title: Limited applicability of 7-methoxy-4-trifluoromethylcoumarin as a CYP2C9-selective substrate.
abstract: Fluorometric substrates selective for various cytochrome P450 isoforms (P450s) have great advantages in in vitro enzyme inhibition and induction studies because they are highly sensitive and suitable for rapid screening. 7-Methoxy-4-trifluoromethylcoumarin (MFC) has been reported as a CYP2C9-selective substrate. The present study investigated the relative catalytic selectivity of several human P450s in the O-demethylation of MFC and the applicability of MFC as a probe substrate for CYP2C9. The CYP2C9-selectivity in liver microsomes was not supported by the correlation analysis within a series of microsomes from individual donors or by studies using chemical inhibitors. MFC O-demethylation of microsomes did not correlate with tolbutamide 4-hydroxylation, the classical CYP2C9-marker activity, suggesting the possible participation of some of the other P450s. Results of inhibition studies using model P450 inhibitors also brought the CYP2C9-selectivity of MFC O-demethylation into question. In microsomes containing cDNA-expressed individual P450s, CYP2B6 and CYP2E1 seemed to be the most active in the O-demethylation of MFC. Our results support the participation of several P450 enzymes (CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4) in MFC O-dealkylation. Therefore, MFC cannot be considered a suitable probe substrate in models that express several P450s, such as liver microsomes or primary hepatocytes. Moreover, MFC is a more potent fluorogenic substrate for CYP2B6 and CYP2E1 than for CYP2C9 in microsomes containing cDNA-expressed P450s.
authors.affiliation: Department of Biochemical Pharmacology, Chemical Research Center, Hungarian Academy of Sciences, Pusztaszeri 59-67, H-1025 Budapest, Hungary.
authors.forename: Pálma
authors.initials: P
authors.lastname: Porrogi
authors.forename: László
authors.initials: L
authors.lastname: Kóbori
authors.forename: Krisztina
authors.initials: K
authors.lastname: Kõhalmy
authors.forename: Judit
authors.initials: J
authors.lastname: Gulyás
authors.forename: László
authors.initials: L
authors.lastname: Vereczkey
authors.forename: Katalin
authors.initials: K
authors.lastname: Monostory
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000502:physiology*; D003374:Coumarins / Q000378:metabolism*; D065702:Cytochrome P-450 CYP2B6; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1 / Q000502:physiology; D003640:Dealkylation; D006801:Humans; D010089:Oxidoreductases, N-Demethylating / Q000502:physiology
chemical_list: C540005:7-methoxy-4-trifluoromethylcoumarin; D003374:Coumarins; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating