Fig. 3: Nec-34 directly inhibits the kinase activity of RIPK1.

a The in vitro kinase activity assay. 293T cells were transfected with HA-tagged FL-RIPK1 (left) or kinase domain of RIPK1 (residues 1–330) (middle) for 24 h. Cells were then lysed with Nonidet P-40 buffer 24 h after transfection. The cell lysates were immunoprecipitated with anti-HA antibody-conjugated agarose and divided equally into five or four parts. Divided immunocomplexes were pretreated with 20 μM Nec-1s, Nec-34, or Nec-34i for 10 min. Kinase reactions were initiated by 20 μM ATP, and the reactions were carried out at 30 °C for 30 min. Recombinant hRIPK1 (residues 1–330, 0.5 μM) purified from Sf-9 insect cells was pretreated with 10 μM Nec-1s or Nec-34 for 30 min as indicated. The kinase reactions were initiated by 200 μM ATP, and the reactions were carried out at 30 °C for 30 min (right). The samples were analyzed by western blotting with indicated antibodies. b The Tm value of recombinant hRIPK1 in Nec-34, Nec-34i, Nec-1s or Nec-1i treatment groups were compared to control group and presented as ΔTm for protein thermal shift assay. Recombinant hRIPK1 (residues 1–330, 2 μM) purified from Sf-9 cells was treated with indicated compounds for 1 h. Protein thermal stability was analyzed through the differential scanning calorimetry by real-time PCR and the Tm values were calculated by Protein Thermal Shift Software. Three replicates for each reaction were performed. c ADP-Glo™ Kinase Assay of hRIPK1. Recombinant hRIPK1 (residues 1–330, 1 μM) purified from Sf-9 cells was pretreated with different concentrations of Nec-1s, Nec-34 for 30 min as indicated. The kinase reactions were initiated by 111 μM, 333 μM, 1 mM and 3 mM ATP as indicated, and the reactions were carried out at 30 °C for 2 h, and then the amount of ADP produced during the kinase reaction was detected by ADP-Glo™ Reagent. d 293T cells were transfected with Flag-tagged WT-RIPK1 (residues 1–330), or S161A mutant for 24 h. Nec-34 or Nec-1s (10 μM) was added at 6 h after transfection. The cells were lysed with Nonidet P-40 buffer and analyzed by western blotting with indicated antibodies. e, f RIPK1-deficient Jurkat cells were infected with retrovirus expressing HA-tagged WT-hRIPK1 and HA-tagged hRIPK1 S161A, respectively, by the Tet-On Advanced Inducible Expression System. Reconstituted Jurkat cells were treated with 1 μg/mL doxycycline for 48 h to induce the expression of RIPK1. The cells were then pretreated with 100 nM SM-164 for 2 h (e) or 100 nM 5Z7 for 1 h (f), and then 100 ng/mL TNFα and 50 μM zVAD.fmk were added for additional 24 h. g Reconstituted hRIPK1 S161A Jurkat cells were pretreated with TNFα/SM-164/zVAD.fmk as indicated, and then were lysed with 0.5% Nonidet P-40 buffer and analyzed by western blotting with indicated antibodies. h, i 293T cells were transfected with HA-tagged RIPK1 (residues 1–330) L70A, or L129M mutant expression plasmids for 24 h. Nec-34 and Nec-1s (10 μM) were added 6 h after transfection. The cells were lysed with Nonidet P-40 buffer and analyzed by western blotting with indicated antibodies. The cell death in e, f were measured by CellTiter-Glo assays. The results shown depict the means ± SEM of n = 3 independent biological experiments. P-values were calculated by two-tailed Student’s t-test (**P < 0.01, ***P < 0.001, ****P < 0.0001).