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

TLDR

Necrostatin‑1 is a widely used inhibitor to probe RIPK1‑mediated cell death and inflammation in disease models. The study compared three Nec‑1 analogues—active Nec‑1, inactive Nec‑1i, and more stable Nec‑1s—to assess their biochemical and in vivo properties. Nec‑1, but not Nec‑1s, also inhibits IDO, and although Nec‑1i is far less potent against RIPK1 in vitro, it matches Nec‑1 and Nec‑1s in mouse necroptosis and high‑dose TNF‑induced mortality protection; however, low doses of Nec‑1 or Nec‑1i sensitize mice to TNF, whereas Nec‑1s does not, underscoring Nec‑1s as the preferred specific RIPK1 inhibitor and highlighting the need to reconsider past Nec‑1‑based studies.

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.