Kinase experiment:

Evaluation of necroptosis inhibitory activity is performed using an FADD-deficient variant of human Jurkat T cells treated for 24 h with TNF-α. Under these conditions the cells underwent necroptosis (the DMSO control had ~40% viability), which is inhibited by Necrostatin 2 (EC50=0.21±0.2 μM) as a positive control. For EC50 value determinations, cells are treated with 10 ng/mL human TNF-α in the presence of increasing concentrations of test compounds (0.029, 0.058, 0.12, 0.23, 0.46, 0.93, 1.9, 3.7, 11.1, 33, and 100 μM) in duplicate for 24 h followed by ATP-based viability assessment. EC50 values±SD are determined from at least two independent experiments[2].

Cell experiment:

L929 cells (100000 cells/mL, 100 μL/well in a 96-well plate) are treated with 10 ng/mL human TNF-α or 100 μM zVAD.fmk in the presence of DMSO (control), 30 μM Necrostatin 2, or 8for 24 h at 37℃ in a humidified incubator with 5% CO2 followed by ATP-based viability assessment as described in the previous experiment. Stock solutions (30 mM) in DMSO are initially prepared and then diluted with DMSO to give testing solutions. Each sample is done in duplicate. The final DMSO concentration is 0.5%. Cell viability values are adjusted to account for nonspecific toxicity, which in most cases is<10%. The reported cell viability values (%)±SD are determined from two independent experiments[2].

Necrostatin 2 is a potent necroptosis inhibitor. EC50 for inhibition of necroptosis in FADD-deficient Jurkat T cells treated with TNF-α is 0.05 μM. Necrostatin 2 is also a RIPK1 inhibitor.

Evaluation of necroptosis inhibitory activity is performed using a FADD-deficient variant of human Jurkat T cells treated with TNF-α. Utilizing these conditions the cells efficiently undergo necroptosis, which is completely and selectively inhibited by Necrostatin 2 (EC50=50 nM). Necrostatin 2 shows activity in a broad range of necroptosis cellular systems[1]. Necrostatin 2 at 30 μM completely protects L929 cells from TNF-α-induced necroptosis. In addition to TNF-α, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD.fmk) has also been found to induce necrosis in L929 cells, which is efficiently inhibited by Necrostatin 2[2]. EC50 for inhibition of necroptosis in FADD-deficient Jurkat T cells treated with TNF-α is 0.05 μM[3].

References:
[1]. Teng X, et al. Structure-activity relationship study of [1,2,3]thiadiazole necroptosis inhibitors. Bioorg Med Chem Lett. 2007 Dec 15;17(24):6836-40.
[2]. Jagtap PG, et al. Structure-activity relationship study of tricyclic necroptosis inhibitors. J Med Chem. 2007 Apr 19;50(8):1886-95.
[3]. Teng X, et al. Structure-activity relationship study of novel necroptosis inhibitors. Bioorg Med Chem Lett. 2005 Nov 15;15(22):5039-44.
[4]. Takahashi N, et al. Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death Dis. 2012 Nov 29;3:e437.