Product Inhibition of UDP-Glucuronosyltransferase (UGT) Enzymes by UDP Obfuscates the Inhibitory Effects of UGT Substrates

Abstract

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 M) and UGT1A4 (K i ‫؍‬ 47 M) 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 l/ 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.UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes that catalyze the formation of glucuronides by the transfer of glucuronic acid from a cofactor uridine 5Ј-diphosphoglucuronic acid (UDPGA) to hydroxyl, carboxyl, or amine groups of endogenous and exogenous substrates (Dutton, 1980). The hydrophilic glucuronides can readily be excreted from the body via bile and urine. Human UGTs are classified into two subfamilies, UGT1 and UGT2, based on similarities between their amino acid sequences and gene organization (Mackenzie et al., 2005). To date, 19 human UGT isoforms, UGT1A1, -1A3, -1A4, -1A5, -1A6, -1A7, -1A8, -1A9, -1A10, -2A1, -2A2, -2A3, -2B4, -2B7, -2B10, -2B11, -2B15, -2B17, and -2B28, have been identified (Mackenzie et al., 2005). Most UGT enzymes are expressed in liver, but some isoforms such as UGT1A7, -1A8, and -1A10 are exclusively expressed in intestine Burchell et al., 2001).Most pharmacokinetic drug-drug interactions occur at the metabolic level and usually involve changes in the activity of the major drug-metabolizing enzymes such as UGT. Identification of the UGT(s) involved in the metabolism of a given compound allows us to predict potential drug-drug ...

Glucuronidation of Dihydrotestosterone and trans-Androsterone by Recombinant UDP-Glucuronosyltransferase (UGT) 1A4: Evidence for Multiple UGT1A4 Aglycone Binding Sites

“…However, such a mechanism in which only one aglycone substrate binding site is incorporated does not adequately explain the activation effect of TAM on DHT and t-AND glucuronidation. UDP, a product of catalysis, has been reported to be an inhibitor for UGT1A4 (IC 50 ϭ 31 M) (Fujiwara et al, 2008). It is also possible that the observed substrate inhibition is due to the increased amount of UDP formation at high substrate concentrations.…”

Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: Advances and open questions

“…This observation has been termed 'albumin effect' [59]. (iii) As mentioned in chapter 2, rapid hydrolysis of UDP in the ER may explain the lack of product inhibition by UDP [15]. In addition, rapid translocation of glucuronides from ER into the cytoplasm may further decrease product inhibition in vivo.…”

“…However, the reversible reaction may only rarely occur in the intact cell since the two products of the UGT reaction are rapidly removed: (i) UDP is rapidly hydrolyzed to UMP by nucleoside diphosphatase in the hepatic ER lumen [14]. Rapid hydrolysis of UDP in microsomes may explain the lack of UDP-dependent inhibition of UGT reactions in liver microsomes in contrast to preparations from expressed UGTs [15]. (ii) Glucuronides appear to be rapidly translocated to the cytosol.…”

Metabolic Drug-Drug Interaction Potential of Macrolactin A and 7- O -Succinyl Macrolactin A Assessed by Evaluating Cytochrome P450 Inhibition and Induction and UDP-Glucuronosyltransferase Inhibition In Vitro

“…Nilotinib, hecogenin, 1-naphthol, niflumic acid, and efavirenz were used as well-known inhibitors for UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7, respectively. All substrates and inhibitors used as positive controls were selected according to published reports (24)(25)(26)(27)(28)(29). On the basis of the observed potency, experiments to determine the K i values of MA and SMA for UGT1A1 were conducted.…”