N Takahashi, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biomedical Molecular Biology, Ghent University
L Duprez, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biomedical Molecular Biology, Ghent University
S Grootjans, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biomedical Molecular Biology, Ghent University
A Cauwels, Department of Biomedical Molecular Biology, Ghent University
W Nerinckx, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biochemistry and Microbiology, Ghent University
J B DuHadaway, Lankenau Institute for Medical ResearchFollow
V Goossens, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biomedical Molecular Biology, Ghent University
R Roelandt, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biomedical Molecular Biology, Ghent University
F Van Hauwermeiren, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biomedical Molecular Biology, Ghent University
C Libert, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biomedical Molecular Biology, Ghent University
W Declercq, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biomedical Molecular Biology, Ghent University
N Callewaert, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biochemistry and Microbiology, Ghent University
G C Prendergast, Lankenau Institute for Medical Research; Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University; Kimmel Cancer Center, Thomas Jefferson University
A Degterev, Department of Biochemistry, School of Medicine, Tufts University
J Yuan, Department of Cell Biology, Harvard Medical School
P Vandenabeele, Department for Molecular Biomedical Research, VIB, Ghent University; Department of Biomedical Molecular Biology, Ghent University

Document Type

Article

Publication Date

11-29-2012

Comments

This article has been peer reviewed. It was published in: Cell Death and Disease.

Volume 3, Issue 11, November 2012, Article number e437.

The published version is available at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542611/. DOI: 10.1038/cddis.2012.176 .

Copyright © 2012 Macmillan Publishers Limited.

Abstract

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.

PubMed ID

23190609

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