Acaudiolide attenuates MASH-related liver fibrosis through covalent engagement of SMAD4 and blockade of p-SMAD2/3 nuclear translocation.

Hepatic stellate cell (HSC) activation is a central driver of fibrosis progression in metabolic dysfunction-associated steatohepatitis (MASH), yet effective antifibrotic pharmacotherapies remain limited. Here, we identify acaudiolide, a sulfur-bridged symmetric dimeric macrodiolide isolated from the fungus Acaulium sp. H-JQSF, as a natural-product lead that suppresses HSC activation and experimental liver fibrosis. In a Western diet (WD)/CCl4 murine model of MASH, acaudiolide reduced collagen deposition, alpha-smooth muscle actin (α-SMA) expression and fibrogenic gene expression. Transcriptomic profiling of HSCs showed that acaudiolide antagonized transforming growth factor-β1 (TGF-β1)-driven profibrotic programs. Mechanistically, acaudiolide did not measurably alter SMAD2/3 phosphorylation but markedly reduced the nuclear accumulation of phosphorylated SMAD2/3 (p-SMAD2/3). Integration of Connectivity Map (CMap) analysis with isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP) chemical proteomics nominated SMAD4 as a candidate covalent target of acaudiolide. Covalent docking suggested Cys363 as a potential engagement site, and co-immunoprecipitation showed that acaudiolide weakened the association of SMAD4 with p-SMAD2/3. These findings support a model in which acaudiolide interferes with SMAD complex assembly, thereby attenuating TGF-β-dependent profibrotic transcription and HSC activation. Collectively, our data nominate acaudiolide as a preclinical antifibrotic lead and SMAD4 as a potentially tractable downstream node for pharmacological modulation of TGF-β signaling in MASH-related fibrosis.