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

## TL;DR

Emestrin-type compounds prevent dangerous cell death in sepsis by blocking a key protein, reducing inflammation and improving survival in animal models.

## Contribution

Emestrin-type epidithiodiketopiperazines are newly identified as inhibitors of Gasdermin D-mediated pyroptosis via caspase-3/7 activation.

## Key findings

- ETPs inhibit GSDMD cleavage and pyroptosis in THP-1-derived macrophages.
- Compound 2 protects against LPS-induced septic shock and reduces lung inflammation in mice.
- ETPs suppress systemic inflammation by inhibiting monocyte-derived dendritic cell maturation.

## 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.

This study demonstrates that emestrin‐type ETPs suppress GSDMD‐mediated pyroptosis through caspase‐3/7 activation, thereby attenuating inflammatory responses and conferring protection against sepsis. In THP‐1 macrophages, ETPs significantly reduced pyroptosis and secretion of the proinflammatory cytokines IL‐1β and IL‐18. In mouse models, compound 2 (emestrin) alleviated acute lung injury, and inhibited systemic inflammation by restraining the maturation of monocyte‐derived dendritic cells.