Fig. 4: Zharp1-163 is a selective inhibitor of RIPK1 kinase activity.

A Binding affinity curve of Zharp1-163 with RIPK1 and RIPK3. B In an in vitro kinase activity assay, recombinant RIPK1 was incubated with Zharp1-163, after which RIPK1 kinase activity was determined by measuring ATP levels via a luciferase reporter assay system. C The kinome selectivity of Zharp1-163 was assessed by establishing a screening model for the evaluation of the selectivity of 81 kinase targets, and the inhibition of Zharp1-163 on the targets was evaluated by ADP-Glo or HTRF methods. Kinases are marked with dots, where the color of each kinase indicates the level of inhibition achieved by Zharp1-163. D Predicted binding conformation of Zharp1-163. E Schematic representation of the interaction patterns between Zharp1-163 and the key residues in the binding pocket of the RIPK1 kinase. F Indicated concentrations of Zharp1-163 did not affect RIPK3 dimerization-induced necroptosis in NIH3T3-RIPK3 cells. NIH3T3-RIPK3 cells were pretreated with the indicated concentrations of Zharp1-163 for 2 h and then treated with AP20187 (100 nM) for 24 h. Cell viability was determined by measuring ATP levels. G Indicated concentrations of Zharp1-163 did not affect MLKL dimerization-induced necroptosis in HeLa-MLKL (1–190) cells. HeLa-MLKL (1–190) cells were pretreated with the indicated concentrations of Zharp1-163 for 2 h and subsequently treated with AP20187 (100 nM) for 24 h. Cell viability was determined by measuring ATP levels. H Viability of IFNβ-primed MEFs at 18 h after stimulation with poly(I:C) plus z-VAD and treatment with Zharp1-163 (10 μM). I Effects of different concentrations of Zharp1-163 on WT or RIPK1 KO HT-29 cells. Cell viability was determined by measuring ATP levels. The data are presented as the mean ± SD of triplicate samples.