Adenanthin exhibits anti-inflammatory effects by covalently targeting the p65 subunit in the NF-κB signaling pathway

Adenanthin is a structurally unique ent-kaurane diterpenoid isolated from Rabdosia adenantha, a traditional Chinese medicinal plant with potent anti-cancer and anti-inflammatory activities. However, its anti-inflammatory molecular mechanism remains largely elusive to date. Here, we developed an affinity-based label-free protein profiling (ALFPP) to identify potential covalent targets of electrophilic natural products with ketone or aldehyde groups. Using ALFPP, we identified 27 potential covalent targets of adenanthin, among which p65 (RelA) has been associated with its anti-inflammatory activities. Through a series of experiments, including LC-MS/MS, molecular docking, electrophoretic mobility shift assays (EMSA), and genome editing, we demonstrated that adenanthin could covalently modify the Cys38 residue of p65 to affect the binding of DNA to p65, thereby inhibiting the NF-κB signaling pathway. ALFPP will facilitate the target identification of electrophilic carbonylated natural products, especially those containing α, β-unsaturated keto groups. Furthermore, the elucidation of the molecular mechanism of adenanthin will contribute to new drug development of adenanthin to treat inflammations and cancers, enhancing the possibility for its clinical application.