Necrostatin-1 Analogues: Critical Issues on the Specificity, Activity and In Vivo Use in Experimental Disease Models
DuHadaway, J B
Van Hauwermeiren, F
Prendergast, G C
Vandenabeele, PNote: Order does not necessarily reflect citation order of authors.
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CitationTakahashi, N., L. Duprez, S. Grootjans, A. Cauwels, W. Nerinckx, J. B. DuHadaway, V. 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: e437.
AbstractNecrostatin-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.
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