Three important signaling proteins, receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), and mixed lineage kinase domain-like protein (MLKL), have been recognized as critical components in the regulated necrotic cell death pathway. RIPA-56 is a highly selective, potent, and metabolically stable RIP1 inhibitor with an IC50 of 13 nM. Notably, RIPA-56 had a half-life of 128 min in human liver microsomal stability assays and an EC50 of 28 nM in TSZ-induced HT-29 necrosis assay. It also demonstrated potency in protection of murine L929 cells from TZ-induced necrosis (EC50 = 27 nM). In C57BL/6 mice, both multiple-dose (0.1, 1, 3 mg/kg body weight (BW), IP, 17 min before mTNFα injection and once every 12 h) and single-dose (6 mg/kg BW, IP, 17 min before mTNFα injection) of RIPA-56 treatment dramatically increased the survival rate of TNFα-treated mice, showing a dose-dependent effect. Mice treated with multiple-dose of RIPA-56 (3 mg/kg) or a single 6 mg/kg dose of RIPA-56 had a survival rate of 100%, much higher than the TNFα/1 group, which had a survival rate of 60%[1].
作用机制
An allosteric back-pocket is isolated from RIP1 ATP binding site and forms an L-shaped hydrophobic pocket. RIPA-56 fits tightly in this pocket[1].
RIPA-56 细胞实验
Cell Line
Concentration
Treated Time
Description
References
Human renal tubular epithelial cells (HK-2)
100 nM
24 hours
To evaluate the inhibitory effect of RIPA-56 on necroptosis in HK-2 cells stimulated by high glucose and palmitic acid. Results showed that RIPA-56 significantly suppressed the expression of p-RIPK1/p-RIPK3/p-MLKL and reduced p-MLKL membrane translocation.
Kidney Dis (Basel). 2023 Mar 17;9(5):408-423.
R28 cells
2 μM, 4 μM, 8 μM
24 hours
To evaluate the protective effect of RIPA-56 on glutamate-induced damage in R28 cells. Results showed that RIPA-56 significantly increased cell viability, reduced apoptosis and mitochondrial membrane potential changes, and decreased ROS levels.
Sci Rep. 2024 Feb 15;14(1):3834.
RIPA-56 动物实验
Species
Animal Model
Administration
Dosage
Frequency
Description
References
Mice (C57BLKS-Leprdb)
High-fat diet-induced diabetic kidney disease model
Oral
300 mg/kg
36 consecutive days
To evaluate the protective effects of RIPA-56 in a high-fat diet-induced diabetic kidney disease mouse model. Results showed that RIPA-56 significantly improved renal function (BUN and ACR), alleviated glomerular and tubulointerstitial pathological damage, and inhibited necroptosis pathway activation (p-RIPK1/p-RIPK3/p-MLKL).
Kidney Dis (Basel). 2023 Mar 17;9(5):408-423.
C57BL/6j mice
NMDA-induced normal tension glaucoma mice model
Intravitreal injection
60 μM, 80 μM, 100 μM
Single injection, observed for 3 days
To evaluate the protective effect of RIPA-56 on NMDA-induced retinal damage. Results showed that RIPA-56 significantly reduced retinal ganglion cell loss, improved retinal structure, and decreased the expression of the inflammatory marker IL-6.
Sci Rep. 2024 Feb 15;14(1):3834.
Mice
Carbon tetrachloride/Galactosamine-induced ACLF model
Intraperitoneal injection
3 mg/kg
Twice daily for 48 hours
To evaluate the protective effect of RIPA56 on hepatocyte necroptosis in the ACLF model. Results showed that RIPA56 significantly reduced liver injury (decreased ALT levels), hepatocyte death (reduced TUNEL staining), and liver RIPK3 and pMLK expression.
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