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Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models

Nozomi Takahashi (UGent) , Linde Duprez, Sasker Grootjans (UGent) , Anje Cauwels (UGent) , Wim Nerinckx (UGent) , JB DuHadaway, Vera Goossens (UGent) , Ria Roelandt (UGent) , Filip Van Hauwermeiren (UGent) , Claude Libert (UGent) , et al.
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Ghent researchers on unfolded proteins in inflammatory disease (GROUP-ID)
Project
Ghent researchers on unfolded proteins in inflammatory disease (GROUP-ID)
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-thiohydantointryptophan, 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 similar to 100 x 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.
Keywords
SIRS, necrostatin, sepsis, HUMAN INDOLEAMINE 2, 3-DIOXYGENASE, NECROTIC CELL-DEATH, REGULATORY T-CELLS, TNF-ALPHA, PROTEIN-KINASE, RIP1 KINASE, NECROSIS, NECROPTOSIS, RECEPTOR, INFLAMMATION, IDO, RIPK1, necroptosis

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Citation

Please use this url to cite or link to this publication:

Chicago
Takahashi, Nozomi, Linde Duprez, Sasker Grootjans, Anje Cauwels, Wim Nerinckx, JB DuHadaway, Vera Goossens, et al. 2012. “Necrostatin-1 Analogues: Critical Issues on the Specificity, Activity and in Vivo Use in Experimental Disease Models.” Cell Death & Disease 3.
APA
Takahashi, Nozomi, Duprez, L., Grootjans, S., Cauwels, A., Nerinckx, W., DuHadaway, J., Goossens, V., et al. (2012). Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. CELL DEATH & DISEASE, 3.
Vancouver
1.
Takahashi N, Duprez L, Grootjans S, Cauwels A, Nerinckx W, DuHadaway J, et al. Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. CELL DEATH & DISEASE. 2012;3.
MLA
Takahashi, Nozomi, Linde Duprez, Sasker Grootjans, et al. “Necrostatin-1 Analogues: Critical Issues on the Specificity, Activity and in Vivo Use in Experimental Disease Models.” CELL DEATH & DISEASE 3 (2012): n. pag. Print.
@article{3083167,
  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-thiohydantointryptophan, 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 similar to 100 x 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.},
  articleno    = {e437},
  author       = {Takahashi, Nozomi and Duprez, Linde and Grootjans, Sasker and Cauwels, Anje and Nerinckx, Wim and DuHadaway, JB and Goossens, Vera and Roelandt, Ria and Van Hauwermeiren, Filip and Libert, Claude and Declercq, Wim and Callewaert, Nico and Prendergast, GC and Degterev, A and Yuan, J and Vandenabeele, Peter},
  issn         = {2041-4889},
  journal      = {CELL DEATH \& DISEASE},
  language     = {eng},
  pages        = {10},
  title        = {Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models},
  url          = {http://dx.doi.org/10.1038/cddis.2012.176},
  volume       = {3},
  year         = {2012},
}

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