Kuroda_2026_Drug.Metab.Pharmacokinet_68_101531

G-Quadruplexes (G4s) are non-canonical DNA structures formed in guanine-rich sequences and recognized as crucial regulators of gene expression. We identified potential G4-forming sequences within the 5'-flanking region of the human carboxylesterase 2 (CES2) gene, which encodes an enzyme that hydrolyses clinical drugs and endogenous lipids, including triacylglycerols. The aim of this study is to elucidate the impact of G4 formation on CES2 expression and metabolic function. qPCR stop assays and chromatin immunoprecipitation (ChIP) assays using an anti-G4 antibody demonstrated that two guanine-rich sequences in the 5'-flanking region of the CES2 form G4 structures. Treatment with the G4-stabilizing ligand pyridostatin (PDS) reduced CES2 expression and hydrolase activity in HepG2, Huh-1, and HepaSH cells. Luciferase and ChIP assays revealed that PDS suppressed CES2 transcription by inhibiting hepatocyte nuclear factor 4alpha (HNF4alpha) binding to the 5'-flanking region of the CES2. PDS also reduced HNF4alpha expression, through G4 formation within the HNF4alpha promoter. Notably, PDS decreased CES2 mRNA levels even under HNF4alpha-overexpressing conditions, indicating that CES2 repression involves both impaired HNF4alpha binding and reduced HNF4alpha expression. PDS treatment accumulated triacylglycerol in hepatic cells. Collectively, these findings indicate that G4 formation suppresses CES2 transcription and function, highlighting G4 structures as potential therapeutic avenues for modulating lipid homeostasis.