title: Product inhibition of UDP-glucuronosyltransferase (UGT) enzymes by UDP obfuscates the inhibitory effects of UGT substrates.
abstract: Substrates that are specific for certain UDP-glucuronosyltransferase (UGT) isoforms are usually used as specific inhibitors to identify UGT isoforms responsible for the glucuronidation of drugs. 1-Naphthol and 4-nitrophenol are probe substrates for human UGT1A6. In the present study, we found that UGT1A1-catalyzed estradiol 3-O-glucuronide formation and UGT1A4-catalyzed imipramine N-glucuronide formation in human liver microsomes were prominently decreased in the presence of 1-naphthol, but those by recombinant human UGT1A1 and UGT1A4, respectively, were not. Interestingly, when recombinant UGT1A6 was added in the reaction mixture, these activities by recombinant UGT1A1 and UGT1A4 were diminished in the presence of 1-naphthol. To interpret this phenomenon, the inhibitory effects of 1-naphthol O-glucuronide and UDP, products of the glucuronidation of 1-naphthol, were investigated. We found that UDP strongly inhibited the UGT1A1 (K(i) = 7 microM) and UGT1A4 (K(i) = 47 microM) activities in a competitive manner for the 5'-diphosphoglucuronic acid binding. These results suggest that UDP produced by UGT1A6-catalyzed 1-naphthol glucuronidation, but not 1-naphthol O-glucuronide and 1-naphthol per se, is the actual inhibition substance. Next, we examined the inhibitory effects of 15 compounds that are substrates of UGTs on estradiol 3-O-glucuronide formation in human liver microsomes compared with those by recombinant UGT1A1. Among them, 4 compounds (1-naphthol, 2-naphthol, 4-nitrophenol, and 4-methylumbelliferone) with high turnover rates (V(max)/K(m) value >200 mul/min/mg) showed more potent inhibition of the activity in human liver microsomes compared with that by the recombinant UGT1A1. Thus, we should pay attention to the inhibitory effects of UDP on UGT, which may cause erroneous evaluations in inhibition studies using human liver microsomes.
authors.affiliation: Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Fujiwara
authors.forename: Miki
authors.initials: M
authors.lastname: Nakajima
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamanaka
authors.forename: Miki
authors.initials: M
authors.lastname: Katoh
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yokoi
mesh_terms: D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D007099:Imipramine / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D009284:Naphthols / Q000494:pharmacology; D015742:Propofol / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D014530:Uridine Diphosphate / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D020719:Glucuronides; D009284:Naphthols; D011994:Recombinant Proteins; C029350:1-naphthol; D012701:Serotonin; D014530:Uridine Diphosphate; D014453:Glucuronosyltransferase; D007099:Imipramine; D015742:Propofol