Fig. 4: PK68 is a highly selective inhibitor of RIPK1 kinase activity.

a In vitro kinase activity assays using recombinant RIPK1 (a) and RIPK3 (b) were performed. Recombinant proteins were incubated with PK6 and PK68 as indicated. Data represent mean value ± standard deviation. c PK68 shows good kinase selectivity against a panel of 369 kinases (without RIPK1) provided by Reaction Biology as viewed in the human kinome phylogenetic tree. d PK6 and PK68 inhibit TNF-induced necroptosis but not RIPK3 dimerization-induced cell death in NIH3T3-RIPK3 cells that stably express RIPK3 fused to FKBP F36V mutant. NIH3T3-RIPK3 cells were pretreated with indicated compounds for 1 h and subsequently treated with T (40 ng/ml)/S (100 nM)/Z (20 μM) or AP20187 (100 nM) for 24 h. Cell viability was determined by measuring ATP levels. e PK6 and PK68 have no effect on MLKL dimerization-induced cell death in HeLa-MLKL (1–190) cells that stably express MLKL (1–190aa) fused to DmrB. HeLa-MLKL (1–190) cells were pretreated with indicated compounds for 1 h and subsequently treated with AP20187 (100 nM) for 24 h. Cell viability was determined by measuring ATP levels. Data are represented as the mean ± standard deviation of triplicates