A label-free magnetic bead affinity approach for direct target identification of herbal extract: Ganoderma lucidum as a case.

Ganoderma lucidum (Chinese name: Lingzhi) has been used for the treatment of diverse liver disorders for centuries in traditional Chinese medicinal practices. Ganoderic acids (GAs) have been well-established as the key bioactive constituents responsible for the hepatoprotective potential and the ability to alleviate hepatocellular carcinoma (HCC). However, the direct molecular targets underlying these therapeutic benefits have not been disclosed, extensively hindering mechanism clarification and further development of GAs as well as G. lucidum. In this study, we developed a label-free affinity fishing workflow to capture and identify direct targets of GAs in HepG2 cells using ganoderic acid-enriched extract (GAE)-modified magnetic beads. LC-MS/MS coupled with bioinformatics analysis revealed 45 high-confidence candidate targets, among which the small ribosomal subunit protein uS3 (RPS3) emerged as particularly promising due to its strong enrichment and centrality in HCC-associated pathways. Surface plasmon resonance (SPR) demonstrated concentration-dependent binding between GAE and RPS3, with a dissociation constant (KD) of 7.49 mg/mL. Six representative GAs also bound to RPS3 in a concentration-dependent manner, with KD values ranging from 92.3 to 120.0 μM. Molecular docking revealed their binding energies between -6.7 and -8.0 kcal/mol with key hydrogen bonds to residues such as Asp-14 and Lys-45. This study establishes a novel label-free affinity fishing workflow for target identification in complex herbal extracts, and RPS3 was identified as a dominant target of GAs mediating the beneficial effects of GAE through integrating diverse assays including magnetic bead-based affinity fishing, bioinformatics, SPR, and molecular docking, providing new insights into the anti-HCC activity of G. lucidum and offering a feasible tool for target discovery of TCM extracts.