In vitro activity: Necrosulfonamide is a potent and highly specific and potent necrosis inhibitor which blocks mixed lineage kinase domain-like protein (MLKL). Necrosulfonamide inhibits MLKL-mediated Necrosis by blocking its N-terminal CC domain function. It blocks necrosis downstream of RIP3 activation. Necrosulfonamide has no effect on apoptosis induced by TNF-α plus Smac mimetic in non-RIP3-expressing Panc-1 cells, even at 5 μM concentration. The receptor-interacting serine-threonine kinase 3 (RIP3) is a key signaling molecule in the programmed necrosis (necroptosis) pathway. This pathway plays important roles in a variety of physiological and pathological conditions, including development, tissue damage response, and antiviral immunity.

Kinase Assay: RIP1 and RIP3 were immunoprecipitated with an anti-Flag antibody. The Flag beads were washed three times with kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 0.02% BSA, 150 μM ATP and 1 mM DTT), then incubated with 2 μCi of [32P]γ-ATP at 37°C for 1 hour with the artificial substrate MBP or purified recombinant MLKL. The reaction mixtures were then subjected to SDS-PAGE followed by autoradiography. we report the identification of a small molecule called (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide--hereafter referred to as necrosulfonamide--that specifically blocks necrosis downstream of RIP3 activation. An affinity probe derived from necrosulfonamide and coimmunoprecipitation using anti-RIP3 antibodies both identified the mixed lineage kinase domain-like protein (MLKL) as the interacting target. MLKL was phosphorylated by RIP3 at the threonine 357 and serine 358 residues, and these phosphorylation events were critical for necrosis.

Cell Assay: Treating cells with necrosulfonamide or knocking down MLKL expression arrested necrosis at a specific step at which RIP3 formed discrete punctae in cells.