The B-RafV600E inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen-induced liver injury

Receptor-interacting protein (RIP)3 is a critical regulator of necroptosis and has been demonstrated to be associated with various diseases, suggesting that its inhibitors are promising in the clinic. However, there have been few RIP3 inhibitors reported as yet. B-Raf^V600E inhibitors are an important anticancer drug class for metastatic melanoma therapy. In this study, we found that 6 B-Raf inhibitors could inhibit RIP3 enzymatic activity in vitro. Among them, dabrafenib showed the most potent inhibition on RIP3, which was achieved by its ATP-competitive binding to the enzyme. Dabrafenib displayed highly selective inhibition on RIP3 over RIP1, RIP2 and RIP5. Moreover, only dabrafenib rescued cells from RIP3-mediated necroptosis induced by the necroptosis-induced combinations, that is, tumor necrosis factor (TNF)α, TNF-related apoptosis-inducing ligand or Fas ligand plus Smac mimetic and the caspase inhibitor z-VAD. Dabrafenib decreased the RIP3-mediated Ser358 phosphorylation of mixed lineage kinase domain-like protein (MLKL) and disrupted the interaction between RIP3 and MLKL. Notably, RIP3 inhibition of dabrafenib appeared to be independent of its B-Raf inhibition. Dabrafenib was further revealed to prevent acetaminophen-induced necrosis in normal human hepatocytes, which is considered to be mediated by RIP3. In acetaminophen-overdosed mouse models, dabrafenib was found to apparently ease the acetaminophen-caused liver damage. The results indicate that the anticancer B-Raf^V600E inhibitor dabrafenib is a RIP3 inhibitor, which could serve as a sharp tool for probing the RIP3 biology and as a potential preventive or therapeutic agent for RIP3-involved necroptosis-related diseases such as acetaminophen-induced liver damage.Necroptosis, also known as programmed necrosis, is a kind of programmed cell death that occurs at conditions that result in blocking the execution of apoptosis.^{1, 2} The protein kinase receptor-interacting protein (RIP)3 is a serine/threonine protein kinase that has recently been demonstrated to be the critical regulator that switches cells from apoptosis to necroptosis.^{3, 4, 5, 6} The death receptor ligands, such as tumor necrosis factor (TNF)α, Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL), are classical inducers of apoptosis or necroptosis. By binding to their respective receptors, they lead to activation of functional caspase-8, which results in apoptosis by activating the effector caspases such as caspase-3 but inactivating the necroptic kinases such as RIP3. When caspase-8 is absent or inhibited by caspase inhibitors such as z-VAD, those death receptor ligands cause necroptosis, which can be augmented by Smac mimetic that promotes degradation of inhibitor of apoptosis proteins.^{3, 4, 5, 6}RIP3 is widely involved in physiological processes and pathological states.⁶ RIP3 deficiency not only rescues the lethality of caspase-8^−/− and FADD^−/− mice⁷ and restores normal proliferation of their T cells,⁶ but also protects hepatocytes from ethanol-induced injury and steatosis,⁸ rescues caspase-8 or FADD deficiency-induced massive inflammation in epithelium,⁹ prevents cerulean-induced acute necrotizing pancreatitis,^{3, 4} inhibits photoreceptor and cone cell death^{10, 11} and alleviates macrophage necrosis in advanced atherosclerosis lesions.¹² Acetaminophen is an extensively used analgesic and antipyretic. When taken in overdose, its most frequent toxicity is hepatotoxicity including fatal centrilobular hepatic necrosis.^{13, 14} Acetaminophen overdose is the most common cause of acute liver failure in the United States and the United Kingdom.¹⁵ It also causes 11.86% of acute liver failure in China.¹⁶ Enhanced levels of high-mobility group box-1 and necrosis keratin-18 marked occurrence of hepatic necrosis.¹⁴ Necrosis has been considered as the predominant mode of cell death in this case, for which RIP3 has been shown to be responsible.¹⁷ In addition, RIP3 might also be associated with carcinogenesis and tumor drug resistance to chemotherapeutics.^{18, 19} These lines of evidence suggest potential extensive uses of small-molecule RIP3 inhibitors in medical prevention or therapy.However, few RIP3 inhibitors have been reported²⁰ and no small-molecule RIP3 inhibitors have been investigated for the potential medical uses. One possible cause is that there lacks a proper RIP3 kinase assay for screening for its inhibitors at molecular levels, which should be highly sensitive, free of radioisotopes, and high throughput. We thus established a non-radioactive luminescent RIP3 kinase assay in this study. By using this assay, we found that 6 B-Raf inhibitors inhibited the RIP3 enzymatic activity in vitro. But only dabrafenib could rescue cells from RIP3-mediated necroptosis induced by TNFα, TRAIL or Fas ligand plus Smac mimetic and the caspase inhibitor z-VAD. Dabrafenib directly and ATP-competitively bound to RIP3 protein and caused highly selective inhibition on RIP3 over RIP1, RIP2 and RIP5. Dabrafenib was demonstrated to ease acetaminophen-induced necrosis in normal human hepatocytes and to prevent acetaminophen-induced liver injury in mice. Our study raises a possibility that the medical indications of the B-Raf^V600E inhibitor dabrafenib might be extended from cancers to RIP3-involved diseases.