(E)-Necrosulfonamide ((E)-Necrosulfonamide) is an inhibitor of necroptosis. Blocks mixed lineage kinase domain-like protein (MLKL), a critical substrate of receptor-interacting serine-threonine kinase 3 (RIP3) during necrosis. Prevents MLKL-RIP1-RIP3 necrosome complex from interacting with downstream necrosis effectors. Binds and inhibits gasdermin D. Inhibits pyroptosis.

Necrosulfonamide 通过阻断其N-terminal CC领域的功能，抑制 MLKL介导的坏死。它在RIP3激活之后阻断坏死过程。在不表达RIP3的Panc-1细胞中，即便是5 μM浓度，Necrosulfonamide 对由TNF-α加Smac模拟剂诱导的凋亡也没有影响。Necrosulfonamide 在人类细胞中有效地阻止坏死，但在小鼠细胞中无效。Necrosulfonamide 物种特异性的原因是，在人类 MLKL中，被Necrosulfonamide 共价修饰的第86位的半胱氨酸，在小鼠 MLKL 中被色氨酸取代[mixed lineage kinase domain-like protein][2]。

PRMT Biochemical Assays: A scintillation proximity assay (SPA) is used for assessing the effect of test compounds on inhibiting the methyl transfer reaction catalyzed by PRMTs. In brief, the tritiated S-adenosyl-L-methionine (3H-SAM) is used as the donor of methyl group. The (3H) methylated biotin labeled peptide is captured in a streptavidin/scintillant-coated microplate, which brings the incorporated 3H-methyl and the scintillant to close proximity resulting in light emission that is quantified by tracing the radioactivity signal (counts per minute) as measured by a TopCount NXT Microplate Scintillation and Luminescence Counter. When necessary, nontritiated SAM is used to supplement the reactions. The IC50 values are determined under balanced conditions at Km concentrations of both substrate and cofactor by titration of test compounds in the reaction mixture.

Necrosis inhibitors induce diverse effects on MLKL phosphorylation. HT-29 cells are treated with T/S/Z with or without necrosis inhibitors for 12 hr or 8 hr. The number of dead cells is determined by measuring released protease activity in culture medium. The whole-cell extracts are prepared and analyzed by western blotting. The final concentrations of 10 μM necrostatin-1 or 1 μM necrosulfonamide are used to block necrosis. (Only for Reference)