M.B. & Choi, D.W. Blockade of glutamate receptors unmasks neuronal apoptosis after oxygen-glucose deprivation in vitro . Neuroscience 68, 615–619 (1995).

    CAS  Article  Google Scholar 

  25. 25

    Rosenbaum, D.M. et al. Fas (CD95/APO-1) plays a role in the pathophysiology of focal cerebral ischemia. J. Neurosci. Res. 61, 686–692 (2000).

    CAS  Article  Google Scholar 

  26. 26

    Martin-Villalba, A. et al. CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons. J. Neurosci. 19, 3809–3817 (1999).

    CAS  Article  Google Scholar 

  27. 27

    Martin-Villalba, A. et al. Therapeutic neutralization of CD95-ligand and TNF attenuates brain damage in stroke. Cell Death Differ. 8, 679–686 (2001).

    CAS  Article  Google Scholar 

  28. 28

    Endres, M. et al. Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family. J. Cereb. Blood Flow Metab. 18, 238–247 (1998).

    CAS  Article  Google Scholar 

  29. 29

    Kitanaka, C. & Kuchino, Y. Caspase-independent programmed cell death with necrotic morphology. Cell Death Differ. 6, 508–515 (1999).

    CAS  Article  Google Scholar 

  30. 30

    Lum, J.J. et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 120, 237–248 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Richard Green, A., Odergren, T. & Ashwood, T. Animal models of stroke: do they have value for discovering neuroprotective agents? Trends Pharmacol. Sci. 24, 402–408 (2003).

    CAS  Article  Google Scholar 

  32. 32

    Takahashi, K. & Greenberg, J.H. The effect of reperfusion on neuroprotection using an inhibitor of poly(ADP-ribose) polymerase. Neuroreport 10, 2017–2022 (1999).

    CAS  Article  Google Scholar 

  33. 33

    Abdelkarim, G.E. et al. Protective effects of PJ34, a novel, potent inhibitor of poly(ADP-ribose) polymerase (PARP) in in vitro and in vivo models of stroke. Int. J. Mol. Med. 7, 255–260 (2001).

    CAS  PubMed  Google Scholar 

  34. 34

    Matsuyama, T. et al. Fas antigen mRNA induction in postischemic murine brain. Brain Res. 657, 342–346 (1994).

    CAS  Article  Google Scholar 

  35. 35

    Eguchi, Y., Shimizu, S. & Tsujimoto, Y. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res. 57, 1835–1840 (1997).

    CAS  PubMed  Google Scholar 

  36. 36

    McCully, J.D., Wakiyama, H., Hsieh, Y.J., Jones, M. & Levitsky, S. Differential contribution of necrosis and apoptosis in myocardial ischemia-reperfusion injury. Am. J. Physiol. Heart Circ. Physiol. 286, H1923–H1935 (2004).

    CAS  Article  Google Scholar 

  37. 37

    Yuan, J., Lipinski, M. & Degterev, A. Diversity in the mechanisms of neuronal cell death. Neuron 40, 401–413 (2003).

    CAS  Article  Google Scholar 

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Acknowledgements

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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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). https://doi.org/10.1038/nchembio711

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