title: Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models.
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.
authors.affiliation: Department for Molecular Biomedical Research, VIB, Ghent University, Ghent, Belgium.
authors.forename: N
authors.initials: N
authors.lastname: Takahashi
authors.forename: L
authors.initials: L
authors.lastname: Duprez
authors.forename: S
authors.initials: S
authors.lastname: Grootjans
authors.forename: A
authors.initials: A
authors.lastname: Cauwels
authors.forename: W
authors.initials: W
authors.lastname: Nerinckx
authors.forename: J B
authors.initials: JB
authors.lastname: DuHadaway
authors.forename: V
authors.initials: V
authors.lastname: Goossens
authors.forename: R
authors.initials: R
authors.lastname: Roelandt
authors.forename: F
authors.initials: F
authors.lastname: Van Hauwermeiren
authors.forename: C
authors.initials: C
authors.lastname: Libert
authors.forename: W
authors.initials: W
authors.lastname: Declercq
authors.forename: N
authors.initials: N
authors.lastname: Callewaert
authors.forename: G C
authors.initials: GC
authors.lastname: Prendergast
authors.forename: A
authors.initials: A
authors.lastname: Degterev
authors.forename: J
authors.initials: J
authors.lastname: Yuan
authors.forename: P
authors.initials: P
authors.lastname: Vandenabeele
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D004358:Drug Therapy; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000737:chemistry; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000008:administration & dosage* / Q000737:chemistry*; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000037:antagonists & inhibitors; D007211:Indoles / Q000008:administration & dosage* / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D015394:Molecular Structure; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D013045:Species Specificity; D018746:Systemic Inflammatory Response Syndrome / Q000188:drug therapy*
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007211:Indoles; C507699:necrostatin-1; D053422:Receptor-Interacting Protein Serine-Threonine Kinases