Identification of a novel HNF4α agonist regulating abnormal metabolism.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a key nuclear receptor involved in liver function and metabolic regulation, making it a potential therapeutic target for lipid-metabolism disorders. Combining time-resolved fluorescence resonance energy transfer (TR-FRET) high-throughput screening with a diffusion-based structure-guided generative design workflow (DiffSBDD), we identified and prioritized a series of compounds with HNF4α agonist-like activity. Among them, (Z)-19 showed good HNF4α activation with an EC50 of 9.2 μM, outperforming the reference agonist N-trans-caffeoyltyramine (NCT; EC50 = 129.6 μM). Surface plasmon resonance (SPR) supported a direct interaction with HNF4α (KD = 4.6 μM). In HepG2 cells, (Z)-19 reduced triglyceride and total cholesterol accumulation with minimal cytotoxicity and exhibited ferroptosis-protective activity, with a response profile differing from that of NCT. In a hyperlipidemia mouse model, (Z)-19 lowered plasma triglyceride and total cholesterol levels, indicating encouraging in vivo efficacy. Additionally, all-atom molecular dynamics (MD) simulations supported a possible binding mode characterized by persistent hydrogen-bonding and hydrophobic contacts within the ligand-binding domain, and yielded favorable binding-free-energy estimates consistent with the experimental potency ranking. Together, these results identify (Z)-19 as a promising starting point for further optimization and mechanistic investigation of compounds that modulate HNF4α.