Figure 6.
Development of a novel RIPK1 kinase inhibitor. (A) Chemical structure of Zharp1-211. (B) Assessment of Zharp1-211 (10 μM) binding against 468 human kinases (DiscoverX kinase panel). TREEspot kinase interaction map of Zharp1-211 with 468 kinases. Red circles indicate kinases that were inhibited by AC002 >65%. Full data are presented in extended Data supplemental Table 2. (C) The binding constant (Kd) of Zharp1-211 with human recombinant RIPK1. (D) In vitro kinase activity assays using recombinant RIPK1. (E-F) Dose response curve and EC50 for Zharp1-211 in TNF-α–induced necroptosis in human HT-29 cells and mouse L929 cells. HT-29 cells were pretreated with Zharp1-211 at the indicated concentration for 2 hours before the treatment with necroptotic stimuli, TNF-α (40 ng/mL), the inhibitor of apoptosis protein (IAP) antagonist Smac mimetic (100 nM), and the pan-caspase inhibitor z-VAD (20 μM), for 48 hours (E). L929 cells were pretreated with Zharp1-211 at the indicated concentration for 2 hours before treatment with TNF-α (40 ng/mL) and z-VAD (20 μM) for 24 hours (F). Cell viability was assessed by measuring ATP levels. (G-H) Small intestinal organoids from WT B6 mice were treated with Zharp1-211 (100 nM) for 2 hours and then treated with TNF-α (40 ng/mL), Smac mimetic (100 nM), and z-VAD (20 μM). Immunoblotting of cell lysates from organoids with antibodies against RIPK1, p-RIPK1, MLKL, p-MLKL, and β-actin was performed at 8 hours after treatment with TNF-α, Smac mimetic, and z-VAD (G). Quantification of p-RIPK1, RIPK1, p-MLKL, or MLKL normalized to β-actin was shown under the band. Cell viability in organoids at 24 hours aftertreatment with TNF-α, Smac mimetic, and z-VAD, assessed by measuring ATP levels (H). (I-J) Small intestinal organoids prepared from B6 mice were pretreated with Zharp1-211 (100 nM) for 2 hours before the treatment with IFN-γ for 12 hours. qPCR measurement of Cxcl9, Cxcl10 expression (I), CIITA, and H2-DMB1 expression (J). (K) Small intestinal organoids prepared from B6 mice were pretreated with Zharp1-211 (100 nM) for 2 hours before the treatment with IFN-γ for 24 hours. The MFI of MHC II was determined by flow cytometer analysis. (L-M) Human intestinal organoids from 5 individual donors (n = 5) were treated with Zharp1-211 (100 nM) for 2 hours and then stimulated with IFN-γ for 12 hours. Expression of the indicated genes was analyzed by qPCR (L) and protein levels were measured by ELISA (M). (N) Intestinal crypt cells isolated from WT B6 mice were treated with DMSO, 100 nM Zharp1-211, or ruxolitinib (RUX) for 2 hours before stimulation with IFN-γ (100 ng/mL) for 1 hour. Cell lysates was collected for immunoblotting with antibodies against p-STAT1, STAT1, and β-actin. Quantification of p-STAT1 or STAT1 normalized to β-actin was shown under the band. Data shown are representative of 3 independent experiments. Data are shown as the mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. Multiple comparisons were evaluated by one-way ANOVA (J-M), two-group comparisons used unpaired t test (two-tailed) (H-I). ANOVA, analysis of variance; ATP, adenosine triphosphate; ELISA, enzyme-linked immunosorbent assay analysis; MFI, mean fluorescence intensity; SD, standard deviation.

Development of a novel RIPK1 kinase inhibitor. (A) Chemical structure of Zharp1-211. (B) Assessment of Zharp1-211 (10 μM) binding against 468 human kinases (DiscoverX kinase panel). TREEspot kinase interaction map of Zharp1-211 with 468 kinases. Red circles indicate kinases that were inhibited by AC002 >65%. Full data are presented in extended Data supplemental Table 2. (C) The binding constant (Kd) of Zharp1-211 with human recombinant RIPK1. (D) In vitro kinase activity assays using recombinant RIPK1. (E-F) Dose response curve and EC50 for Zharp1-211 in TNF-α–induced necroptosis in human HT-29 cells and mouse L929 cells. HT-29 cells were pretreated with Zharp1-211 at the indicated concentration for 2 hours before the treatment with necroptotic stimuli, TNF-α (40 ng/mL), the inhibitor of apoptosis protein (IAP) antagonist Smac mimetic (100 nM), and the pan-caspase inhibitor z-VAD (20 μM), for 48 hours (E). L929 cells were pretreated with Zharp1-211 at the indicated concentration for 2 hours before treatment with TNF-α (40 ng/mL) and z-VAD (20 μM) for 24 hours (F). Cell viability was assessed by measuring ATP levels. (G-H) Small intestinal organoids from WT B6 mice were treated with Zharp1-211 (100 nM) for 2 hours and then treated with TNF-α (40 ng/mL), Smac mimetic (100 nM), and z-VAD (20 μM). Immunoblotting of cell lysates from organoids with antibodies against RIPK1, p-RIPK1, MLKL, p-MLKL, and β-actin was performed at 8 hours after treatment with TNF-α, Smac mimetic, and z-VAD (G). Quantification of p-RIPK1, RIPK1, p-MLKL, or MLKL normalized to β-actin was shown under the band. Cell viability in organoids at 24 hours aftertreatment with TNF-α, Smac mimetic, and z-VAD, assessed by measuring ATP levels (H). (I-J) Small intestinal organoids prepared from B6 mice were pretreated with Zharp1-211 (100 nM) for 2 hours before the treatment with IFN-γ for 12 hours. qPCR measurement of Cxcl9, Cxcl10 expression (I), CIITA, and H2-DMB1 expression (J). (K) Small intestinal organoids prepared from B6 mice were pretreated with Zharp1-211 (100 nM) for 2 hours before the treatment with IFN-γ for 24 hours. The MFI of MHC II was determined by flow cytometer analysis. (L-M) Human intestinal organoids from 5 individual donors (n = 5) were treated with Zharp1-211 (100 nM) for 2 hours and then stimulated with IFN-γ for 12 hours. Expression of the indicated genes was analyzed by qPCR (L) and protein levels were measured by ELISA (M). (N) Intestinal crypt cells isolated from WT B6 mice were treated with DMSO, 100 nM Zharp1-211, or ruxolitinib (RUX) for 2 hours before stimulation with IFN-γ (100 ng/mL) for 1 hour. Cell lysates was collected for immunoblotting with antibodies against p-STAT1, STAT1, and β-actin. Quantification of p-STAT1 or STAT1 normalized to β-actin was shown under the band. Data shown are representative of 3 independent experiments. Data are shown as the mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. Multiple comparisons were evaluated by one-way ANOVA (J-M), two-group comparisons used unpaired t test (two-tailed) (H-I). ANOVA, analysis of variance; ATP, adenosine triphosphate; ELISA, enzyme-linked immunosorbent assay analysis; MFI, mean fluorescence intensity; SD, standard deviation.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal