Anisodine Activates SIK1 to Drive RIPK1-dependent Necroptosis in TNBC.

INTRODUCTION

Triple-Negative Breast Cancer (TNBC) has limited targeted therapeutic options. SIK1 is a tumor-suppressive kinase in breast cancer, but pharmacological SIK1 activators remain poorly defined. Although recent anticancer patents have disclosed SIK modulators and RIPK1/necroptosis-targeting agents, most SIK-related inventions focus on kinase inhibition rather than therapeutic SIK1 activation.

METHODS

We performed structure-based virtual screening of 23,006 traditional Chinese medicine- derived compounds against human SIK1 and validated anisodine binding and activity using molecular docking, CETSA, DARTS, immunoblotting, kinase assays, rescue experiments, RIPK1 mutagenesis, and xenograft models. Female BALB/c nude mice (4 weeks old) were used for the xenograft experiments.

RESULTS

Anisodine directly bound the SIK1 ligand-binding pocket, with S247 required for anisodine- mediated SIK1 activation. Anisodine increased pSIK1-T182 and SIK1 substrate phosphorylation, suppressed TNBC clonogenic growth and invasion, and reduced xenograft tumor growth. Pharmacological rescue showed that Necrostatin-1, but not inhibitors of apoptosis, ferroptosis, or copper chelation, reversed anisodine-induced cytotoxicity. Mechanistically, SIK1 interacted with RIPK1 and directly phosphorylated RIPK1 at Ser316, thereby activating the RIPK1(S316)-RIPK3-MLKL necroptosis cascade. RIPK1-S316A attenuated, whereas RIPK1- S316D enhanced, necroptotic signaling and tumor suppression.

DISCUSSION: These findings position anisodine as a mechanistically defined SIK1 activator and distinguish this strategy from existing SIK inhibitor-centered patent landscapes. The SIK1-RIPK1(S316) axis also provides a druggable framework that links kinase activation to regulated necroptosis in TNBC.

CONCLUSION

This study identifies anisodine as a small-molecule SIK1 activator and defines a SIK1-RIPK1(S316) signaling axis that induces necroptosis in TNBC, supporting a patentrelevant therapeutic concept for kinase-directed and regulated-cell-death-based anticancer drug discovery.