Cell experiment:

Human colorectal adenocarcinoma (HT)-29 cells are incubated with compound 12 (1 mM) or DMSO for 2 h. For the binding competition experiment samples, cells are pre-incubated with 5 mM NSA or 100 nM TC13172 for 2 h, 1 mM compound 12 is then added and incubated for an additional 2 h. The click reaction (Biotin-C2H4-N3 10 mM, TBTA 10 mM, CuSO4 50 mM, Sodium ascorbate 50 mM) is carried out with cell lysates for 2 h. The biotinmodified proteins are enriched and analysed by western blotting using antibodies against flag and GAPDH[1].

TC13172 is a mixed lineage kinase domain-like protein (MLKL) inhibitor with an EC50 value of 2 nM for HT-29 cells.

The anti-necroptosis potency of TC13172 is evaluated in the HT-29 cell line, the EC50 value is 2±0.6 nM. TC13172 has an inhibition potency of 2 nM against cell necroptosis. TC13172 inhibits MLKL by directly binding to Cys-86. TC13172 does not disrupt the phosphorylation of MLKL, but do decrease the level of MLKL in the membrane phase, demonstrating that these MLKL inhibitors block the translocation of MLKL to the cell membrane, thereby protecting cells from necroptosis[1].

[1]. Bo Yan, et al. Discovery of a new class of highly potent necroptosis inhibitors targeting the mixed lineage kinase domain-like protein.ChemCommun (Camb). 2017 Mar 28;53(26):3637-3640.