Comments from the Award Winners

On receiving the Best Poster Award Laboratory of Drug Metabolism and Safety, Kanazawa University Graduate School of Pharmaceutical Sciences Laboratory of Drug Metabolism and Safety Seiya Takemoto

　I would like to express my sincere appreciation to all the judges and participants who had meaningful discussions with me for the Best Poster Award at the 37th Annual Meeting of the Japanese Society of Pharmacokinetics and Pharmacodynamics held in Yokohama, Japan.

　I made a presentation entitled “Stabilization of DNA G-quadruplexes on the upstream region of CYP3A4 gene promotes the transcription” at the annual meeting. Genomic DNA forms a double helix structure by Watson-Crick base pairing, and guanine-rich DNA sequences are known to adopt a guanine quadruplex (G4) structure via a Hugestein base pair. The G4 structure plays an important role in the regulation of gene expression because it exists in the upstream region of genes and regulates gene transcription. In this study, we focused on the presence of three potential G4 forming sequences in the upstream region of the CYP3A4 gene and aimed to clarify the effect of G4 structure formation by these sequences on CYP3A4 expression. First, we demonstrated that the G4-forming sequences upstream of the CYP3A4 gene actually form the G4 structure by qPCR stop assay and chromatin immunoprecipitation using anti-G4 structure antibody. Treatment of primary cultured human hepatocytes and human hepatocellular carcinoma-derived HepG2 cells with pyridostatin, a global G4 structure stabilizer, resulted in increased CYP3A4 mRNA expression, indicating that G4 structure stabilization induces CYP3A4 expression. The results of the formaldehyde-assisted enrichment of regulatory elements assay, which evaluates the degree of relaxation of chromatin structure, and chromatin immunoprecipitation using anti-PXR antibody showed that stabilization of G4 structure in the presence or absence of the PXR ligand The results of chromatin immunoprecipitation with anti-PXR antibodies showed that stabilization of the G4 structure in the presence or absence of the PXR ligand relaxes the chromatin structure in the upstream region of the CYP3A4 gene and enhances the binding of PXR, resulting in transcriptional activation of CYP3A4. Finally, we hypothesized that there are drugs that induce CYP3A4 induction via stabilization of the G4 structure upstream of the CYP3A4 gene, and attempted to search for CYP3A4 G4 stabilizers in a library of 765 FDA-approved drugs by qPCR stop assay. The results showed that digoxin, a PXR non-ligand, selectively stabilizes one of the three G4-forming sequences. Interestingly, treatment of HepG2 cells with digoxin actually increased CYP3A4 mRNA expression. This study has revealed for the first time that the formation of G4 structures is involved in the regulation of drug-metabolizing enzyme expression, and has also demonstrated the possibility that digoxin induces CYP3A4 expression via a novel mechanism of stabilization of G4 structures.

　Finally, we would like to take this opportunity to express our sincere gratitude to Assistant Professor Masataka Nakano, Associate Professor Tatsumi Fukami, and Professor Miki Nakajima for their guidance during the course of this research. The study of the role of guanine quadruplex structure in pharmacokinetics has just started, and it is a continuous process of trial and error.