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

N Takahashi, L Duprez, S Grootjans, A Cauwels… - Cell death & …, 2012 - nature.com
N Takahashi, L Duprez, S Grootjans, A Cauwels, W Nerinckx, JB DuHadaway, V Goossens…
Cell death & disease, 2012nature.com
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 …
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.
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