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

Abstract

ABSTRACT:UDP-glucuronosyltransferase (UGT) 1A4-catalyzed glucuronidation is an important drug elimination pathway. Although atypical kinetic profiles (nonhyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucuronidation have been reported occasionally, systematic kinetic studies to explore the existence of multiple aglycone binding sites in UGT1A4 have not been conducted. To this end, two positional isomers, dihydrotestosterone (DHT) and trans-androsterone (t-AND), were used as probe substrates, and their glucuronidation kinetics with HEK293-expressed UGT1A4 were evaluated both alone and in the presence of a UGT1A4 substrate [tamoxifen (TAM) or lamotrigine (LTG)]. Coincubation with TAM, a high-affinity UGT1A4 substrate, resulted in a concentrationdependent activation/inhibition effect on DHT and t-AND glucuronidation, whereas LTG, a low-affinity UGT1A4 substrate, noncompetitively inhibited both processes. The glucuronidation kinetics of TAM were then evaluated both alone and in the presence of different concentrations of DHT or t-AND. TAM displayed substrate inhibition kinetics, suggesting that TAM may have two binding sites in UGT1A4. However, the substrate inhibition kinetic profile of TAM became more hyperbolic as the DHT or t-AND concentration was increased. Various two-site kinetic models adequately explained the interactions between TAM and DHT or TAM and t-AND. In addition, the effect of TAM on LTG glucuronidation was evaluated. In contrast to the mixed effect of TAM on DHT and t-AND glucuronidation, TAM inhibited LTG glucuronidation. Our results suggest that multiple aglycone binding sites exist within UGT1A4, which may result in atypical kinetics (both homotropic and heterotropic) in a substrate-dependent fashion.

The UGT1 locus is a determinant of prostate cancer recurrence after prostatectomy

“…On the basis of the substratespecificity of the nine functional UGT1As involved in estrogen catabolism, we expected to find little if any influence on formation of androgen glucuronides associated with htSNPs. In fact, few reported results support the ability of recombinant UGT1As to conjugate androgens, with the exception of UGT1A3 and UGT1A4, which have weak activity toward DHT and its metabolite ADT compared with UGT2B enzymes (Gall et al 1999, Zhou et al 2010. This is consistent with our observation that no significant changes in circulating androgen glucuronides are observed for carriers of UGT1-related risk haplotypes.…”

“…In contrast to UGT2B enzymes, UGT1As are mainly regulators of tissue exposure to estrogens, because most of them are involved in the glucuronidation of parental estrogen (estradiol (E 2 ) and estrone (E 1 )), and inactivation of their hydroxyl moieties at position 2 or 4 as well as methoxy catechol estrogens (Gall et al 1999, Lepine et al 2004, Murai et al 2006, Starlard-Davenport et al 2007, Zhou et al 2010, Sneitz et al 2013. Conversely, there is little evidence to support them having a role in the inactivation of androgens, e.g., the potent androgen, dihydrotestosterone (DHT; Gall et al 1999, Zhou et al 2010. UGT1A1, UGT1A3, UGT1A8, UGT1A9, and UGT1A10 conjugate E 1 and E 2 most efficiently, whereas UGT1A4 is less efficient.…”

Correlation between Bilirubin Glucuronidation and Estradiol-3-Gluronidation in the Presence of Model UDP-Glucuronosyltransferase 1A1 Substrates/Inhibitors

“…Atypical kinetic profiles, classified by homotropic and heterotropic cooperativity, have been increasingly reported with UGTs. Similar to the cytochromes P450, UGTs have been described to exhibit substrate-dependent autoactivation (Udomuksorn et al, 2007), substrate inhibition (Zhou et al, 2010b), and biphasic kinetics (Stone et al, 2003). Because of this propensity of the UGTs to exhibit atypical kinetic properties, a compound may activate the glucuronidation of one substrate but inhibit or have no effect on the glucuronidation of a second substrate catalyzed by the same UGT (Uchaipichat et al, 2008;Zhou et al, 2010b).…”

“…Similar to the cytochromes P450, UGTs have been described to exhibit substrate-dependent autoactivation (Udomuksorn et al, 2007), substrate inhibition (Zhou et al, 2010b), and biphasic kinetics (Stone et al, 2003). Because of this propensity of the UGTs to exhibit atypical kinetic properties, a compound may activate the glucuronidation of one substrate but inhibit or have no effect on the glucuronidation of a second substrate catalyzed by the same UGT (Uchaipichat et al, 2008;Zhou et al, 2010b). In a recent study, we investigated the effect of tamoxifen on UGT1A4-catalyzed dihydrotestosterone, trans-androsterone, and lamotrigine glucuronidation and discovered that tamoxifen exhibited a concentration-dependent activation/inhibition of dihydrotestosterone and trans-androsterone glucuronidation, but only inhibited lamotrigine glucuronidation (Zhou et al, 2010b).…”

“…Because of this propensity of the UGTs to exhibit atypical kinetic properties, a compound may activate the glucuronidation of one substrate but inhibit or have no effect on the glucuronidation of a second substrate catalyzed by the same UGT (Uchaipichat et al, 2008;Zhou et al, 2010b). In a recent study, we investigated the effect of tamoxifen on UGT1A4-catalyzed dihydrotestosterone, trans-androsterone, and lamotrigine glucuronidation and discovered that tamoxifen exhibited a concentration-dependent activation/inhibition of dihydrotestosterone and trans-androsterone glucuronidation, but only inhibited lamotrigine glucuronidation (Zhou et al, 2010b). Similar context-dependent heterotropic effects were also reported by Uchaipichat et al (2008), who reported that 4-methylumbelliferone-activated UGT2B7 catalyzed 1-naphthol glucuronidation but inhibited zidovudine glucuronidation.…”