Multi-layered target identification strategy of natural products: Daphnetin targets NQO1 and OPLAH in silicosis alleviation

Pneumoconiosis is a common occupational lung disease with unclear pathogenesis and no effective targeted therapies. Emerging evidence suggests that daphnetin is a promising therapeutic candidate for silicosis, yet its efficacy targets remain poorly characterized. In this study, we propose and implement a multi-layered target identification strategy that integrates transcriptomics, clinical metabolomics, public database mining, an artificial intelligence (AI)-based target prediction model, and tissue-based thermal proteome profiling (Tissue-TPP) technology. This strategy enables stepwise elucidation, from pneumoconiosis-related targets to daphnetin efficacy targets and ultimately to direct targets. Using this approach, we validated Nicotinamide adenine dinucleotide phosphate hydrogen (NAD(P)H) quinone oxidoreductase 1 (NQO1) and 5-oxoprolinase (OPLAH) as direct targets of daphnetin, and identified Kelch-like ECH-associated protein 1 (KEAP1), nuclear factor erythroid 2-related factor 2 (NRF2), and arginase 1 (ARG1) as indirect targets. Notably, OPLAH is reported here as a direct target of daphnetin for the first time. Furthermore, the binding of daphnetin to NQO1 and OPLAH helps explain its effects on cellular thiol levels. Using a thiol-responsive probe, we observed that daphnetin significantly increased intracellular thiol levels and reduced silica-induced reactive oxygen species (ROS) accumulation in both A549 and THP-1 cells, thereby contributing to its antioxidant effects. Additionally, overexpression (OE) of OPLAH significantly increased reduced glutathione (GSH) levels and alleviated silica-induced epithelial-mesenchymal transition (EMT). Conversely, OPLAH knockdown partially inhibited the efficacy of daphnetin. Together, these findings clarify the anti-fibrotic mechanism of daphnetin, support its therapeutic potential for silicosis, and provide a practical framework for the target discovery of natural products (NPs). • We propose and implement a multi-layered strategy for identifying the targets of natural products. • The antifibrotic effects of daphnetin are partially mediated by OPLAH. • Daphnetin upregulates NQO1 through an NRF2-independent mechanism. • OPLAH plays a key role in mediating daphnetin’s regulation of thiol levels and ROS accumulation.