Journal: Cell Death Discovery

Article Title: Discovery of Zharp1-163 as a dual inhibitor of ferroptosis and necroptosis for the treatment of inflammatory disorders and kidney injury

doi: 10.1038/s41420-025-02693-5

Figure Lengend Snippet: A , B HT-29 and MEF cells were pretreated with DMSO or the indicated concentrations of Zharp1-163 for 2 h, followed by treatment with T (40 ng/ml), S (100 nM) and Z (20 μM) for 8 h. Cell lysates were harvested and then subjected to Western blot analysis for the phosphorylation of RIPK1, RIPK3, and MLKL. T, TNF-α; S, Smac mimetic; Z, z-VAD ( A , B ). C , D Effects of Zharp1-163 on RIPK1 phosphorylation. HT-29 cells were pretreated with Zharp1-163 for 2 h, followed by treatment with T (40 ng/ml), S (100 nM) and Z (20 μM) for an additional 8 h. The phosphorylation of RIPK1 was detected using immunofluorescence and subsequently quantified and analyzed ( C , D ). Scale bar, 25 μm. E , F Effects of Zharp1-163 on the formation of RIPK3 puncta. Prior to the addition of T (40 ng/ml), S (100 nM) and Z (20 μM) for an additional 8 h, HT-29 cells stably expressing Flag-tagged RIPK3 were preincubated with the specified compounds for 2 h ( E , F ). Scale bar, 25 μm. G Effects of Zharp1-163 on the oligomerization of MLKL. HT-29 cells were pretreated with the indicated concentrations of Zharp1-163 for 2 h, followed by treatment with T (40 ng/ml), S (100 nM) and Z (20 μM) for an additional 8 h. The data presented are representative of three independent experiments. The data are presented as the mean ± SD. n.s., not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following antibodies were used: anti-RIPK1 monoclonal antibody (BD Bioscience, 610459), anti-mouse-phospho-RIPK1 monoclonal antibody (Biolynx, BX60008), anti-human-phospho-RIPK1 monoclonal antibody (Cell Signaling, #65746), anti-human-RIPK3 monoclonal antibody (Cell Signaling, #13526), anti-human-phospho-RIPK3 monoclonal antibody (Abcam, ab209384), anti-mouse-RIPK3 monoclonal antibody (Prosci, 2283), anti-mouse-phospho-RIPK3 monoclonal antibody (Cell Signaling, #91702), anti-human-MLKL monoclonal antibody (Abcam, ab184718), anti-human-phospho-MLKL monoclonal antibody (Abcam, ab187091), anti-mouse-MLKL monoclonal antibody (Abgent, AP14272b), anti-mouse-phospho-MLKL monoclonal antibody (Abcam, ab196436), anti-GPX4 monoclonal antibody (Cell Signaling, #52455) and anti-β-actin monoclonal antibody (Sigma-Aldrich, A2066).

Techniques: Western Blot, Phospho-proteomics, Immunofluorescence, Stable Transfection, Expressing

Journal: Cell Death Discovery

Article Title: Discovery of Zharp1-163 as a dual inhibitor of ferroptosis and necroptosis for the treatment of inflammatory disorders and kidney injury

doi: 10.1038/s41420-025-02693-5

Figure Lengend Snippet: 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.

Article Snippet: The following antibodies were used: anti-RIPK1 monoclonal antibody (BD Bioscience, 610459), anti-mouse-phospho-RIPK1 monoclonal antibody (Biolynx, BX60008), anti-human-phospho-RIPK1 monoclonal antibody (Cell Signaling, #65746), anti-human-RIPK3 monoclonal antibody (Cell Signaling, #13526), anti-human-phospho-RIPK3 monoclonal antibody (Abcam, ab209384), anti-mouse-RIPK3 monoclonal antibody (Prosci, 2283), anti-mouse-phospho-RIPK3 monoclonal antibody (Cell Signaling, #91702), anti-human-MLKL monoclonal antibody (Abcam, ab184718), anti-human-phospho-MLKL monoclonal antibody (Abcam, ab187091), anti-mouse-MLKL monoclonal antibody (Abgent, AP14272b), anti-mouse-phospho-MLKL monoclonal antibody (Abcam, ab196436), anti-GPX4 monoclonal antibody (Cell Signaling, #52455) and anti-β-actin monoclonal antibody (Sigma-Aldrich, A2066).

Techniques: Binding Assay, In Vitro, Kinase Assay, Recombinant, Incubation, Activity Assay, Luciferase, Reporter Assay, Inhibition