Thiophene derivatives as new mechanism-based inhibitors of cytochromes P-450: inactivation of yeast-expressed human liver cytochrome P-450 2C9 by tienilic acid.
Oxidation of tienilic acid (TA) by microsomes of yeast expressing two closely related human liver cytochrome P-450s (P450), P450 2C9 and 2C10, led to catalysis-dependent loss of activity of these P450s. Under identical conditions, oxidation of a tienilic acid isomer (TAI) failed to give any P450 inactivation. The loss of P450 activity during TA oxidation was concomitant with product (5-hydroxytienilic acid, 5-OHTA) formation, showed pseudo-first-order and saturation kinetics, and was inhibited by an alternative substrate, tolbutamide. Covalent binding of TA metabolites to microsomal proteins occurred in parallel with enzyme inactivation and was partially inhibited by the presence of glutathione in the reaction medium. However, glutathione did not protect P450 enzyme from inactivation. Thus, TA exhibited all of the characteristics of a mechanism-based inactivator for P450 2C9 and 2C10 enzymes. The following kinetic parameters were determined in the case of P450 2C10: t1/2,max = 3.4 min, k(inact) = 3.6 10(-3) s-1, KI = 4.3 microM, k(inact)/KI = 813 L mol-1 s-1, and partition ratio = 11.6. Moreover, a specific covalent binding of 0.9 mol of TA metabolite per mole of P450 2C10 was found to occur before the complete loss of enzyme activity (in incubations performed in the presence of glutathione). A plausible mechanism for P450 2C10 (2C9) inactivation during TA oxidation is proposed. It involves the intermediate formation of an electrophilic thiophene sulfoxide, which may react at position 5 of its thiophene ring either with H2O to give 5-OHTA or with a nucleophilic group of an amino acid residue of the P450 active site, which results in its covalent binding to P450 protein. This alkylation and inactivation of P450 2C9 (2C10) by TA could be a starting point for the appearance of anti-P450 2C antibodies detected in patients treated with TA and suffering from immunoallergic hepatitis.
López-Garcia
M P
MP
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, URA 400 CNRS, Université René Descartes, Paris, France.
Dansette
P M
PM
Mansuy
D
D
D001189:Aryl Hydrocarbon Hydroxylases; D003001:Cloning, Molecular; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008962:Models, Theoretical; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D013989:Ticrynafen / Q000494:pharmacology*; D013997:Time Factors; D014044:Tolbutamide / Q000494:pharmacology
D065607:Cytochrome P-450 Enzyme Inhibitors; D018076:DNA, Complementary; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D005978:Glutathione; D013989:Ticrynafen