Oriented Immobilization Coupled With Ligand Fishing Strategy for Rapid Discovery of Direct HMGB1 Inhibitors in Taxus wallichiana var. mairei.

High Mobility Group Box 1 (HMGB1), a non-histone chromatin protein, is a key inflammatory cytokine. Direct HMGB1 inhibitors have gained attention for their therapeutic potential in age-related diseases by blocking the pro-inflammatory activity of extracellular HMGB1, but remain limited. Here, an immobilized HMGB1 was synthesized in one step using an oriented immobilization approach and applied to efficiently acquire direct HMGB1 inhibitors from a highly complex matrix. The oriented immobilization method employed a Ni² ⁺-modified metal-organic framework combined with cell lysate containing engineered His-tagged HMGB1, bypassing the need for pre-purified proteins. The resulting immobilized HMGB1 demonstrated high protein loading capacity (110.2 mg/g) while retaining its cytokine activity. The immobilized HMGB1 was employed to rapidly identify its direct inhibitors from the Taxus wallichiana var. mairei twig. The application of this immobilized HMGB1 enabled efficient capture of 11 HMGB1 ligands from this plant. Among the 11 ligands, seven compounds were structurally characterized by HPLC-Q-TOF-MS/MS. Among these compounds, sciadopitysin was selected due to its higher HMGB1 inhibitory activity and predicted binding affinity to HMGB1. Through fluorescence quenching and surface plasmon resonance analyses, sciadopitysin was experimentally validated, where the surface plasmon resonance data demonstrated direct binding to HMGB1 with a dissociation constant (K_d) of 2.77 × 10^-4 M, and the observed intrinsic fluorescence quenching provided additional evidence of the ligand-protein interaction. Furthermore, validation in RAW264.7 macrophages demonstrated its dose-dependent suppression of HMGB1-stimulated inflammation (IC₅₀ = 27.25 ± 3.31 µM). Collectively, a highly efficient ligand fishing strategy for identifying direct HMGB1 inhibitors was established. Moreover, our findings illustrated the correlation between Taxus wallichiana var. mairei and HMGB1 inhibition.