Pharmacological and Mechanism Study of a New RIPK1 Inhibitor Via Necroptosis to Treat Hemophagocytic Lymphohistiocytosis

Background: Hemophagocytic lymphohistiocytosis (HLH) is a cytokine-release syndrome with high mortality, primarily treated with corticosteroids, though 30% of patients exhibit resistance. Necroptosis, a caspase-independent programmed cell death, is overly activated in autoimmune diseases, severe COVID-19, and SIRS, leading to sterile inflammation and cytokine storm. Receptor-interacting serine/threonine protein kinase 1 (RIPK1) regulates necroptosis, and inhibitors of RIPK1 show potential as treatments for inflammatory diseases, sepsis, cancer, and neurodegenerative diseases. In this study, we hypothesize that RIPK1 inhibitors can treat HLH by inhibiting necroptosis and sensitizing cells to corticosteroid-induced apoptosis.

Material and method: This study utilizes AI-assisted drug design and deep learning models based on conditionally recurrent neural networks for decentralized learning to develop a novel, highly selective, and potent RIPK1 inhibitor—SKLB-Z30. SKLB-Z30, with a new scaffold, demonstrates superior pharmacodynamics and safety compared to the reported RIPK1 inhibitor GSK2982772. We induced necroptosis in murine macrophage and T cell lines in vitro to explore the inhibitory effects of SKLB-Z30, both as monotherapy and in combination therapy. In secondary and primary murine models of HLH, we used SKLB-Z30 alone or with dexamethasone and preliminarily analyzed the mechanisms of action.

Result: This study successfully induced necroptosis in murine macrophages and T cells in vitro. PI/Hoechst results showed that SKLB-Z30 monotherapy inhibited necroptosis at low concentrations (10 nM), significantly outperforming GSK2982772. SKLB-Z30 dose-dependently inhibited the phosphorylation of key proteins (RIPK1, RIPK3, MLKL) in necroptotic macrophages and T cells. SKLB-Z30 also reduced TNFα and IL-6 levels in the supernatant of necroptotic cells in a concentration-dependent manner. In vivo, macrophage activation syndrome (MAS) and primary HLH animal models were successfully established. MAS and HLH mice exhibited significant anemia, thrombocytopenia, liver dysfunction, elevated body temperature, hepatosplenomegaly, increased ferritin, and bone marrow hemophagocytosis. Transcriptome sequencing revealed that the TNFα-RIPK1-RIPK3 signaling pathway was upregulated in model mice compared to normal mice. Immunohistochemistry showed significantly elevated p-RIPK1 and p-MLKL protein expression both in MAS and HLH mice. SKLB-Z30 monotherapy and combination therapy with dexamethasone significantly reduced liver and spleen size, improved anemia, increased platelet counts, reduced serum ferritin, and decreased levels of TNFα, IL-6, and other cytokines.

Conclusion: The novel RIPK1 inhibitor SKLB-Z30 demonstrated effective therapeutic action against HLH in both in vivo and in vitro experiments. RIPK1 inhibitors represent a promising new approach for the clinical treatment of HLH patients.