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Identification of RIP1 kinase as a specific cellular target of necrostatins

Abstract

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.

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Figure 1: Nec-1 (1) is an inhibitor of RIP1 kinase.
Figure 2: SAR analysis of RIP1 inhibition by necrostatins.
Figure 3: Effect of Ser161 and Phe162 mutations on necroptosis and inhibition by 1.
Figure 4: Molecular model of the RIP1–11 complex.
Figure 5: Necrostatin-3 and necrostatin-5 inhibit RIP1 kinase activity.

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Acknowledgements

The authors thank J. Tschopp (University of Lausanne), I. Verma (Salk Institute) and G. Nunez (University of Michigan) for the generous gifts of the plasmids. This work was supported in part by grants from the National Institute of General Medicine (R01 GM64703), the National Institute on Aging (R37 AG012859) and the National Institute of Neurological Disorders and Stroke (UO1 NS050560) to J.Y., and by funding from the Harvard NeuroDiscovery Center to X.T. and G.D.C. A.D. is a recipient of a National Institute on Aging Mentored Research Scientist Career Development Award and a Massachusetts Medical Foundation Smith Family New Investigator Award.

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A.D., J.H., I.L.C., O.K. and S.M.H. analyzed inhibition of RIP1 kinase and Hsp90 by necrostatins. A.D., M.G. and O.K. performed RIP1 mutagenesis and characterized cellular and in vitro kinase activity of the mutants. A.D. and J.H. generated samples for mass spectrometry analysis, which was carried out by S.A.G. A.L. and G.W. performed molecular modeling of RIP1. X.T., G.D.C. and C.Y. designed and synthesized the necrostatin analogs. D.A. prepared the baculoviral RIP1 expression vector and optimized expression conditions. The project was designed and coordinated by A.D. and J.Y. A.D. and J.Y. wrote the manuscript. All authors discussed the results and contributed to discussion of relevant experimental results in the manuscript.

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Correspondence to Alexei Degterev or Junying Yuan.

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Harvard Medical School and Brigham and Women's Hospital have filed patent applications including results from this study.

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Degterev, A., Hitomi, J., Germscheid, M. et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 4, 313–321 (2008). https://doi.org/10.1038/nchembio.83

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