Necrosulfonamide ((E)-Necrosulfonamide) is a necroptosis inhibitor that targets MLKL and is selective. Necrosulfonamide prevents the MLKL-RIP1-RIP3 necrosomal complex from interacting with its downstream effectors.

PI4Kα:8.0 (pIC50), PI3Kγ:5.9 (pIC50), PI4Kβ:5.9 (pIC50), PI3Kδ:6.4 (pIC50), PI4KA:8.0 (pIC50), PI4KB:5.9 (pIC50), PI3Kα:5.8 (pIC50), PI3Kβ:5.9 (pIC50)

METHODS: FLAG-tagged RIP3-HT-29 cells were treated with Necrosulfonamide for 6 h, and the interaction between RIP1 and RIP3 was examined using a FLAG-tagged RIP3 pull-down assay.
RESULTS: Necrosulfonamide, an inhibitor of necrosis downstream of RIP3, did not block the necrosis-induced interaction between RIP1 and RIP3. [1]

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)