Lys31/Lys90-dependent stabilization of GPX4 by Schisandrin lignans suppresses ferroptosis in drug-induced liver injury

Abstract Drug-induced liver injury (DILI), particularly from isoniazid (INH), is a major clinical concern. Ferroptosis is implicated in DILI, yet direct GPX4 stabilizers with defined binding sites remain undiscovered. While Schisandra chinensis lignans exhibit hepatoprotective potential, their mechanistic interplay with ferroptosis remains unexplored. Here, we delineate a novel molecular axis by which Schisandrin A (SinA) and Schisandrin B (SinB) mitigate INH-induced hepatotoxicity through ferroptosis suppression. We demonstrate that SinA/SinB significantly attenuates hepatic injury markers (ALT/AST), iron overload, lipid peroxidation, and glutathione depletion in vitro and in vivo. Strikingly, GPX4 knockout abolished their protective effects, underscoring GPX4 as the pivotal target. ITC and SPR revealed high-affinity binding of SinA/SinB to GPX4, while molecular docking identified K31 and K90 as critical residues for GPX4 interaction. Mutagenesis studies confirmed that K31/K90 substitutions abolished SinA/SinB’s efficacy, highlighting a structure-dependent mechanism. Compound binding reduced the interaction between GPX4 and TRIM25, inhibited the ubiquitination of GPX4, as evidenced by Co-IP and MD. This stabilizes GPX4, suppresses lipid peroxidation/iron accumulation, and rescues INH-induced ferroptosis in vitro and in vivo. These findings establish a novel mechanism of GPX4 regulation and provide a structural blueprint for anti-ferroptotic drug design.