Emestrin‐Type Epidithiodiketopiperazines Inhibited Gasdermin D‐Mediated Pyroptosis via Caspase‐3/7 Activation

ABSTRACT Sepsis, a life‐threatening dysregulated host response to infection, is frequently exacerbated by pyroptosis—a programmed, proinflammatory cell death process mediated by Gasdermin D (GSDMD) activation. Using high‐throughput screening, we identified emestrin‐type epidithiodiketopiperazines (ETPs) as potent inhibitors of GSDMD cleavage during pyroptosis in Tohoku Hospital Pediatrics‐1 (THP‐1, a human acute monocytic Leukemia cell line)‐derived macrophages. Combined surface plasmon resonance and western blotting analyses demonstrated that these ETPs activate caspase‐3/7, which in turn cleaves GSDMD at aspartic acid residue 87 to generate a p10 fragment. This process prevents the formation of the pore‐forming p30 fragment, thereby mitigating its associated inflammatory effects. Building on these results, in vivo studies showed that a low dose of the lead emestrin‐type ETP (compound 2 ) protected against lethal lipopolysaccharide (LPS)‐induced septic shock and attenuated lung inflammation. This protective effect was further validated in the clinically relevant cecal ligation and puncture (CLP) model, where compound 2 significantly enhanced survival by suppressing the infiltration of GSDMD‐positive neutrophils and monocytes. scRNA‐seq of murine lung tissue showed that compound 2 suppressed LPS‐induced systemic inflammation by inhibiting moDC maturation. Collectively, these findings establish the therapeutic potential of targeting GSDMD‐driven pyroptosis with ETPs in sepsis and suggest their promise for clinical translation.