Adenosine triphosphate-binding pocket inhibitor for mixed lineage kinase domain-like protein attenuated alcoholic liver disease via necroptosis-independent pathway

BACKGROUND Mixed lineage kinase domain-like protein (MLKL) serves as a critical mediator in necroptosis, a form of regulated cell death linked to various liver diseases. This study aims to specifically investigate the role of MLKL’s adenosine triphosphate (ATP)-binding pocket in facilitating necroptosis-independent pathways that may contribute to liver disease progression. By focusing on this mechanism, we seek to identify potential therapeutic targets that can modulate MLKL activity, offering new strategies for the prevention and treatment of liver-related pathologies. AIM To investigate the possibility of using the ATP-binding pocket-associated, necroptosis-independent MLKL pathway as a target for liver diseases. METHODS Cell death following necroptosis stimuli was evaluated using cell proliferation assays, flow cytometry, and electron microscopy in various cells. The human liver organoid system was used to evaluate whether the MLKL ATP pocket-binding inhibitor could attenuate inflammation. Additionally, alcoholic and non-alcoholic fatty liver diseases animal models were used to determine whether MLKL ATP pocket inhibitors could attenuate liver injury. RESULTS While an MLKL ATP pocket-binding inhibitor did not prevent necroptosis-induced cell death in RAW 264.7 cells, it did reduce the necroptosis-led expression of CXCL2 , ICAM , and VCAM . Notably, MLKL ATP pocket inhibitor diminishes the expression of CXCL2 , ICAM , and VCAM by inhibiting the IκB kinase and nuclear factor kappa-B pathways without inducing necroptosis-induced cell death in two-dimensional cell culture as well as the human-derived liver organoid system. Although MLKL ATP-binding inhibitor was ineffective in non-alcoholic fatty liver disease animal models, MLKL ATP-binding inhibitor attenuated hepatic inflammation in the alcoholic liver disease model. CONCLUSION MLKL ATP pocket-binding inhibitor exerted anti-inflammatory effects through the necroptosis-independent MLKL pathway in an animal model of alcoholic liver disease.