DeepSAT-guided discovery of sesquiterpenoid dimers from Inula japonica and identification of a natural pyroptosis inducer targeting the ROS-caspase-3-GSDME pathway.

Sesquiterpenoid dimers (SDs) from the genus Inula are well recognized for their structural diversity and antitumor potential. However, their ability to induce pyroptosis remains unexplored. In this work, DeepSAT-guided HSQC analysis was employed for the phytochemical investigation of I. japonica, leading to the isolation and characterization of 21 SDs, including 18 new compounds belonging to three skeleton classes. Among them, neoinujaponolide A (1) possesses an unprecedented 6/7/5 tricyclic skeleton, representing a structurally distinctive guaianolide-guaianolide dimer fused through C-2/C-2' and C-4/C-3' single bonds. While compound 1 represents the major structural novelty of this study, biological screening identified compound 17 as the most potent cytotoxic compound among the tested isolates; therefore, it was selected for further mechanistic investigation. Preliminary structure-activity relationship analysis suggested that the α,β-unsaturated lactone moiety, the Δ5',6' double bond, and the 1'-acetyl group may be potentially associated with cytotoxic activity. Eighteen compounds induced lytic cell death, and the hallmark pyroptotic features were confirmed with compound 17. Compound 17 activated gasdermin E (GSDME) and elicited membrane rupture. Further mechanistic studies revealed that the ROS-caspase-3-GSDME pathway is associated with compound 17-induced pyroptosis. Taken together, these findings identify compound 17 as a natural pyroptosis inducer that may serve as a scaffold for the design and development of novel pyroptosis inducers.