Fig. 4. AZD4547 is a highly selective RIPK1 inhibitor.

a In vitro kinase activity was performed with recombinant RIPK1, RIPK2 or RIPK3. b The IC₅₀ values of AZD4547 in three RIPKs family members. c 293 T cells were transfected with RIPK1 (left panel) or RIPK3 (right panel). After 12 h, the cells were treated with AZD4547 at the indicated concentrations for 4 h. Cell lysates were then analyzed by immunoblotting. d L929 cells were primed with IFN-β (50 units/mL) for 24 h, then treated with 10 μg/mL poly(I:C) plus 20 μM zVAD and different concentrations of compounds indicated for 24 h. Cell viability was analyzed by CellTiter‐Glo assay with DMSO(D)as control. e Kinetic study of AZD4547 against RIPK1 with varied concentrations (5 nM, 10 nM, 20 nM and 30 nM respectively) of ATP. Michaelis–Menten plot (left panel) and Lineweaver-Burk plot (right panel) for RIPK1 with AZD4547. f 293 T cells were transfected with RIPK1-WT or S161A mutant for 24 h. AZD4547 or Nec-1 was added post 6 h transfection. Cell lysates were harvested and subjected to Western blot analysis. g Molecular modeling of the binding mode of AZD4547 with kinase domain of human RIPK1 (PDB ID: 6NYH).