BackgroundPyruvate kinase liver and red blood cells (PKLR) is linked to metabolic dysfunction-associated steatotic liver disease (MASLD). Previous study, we identified JNK-IN-5A, a c-Jun N-terminal kinase (JNK) inhibitor that suppresses PKL expression in HepG2 cells using computational drug repurposing and screened out four hit JNK-IN-5A derivatives (SET-151, SET-152, SET-162, SET-130).Materials and MethodsWe validated therapeutic efficacy of JNK-IN-5A and four derivative (SET-151, SET-152, SET-162, SET-130). HepG2 de novo lipogenesis (DNL) steatosis model was used in vitro validation. RNA sequencing data were analysed using systems biology approaches, including transcriptomic profiling and COMPASS analysis. GLP-like toxicity assessment in rat model shows in vivo safety and MASLD rat model revealed in vivo therapeutic effect to MASLD and MASH.ResultsIn a HepG2 DNL steatosis model, all compounds reduced intracellular triacylglycerol (TAG) and inhibited key DNL proteins (PKL, FASN, ACACA, SCD1, SREBP1-c, ChREBP). Transcriptomic profiling revealed stronger anti-steatotic effects with SET-151, SET-152, and SET-162, which uniquely downregulated genes in pyruvate metabolism, bile acid synthesis, fatty acid metabolism, and glycolysis. Compass analysis showed these derivatives significantly altered lipid-related metabolic reactions, unlike JNK-IN-5A. In a high-sucrose, high-fat diet-induced MASLD rat model, JNK-IN-5A and SET-152 reduced hepatic lipid accumulation, liver stiffness, and MASLD biomarkers.ConclusionOur findings identify PKLR as a promising therapeutic target for MASLD and MASH. SET-152 suppressing PKLR through JNK inhibition highlights its potential as a new drug for MASLD and MASH therapy.