Structure-based identification of HNF4α agonists: Rosmarinic acid as a promising candidate for NAFLD treatment.

The prevention and treatment of metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD), have emerged as critical global health challenges. Current lipid-lowering pharmacotherapies are associated with side effects, including hepatotoxicity, rhabdomyolysis, and decreased erythrocyte counts, underscoring the urgent need for safer therapeutic alternatives. Hepatocyte nuclear factor 4α (HNF4α) has been identified as a pivotal regulator of lipid metabolism, making it an attractive target for drug development. In this study, we investigated the structural characteristics and binding interactions of four HNF4α agonists: Alverine, Benfluorex, N-trans caffeoyltyramine (NCT), and N-trans feruloyltyramine (NFT). Our results indicate that the conjugated structure formed by the amide bond and the aromatic ring in NCT and NFT enhances electron density, potentially contributing to their increased specificity for HNF4α relative to Alverine and Benfluorex. Additionally, electrostatic interactions between the aromatic moieties of the compounds and HNF4α residues were found to play a crucial role in ligand binding. Leveraging these insights, we performed a high-throughput virtual screening of 2131 natural compounds, using the binding modes of NCT and NFT as reference templates. Rosmarinic acid emerged as a promising HNF4α agonist, exhibiting a high consensus score and favorable binding affinity. Subsequent biological assays demonstrated that rosmarinic acid significantly inhibited HepG2 cell proliferation which related to the enhancement of autophagy. After the knockdown of P2 isoform of HNF4α, HepG2 was more sensitive to the administration of NCT and rosmarinic acid. Furthermore, the proliferation of DLD-1 cell, which only expresses the P2 isoform of HNF4α, was not significantly inhibited by the administration of NCT and rosmarinic acid. Collectively, these findings suggest that rosmarinic acid is a promising HNF4α agonist which is more effective to activate the P1 isoform of HNF4α and holds potential as an effective treatment for NAFLD, providing a foundation for the development of novel lipid-lowering drugs with enhanced efficacy and reduced side effect. Data Availability: Data will be made available on request.