Zharp1-163 is a dual inhibitor of ferroptosis and necroptosis. It effectively blocks ferroptosis by reducing reactive oxygen species (ROS) levels and inhibits necroptosis by potently and selectively targeting RIPK1 kinase activity (KD = 240 nM; IC50 = 406.1 nM). Zharp1-163 suppresses the cellular activation of RIPK1, RIPK3, and MLKL induced by necroptosis stimuli. The compound significantly alleviates TNF-α-induced systemic inflammatory syndrome, preventing TNF-α-induced lethality and hypothermia in mice. Additionally, Zharp1-163 mitigates acute kidney injury related to necroptosis and ferroptosis in models induced by cisplatin and ischemia-reperfusion. Zharp1-163 is applicable for studying diseases linked to cellular death pathways, such as kidney diseases.

RIPK1:240 nM (Kd)

Zharp1-163, ranging from concentrations of 0.01 to 10 μM, shows significant inhibition of ferroptosis induced by Erastin or RSL3 in HT-1080 cells (EC50 = 0.95 μM; EC50 = 1.33 μM) and mouse embryonic fibroblasts (MEF) (EC50 = 1.93 μM; EC50 = 1.39 μM) over 18 hours. It also blocks necroptosis induced by TNF-α, Smac mimetic, and Z-VAD in HT-29 cells (EC50 = 0.1 μM) and MEF (EC50 = 0.11 μM) within 14-18 hours. Furthermore, Zharp1-163 effectively suppresses necroptosis in murine L929 fibroblasts triggered by TNFα and Z-VAD over 14 hours. At 0.3-3 μM for 26 hours, it inhibits apoptosis induced by TNFα combined with a Smac mimetic in MEF cells. However, at 0.3-3 μM for 8 hours, it does not affect pyroptosis in THP-1 cells. At a concentration of 10 μM over 7 hours, Zharp1-163 significantly reduces lipid ROS generation during ferroptosis and decreases CHAC1 and PTGS2 expression in RSL3-induced HT-1080 cells. It eliminates the phosphorylation of RIPK1, RIPK3, and MLKL in human HT-29 cells at 0.1-3 μM for 10 hours and blocks their phosphorylation in MEF cells. Additionally, at 0.3-3 μM over 10 hours, it inhibits the formation of RIPK3 puncta and MLKL oligomerization in HT-29 cells.

Zharp1-163 (5 mg/kg, administered intraperitoneally, once) acts as an effective RIPK1 inhibitor, providing significant protection to C57BL/6 mice against TNF-induced SIRS. It also markedly reduces acute kidney injury associated with necroptosis and ferroptosis in models induced by Cisplatin and ischemia-reperfusion in C57BL/6 mice.