A high-throughput reporter assay for screening potential gasdermin D inhibitors from natural products

Pyroptosis, an inflammatory programmed cell death, is typically initiated by inflammasomes and executed by gasdermin proteins. Gasdermin D (GSDMD)-mediated pyroptotic cell death and downstream inflammation cascades are crucial innate immune mechanisms implicated in infectious and inflammatory diseases, thus GSDMD is regarded as a novel therapeutic target of various diseases. Inhibiting the activation of GSDMD is an attractive strategy to curb pyroptosis and inflammation. To facilitate pharmacological discovery of GSDMD inhibitors, we aimed to develop a novel high-throughput hGLuc-mGSDMD-PCA screening system with coelenterazine as the substrate to analyze the GSDMD N-terminal (GSDMD-N) oligomerization sensitively. The system was based on lipopolysaccharides (LPS) and nigericin (Nig)-induced pyroptosis model, where luciferase signal intensity was correlated with pyroptotic progression and verified by further experiments. Based on this system, 64 natural products were subjected to screening. 17 candidate compounds of them were identified to suppress the cleavage and aggregation of GSDMD-N. Baohuoside I (BI) and andrographolide (AG) possessed the most potent inhibitory effects on pyroptotic cell death. More importantly, in addition to chlorogenic acid, forsythoside A and other active compounds that had been reported to inhibit pyroptotic possess, isochlorogenic acid A (ICGA), epigoitrin (EPI), and calycosin-7-O-glucoside (CG) were also screened out and firstly confirmed to inhibit pyroptosis via regulating NLRP3/ASC/Caspase-1/GSDMD-dependent pyroptotic process in vitro and in vivo. We have established a useful tool to detect GSDMD-mediated pyroptosis, and systematically investigated and optimized usage conditions. The ease of use and applicability in vitro makes the hGLuc-mGSDMD-PCA system an attractive tool to monitor GSDMD-N oligomerization, which is of great significance for the screening of drugs with anti-pyroptotic and inflammatory activity.