Identification of 1,2,3,4,6-O-Pentagalloylglucose as a novel ASNS inhibitor for MASLD amelioration in mice by increasing l-aspartate levels alongside LKB1/AMPK metabolic axis activation.

l-aspartate is a nonessential amino acid involving tricarboxylic acid cycle, amplifying hepatic l-aspartate level is a practicable and promising therapeutic approach in treating metabolic dysfunction-associated steatotic liver disease (MASLD) and liver injury-induced liver fibrosis. However, fewer compounds have been reported to increase hepatic l-aspartate level for ameliorating MASLD in vivo. Asparagine synthetase (ASNS) catalyzes the conversion of l-aspartate into asparagine, here, we identified a natural molecule, named 1, 2, 3, 4,6-O-Pentagalloylglucose (PGG), from the compound library (∼7133 compounds) using the free energy perturbation (FEP)-based virtual screening strategy. PGG showed strong binding affinity (K = 8.8 μM) against recombinant human ASNS and inhibited its enzymatic activity (IC = 7.1 μM), subsequently increased cellular l-aspartate level and activated LKB1/AMPK metabolic axis and enhanced lipid oxidation, leading to lipid accumulation suppression in hepatocytes. Correspondingly, treating PGG (10 mg/kg/per 2 days, i. p.) in mice for 6 weeks efficiently corrected high-fat and high-cholesterol (HFC) diet induced bodyweight gained, glucose tolerance impairment, insulin resistance, and all the typical manifestations of MASLD, including hepatic steatosis, liver injury, and inflammation. These therapeutics were associated with decreases in ASNS expression level in liver, leading to increases in hepatic l-aspartate level, activation of LKB1/AMPK axis, and improvement of mitochondrial oxidation. These data indicate that increasing hepatic l-aspartate level would be a promising therapeutic strategy in treating MASLD, and ASNS would be a novel target for developing anti-MASLD agents.