Simultaneous Harvesting of PINK1, Parkin, and FUNDC1 Ligands from Herbal Medicines Using “Net Fishing” Strategy

Objective: Targeting mitophagy represents a promising therapeutic strategy for human diseases. This study aimed to develop an efficient approach to simultaneously identify natural ligands for PTEN-induced kinase 1 (PINK1), Parkin, and FUN14 domain containing 1 (FUNDC1) – core regulators of mitophagy – from complex herbal medicine (HM) extracts. Materials and Methods: We established a novel capture strategy where PINK1, Parkin, and FUNDC1 proteins were individually and physically adsorbed on the surface of porous cellulose microspheres were used as “bait” for the recognition and separation of ligands, and ultra-high-performance liquid chromatography/mass spectrometry (UPLC/MS) was used for ligand identification. After evaluating the reliability of the strategy using kushenol N (KSN), kushenol L (KSL), kushenol X (KSX), and genistein as the positive controls, our strategy was successfully applied to “fish” out ligands from HMs (including Sophorae Tonkinensis Radix et Rhizoma, Poria, and Sophorae Flavescentis Radix). Results: UPLC/MS revealed 7, 3, and 4 novel PINK1-, Parkin-, and FUNDC1-targeting compounds, respectively. The affinities of these compounds to their targeting proteins were confirmed using molecular docking, molecular dynamics simulation, surface plasmon resonance, and microscale thermophoresis analysis. The antisteatotic activity of the 9 ligands (KSN, genistein, KSX, KSL, kushenol W, maackiain, kushenol F, poricoic acid AM, and dehydropachymic acid) were validated in palmitic acid–damaged HepG2 cells. Conclusions: Our results confirm the effectiveness of the established strategy for simultaneously discovering PINK1, Parkin, and FUNDC1 ligands from complex herbal preparations and may serve as a useful means in advancing medicinal plant–related drug discovery.