Addendum: Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury

Abstract

In this Article 1 , we described a small-molecule inhibitor of necroptosis, termed Necrostatin-1 (Nec-1). Since the original publication, additional data regarding the properties of Nec-1 have been reported, including off-target activity and metabolic stability in mice, that are important in designing in vitro and, especially, in vivo experiments with Nec-1.Teng et al. 2 reported an optimized derivative of Nec-1, termed 7-Cl-O-Nec-1 (66 in ref.2), that was used in ref. 1 to demonstrate the protection in an ischemic brain injury model. This molecule showed higher activity in inhibiting necroptosis in Jurkat cells than Nec-1 (EC 50 = 210 nM versus EC 50 = 490 nM), no nonspecific cytotoxicity at high concentrations (~100 mM) and reasonable pharmacokinetic characteristics following intravenous administration in mice. Degterev et al. 3 subsequently reported that Nec-1 shows limited metabolic stability, which is substantially improved with 7-Cl-O-Nec-1. Takahashi et al. 4 also reported that Nec-1 showed paradoxical toxicity at lower, but not higher, doses in a mouse model of systemic inflammatory stress syndrome (SIRS). No such toxicity was observed with 7-Cl-O-Nec-1. Thus, for in-cell and in vivo experiments, we recommend the use of 7-Cl-O-Nec-1.Muller et al. 5 reported that Nec-1, also known by its chemical name of methylthiohydantoin-tryptophan, is a micromolar inhibitor of indolamine 2,3-deoxygenase (IDO) with EC 50 = 11.4 mM in a cell-based assay. Thus, given the ~20-fold higher activity of Nec-1 in a necroptotic assay, the use of lower concentrations of this molecule could be helpful in distinguishing between inhibition of necroptosis and IDOrelated processes. Another known inhibitor of IDO, 1-methyl-dl-tryptophan, lacks activity against necroptosis as reported by both Degterev et al. 3 and Takahashi et al. 4 Notably, both reports show that optimized 7-Cl-O-Nec-1 lacks activity against IDO. Overall, potential nonspecific toxicity, inhibition of IDO and limited stability of Nec-1 should be taken into account when the molecule is used in vivo, whereas 7-Cl-ONec-1 lacks these liabilities and thus represents a superior choice for in vivo studies.

Membrane Disruption by Very Long Chain Fatty Acids during Necroptosis

“…We recently conducted the first untargeted lipidomic analysis of necroptotic cells and identified lipid participants of this process . Necroptosis is a form of regulated cell death that exhibits phenotypic hallmarks of necrotic cell death including cell and organelle swelling and the permeabilization of cellular membranes upon activation of the death pathway . The permeabilization of the plasma membrane, in particular, results in the release of cytokines and other molecules to the extracellular matrix, which can result in an inflammatory response at the organismal level. , In an effort to identify lipids involved in membrane permeabilization during necroptosis, we analyzed the changes in lipid composition in multiple cell lines during this process.…”

Effects of Modified Melatonin Release on Human Colostrum Neutrophils to Induce Death in the MCF-7 Cell Line

“…Programmed necrosis is cell death independent of caspase and is always triggered as a backup mechanism for apoptosis when caspases are inactivated. In addition, programmed necrosis is accompanied by autophagy, but the specific relationship remains an enigma [ 60 ].…”

Mechanisms of Regulated Cell Death: Current Perspectives

“…In this context, it was demonstrated that this cell death process is dependent on receptor-interacting serine/threonine kinase 1 (RIPK1) and can be inhibited by necrostatin 1 (Nec-1). 62,63 Subsequently, necroptosis was better defined as a RIPK3-dependent molecular cascade promoting regulated necrosis 218 based on the discovery of a close interaction between RIPK1 and its homolog RIPK3 in transducing pronecrotic signals, 47,134,344 and on the identification of multiple RIPK3-dependent but RIPK1-independent instances of regulated necrosis such as in murine cytomegalovirus infections, nitric oxide (NO)-induced pancreatic beta-cell death, and cell death in fibroblasts. 156,282,291,297 This understanding was further enhanced by the discovery of mixed lineage kinase domain-like pseudokinase (MLKL) as the effector of necroptosis (Figs.…”