Fig. 2: Structure-based design of R69H-mimicking inhibitors and biochemical evaluation of their potency.

a Strategy for the design of R69H-mimicking inhibitors based on GSK'872. b Chemical structures of inhibitors with their IC₅₀ values determined by the ADP-Glo assay and EC₅₀ values against TNF-α-induced necroptosis in L929 and HT29 cell lines determined by the CCK-8 assay. c–e Representative inhibitory profiles of GSK'872 (magenta solid circle), LK01001 (green solid triangle), LK01002 (blue solid inverted triangle), LK01003 (purple solid diamond), and LK01045 (cyan open inverted triangle) against mRIPK3^KD in the ADP-Glo assay (c), and against TNF-α-induced necroptosis in L929 (d) and HT29 (e) cells. In L929 cells, necroptosis is induced by treatment with mouse TNF-α (20 ng/mL) and zVAD (10 μM). Necroptosis in HT29 cells is induced by treatment with zVAD (20 μM), human TNF-α (40 ng/mL), and Smac mimetic SM-164 (100 nM). f, g Western blot analysis of RIPK3 and MLKL phosphorylation in L929 (f) and HT29 (g) cells with or without RIPK3 inhibitors. Cells are treated with inhibitors at concentrations of 3 and 10 µM, followed by induction of necroptosis for 3 hours. h Overlaid size-exclusion chromatograms (Superdex 200) of mRIPK3^KD with GSK'872 (magenta lines), LK01002 (blue lines), LK01003 (purple lines), and LK01045 (cyan lines), alongside mRIPK3^KD R69H mutant with GSK'872 (green lines). The protein is concentrated to 10 mg/mL and the inhibitors are added to a final compound concentration of 1 mM. The data in panel c are plotted as the mean ± SD of triplicate wells. Three independent experiments are performed for (d, e), each with three technical replicates, and data are shown as mean ± SD. Data shown in panels (f–h) represent one of the three independent experiments, all of which showed similar results. Source data are provided as a Source Data file.