Oxidation of tienilic acid by human yeast-expressed cytochromes P-450 2C8, 2C9, 2C18 and 2C19. Evidence that this drug is a mechanism-based inhibitor specific for cytochrome P-450 2C9.
Oxidation of tienilic acid by human cytochromes P-450 (CYP) 2C9, 2C18, 2C8 and 2C19 was studied using recombinant enzymes expressed in yeast. CYP 2C9 was the best catalyst for 5-hydroxylation of tienilic acid (K(m) = 5 +/- 1 microM, kcat = 1.7 +/- 0.2 min-1), 30-fold more potent in terms of kcat/K(m) than CYP 2C18 (K(m) = 150 +/- 15 microM, kcat = 1.8 +/- 0.2 min-1) and 300-fold more potent than CYP 2C8 (K(m) = 145 +/- 15 microM, kcat = 0.2 +/- 0.1 min-1). CYP 2C19 was unable to catalyze this hydroxylation under our experimental conditions. During this study, a marked effect of the ionic strength on the activities (hydroxylations of tienilic acid and tolbutamide) of these cytochromes P-450 expressed in the yeast strain 334 was observed. The effect was particularly great in the case of CYP 2C18, with a tenfold decrease of activity upon increasing ionic strength from 0.02 to 0.1. Specific-covalent binding of tienilic acid metabolites to cytochrome P-450 (incubations in the presence of 5 mM glutathione) was markedly higher upon tienilic acid oxidation by CYP 2C9 than by CYP 2C18 and CYP 2C8. Mechanism-based inactivation of cytochrome P-450 during tienilic acid oxidation was observed in the case of CYP 2C9 but was not detectable with CYP 2C18 and CYP 2C8. Tienilic acid thus appears to be a mechanism-based inhibitor specific for CYP 2C9 in human liver. Experiments performed with human liver microsomes confirmed that tienilic acid 5-hydroxylase underwent a time-dependent inactivation (apparent t1/2 = 10 +/- 5 min) during 5-hydroxylation of tienilic acid.
Jean
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P
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, URA 400 CNRS, Université Paris V, France.
Lopez-Garcia
P
P
Dansette
P
P
Mansuy
D
D
Goldstein
J L
JL
D001189:Aryl Hydrocarbon Hydroxylases; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D039181:Steroid 16-alpha-Hydroxylase; D013989:Ticrynafen / Q000378:metabolism* / Q000494:pharmacology; D014044:Tolbutamide / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D013989:Ticrynafen