In vitro glucuronidation of 7-hydroxycoumarin derivatives in intestine and liver microsomes of Beagle dogs.

Beagle dog is a standard animal model for evaluating nonclinical pharmacokinetics of new drug candidates. Glucuronidation in intestine and liver is an important first-pass drug metabolic pathway, especially for phenolic compounds. This study evaluated the glucuronidation characteristics of several 7-hydroxycoumarin derivatives in beagle dog's intestine and liver in vitro. To this end, glucuronidation rates of 7-hydroxycoumarin (compound 1), 7-hydroxy-4-trifluoromethylcoumarin (2), 6-methoxy-7-hydroxycoumarin (3), 7-hydroxy-3-(4-tolyl)coumarin (4), 3-(4-fluorophenyl)coumarin (5), 7-hydroxy-3-(4-hydroxyphenyl)coumarin (6), 7-hydroxy-3-(4-methoxyphenyl)coumarin (7), and 7-hydroxy-3-(1H-1,2,4-tirazole)coumarin (8) were determined in dog's intestine and liver microsomes, as well as recombinant dog UGT1A enzymes. The glucuronidation rates of 1, 2 and 3 were 3-10 times higher in liver than in small intestine microsomes, whereas glucuronidation rates of 5, 6, 7 and 8 were similar in microsomes from both tissues. In the colon, glucuronidation of 1 and 2 was 3-5 times faster than in small intestine. dUGT1A11 glucuronidated efficiently all the substrates and was more efficient catalyst for 8 than any other dUGT1A. Other active enzymes were dUGT1A2 that glucuronidated efficiently 2, 3, 4, 5, 6 and 7, while dUGT1A10 glucuronidated efficiently 1, 2, 3, 4, 5 and 7. Kinetic analyses revealed that the compounds' Km values varied between 1.1 (dUGT1A10 and 2) and 250 µM (dUGT1A7 and 4). The results further strengthen the concept that dog intestine has high capacity for glucuronidation, and that different dUGT1As mediate glucuronidation with distinct substrates selectivity in dog and human.

Data on biosynthesis of BPAF glucuronide, enzyme kinetics of BPAF glucuronidation, and molecular modeling.

Bisphenol AF (BPAF) is in the body mainly metabolized to the corresponding bisphenol AF glucuronide (BPAF-G). While BPAF-G is not commercially available, enzyme-assisted synthesis of BPAF-G using the human recombinant enzyme UGT2A1, purification of BPAF-G by solid phase extraction and semi-preparative HPLC and chemical characterization of BPAF-G by NMR and LC-MS/MS were performed and are described here. Furthermore, BPAF glucuronidation kinetics with the UGT enzymes that showed the highest glucuronidation activity in previous studies (i.e hepatic UGTs 1A3, 2B7, and 2B17, intestinal UGT 1A10 and UGT 2A1 that is present in airways) was performed and data is presented. Hepatic enzymes exhibited high affinities toward BPAF, while extrahepatic UGTs 2A1 and 1A10 showed the high vmax values (3.3 and 3.0 nmol/min/mg, respectively). To understand molecular interactions of BPA, BPAF and BPAF-G with ligand biding sites of several nuclear receptors, molecular modeling was performed and data on the binding modes of BPAF, BPA, and BPAF-G in the ligand-binding sites of nuclear receptors are presented. This article is related to "Endocrine activities and adipogenic effects of bisphenol AF and its main metabolite" (Skledar et al., 2019).

Endocrine activities and adipogenic effects of bisphenol AF and its main metabolite.

Bisphenol AF (BPAF) is a fluorinated analog of bisphenol A (BPA), and it is a more potent estrogen receptor (ER) agonist. BPAF is mainly metabolized to BPAF-glucuronide (BPAF-G), which has been reported to lack ER agonist activity and is believed to be biologically inactive. The main goal of the current study was to examine the influence of the metabolism of BPAF via glucuronidation on its ER activity and adipogenesis. Also, as metabolites can have different biological activities, the effects of BPAF-G on other nuclear receptors were evaluated. First, in-vitro BPAF glucuronidation was investigated using recombinant human enzymes. Specific reporter-gene assays were used to determine BPAF and BPAF-G effects on estrogen, androgen, glucocorticoid, and thyroid receptor pathways, and on PXR, FXR, and PPARγ pathways. Their effects on lipid accumulation and differentiation were determined in murine 3T3L1 preadipocytes using Nile Red, with mRNA expression analysis of the adipogenic markers adiponectin, Fabp4, Cebpα, and PPARγ. BPAF showed strong agonistic activity for hERα and moderate antagonistic activities for androgen and thyroid receptors, and for PXR. BPAF-G was antagonistic for PXR and PPARγ. BPAF (0.1 µM) and BPAF-G (1.0 µM) induced lipid accumulation and increased expression of key adipogenic markers in murine preadipocytes. BPAF-G is therefore not an inactive metabolite of BPAF. Further toxicological and epidemiological investigations of BPAF effects on human health are warranted, to provide better understanding of the metabolic end-elimination of BPAF.

Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10.

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4-(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.

Identification and characterization of human UDP-glucuronosyltransferases responsible for xanthotoxol glucuronidation.

1. Xanthotoxol is a furanocoumarin that possesses many pharmacological activities and in this study its in vitro glucuronidation was studied. 2. Xanthotoxol can be rapidly metabolized to a mono-glucuronide in both human intestine microsomes (HIM) and human liver microsomes (HLM); the structure of the metabolite was confirmed by NMR spectroscopy. 3. Reaction phenotyping with 12 commercial recombinant human UGTs, as well as with the Helsinki laboratory UGT1A10 that carry a C-terminal His-tag (UGT1A10-H), revealed that UGT1A10-H catalyzes xanthotoxol glucuronidation at the highest rate, followed by UGT1A8. The other enzymes, namely UGT1A3, UGT1A1, UGT1A6, UGT1A10 (commercial), and UGT2B7 displayed moderate-to-low reaction rates. 4. In kinetic analyses, HIM exhibited much higher affinity for xanthotoxol, along with high Vmax and mild substrate inhibition, whereas the kinetics in HLM was biphasic. UGT1A1 (high Km value), UGT1A10-H (low Km value), and UGT1A8 exhibited mild substrate inhibition. 5. Considering the above findings and the current knowledge on UGTs expression in HIM, it is likely that UGT1A10 is mainly responsible for xanthotoxol glucuronidation in the human small intestine, with some contribution from UGT1A1. In the liver, this reaction is mainly catalyzed by UGT1A1 and UGT2B7. 6. Glucuronidation appears to be the major metabolic pathway of xanthotoxol in human.

Selectivity in the Efflux of Glucuronides by Human Transporters: MRP4 Is Highly Active toward 4-Methylumbelliferone and 1-Naphthol Glucuronides, while MRP3 Exhibits Stereoselective Propranolol Glucuronide Transport.

Xenobiotic and endobiotic glucuronides, which are generated in hepatic and intestinal epithelial cells, are excreted via efflux transporters. Multidrug resistance proteins 2-4 (MRP2-MRP4) and the breast cancer resistance protein (BCRP) are efflux transporters that are expressed in these polarized cells, on either the basolateral or apical membranes. Their localization, along with expression levels, affects the glucuronide excretion pathways. We have studied the transport of three planar cyclic glucuronides and glucuronides of the two propranolol enantiomers, by the vesicular transport assay, using vesicles from baculovirus-infected insect cells expressing human MRP2, MRP3, MRP4, or BCRP. The transport of estradiol-17ß-glucuronide by recombinant MRP2-4 and BCRP, as demonstrated by kinetic values, were within the ranges previously reported. Our results revealed high transport rates and apparent affinity of MRP4 toward the glucuronides of 4-methylumbelliferone, 1-naphthol, and 1-hydroxypyrene (Km values of 168, 13, and 3 µM, respectively) in comparison to MRP3 (Km values of 278, 98, and 8 µM, respectively). MRP3 exhibited lower rates, but stereoselective transport of propranolol glucuronides, with higher affinity toward the R-enantiomer than the S-enantiomer (Km values 154 vs 434 µM). The glucuronide of propranolol R-enantiomer was not significantly transported by either MRP2, MRP4, or BCRP. Of the tested small glucuronides in this study, BCRP transported only 1-hydroxypyrene glucuronide, at very high rates and high apparent affinity (Vmax and Km values of 4400 pmol/mg/min and 11 µM). The transport activity of MRP2 with all of the studied small glucuronides was relatively very low, even though it transported the reference compound, estradiol-17ß-glucuronide, at a high rate (Vmax = 3500 pmol/mg/min). Our results provide new information, at the molecular level, of efflux transport of the tested glucuronides, which could explain their disposition in vivo, as well as provide new tools for in vitro studies of MRP3, MRP4, and BCRP.

UGT1A10 Is a High Activity and Important Extrahepatic Enzyme: Why Has Its Role in Intestinal Glucuronidation Been Frequently Underestimated?

The aim of this work was to highlight a considerable and broad problem in UGT1A10 activity assessment that has led to underestimation of its role in intestinal glucuronidation of drugs and other xenobiotics. The reason appears to be poor activity of the commercial UGT1A10 that is used by many laboratories, and here we have tested it by comparison with our recombinant His-tagged UGT1A10 (designated as UGT1A10-H), both expressed in insect cells. The glucuronidation rates of morphine, estradiol, estrone, SN-38, diclofenac, 4-methylumbelliferone, 7-amino-4-methylcoumarin, N-(3-carboxypropyl)-4-hydroxy-1,8-naphthalimide, and bavachinin were assayed. The results revealed that the activity of commercial UGT1A10 was low, very low, and in the cases of morphine, estrone, 7-methyl-4-aminocoumarin, and bavachinin it was below the detection limit. On the other hand, under the same conditions, UGT1A10-H exhibited high glucuronidation rates toward all these compounds. Moreover, using estradiol, morphine, and estrone, in the presence and absence of suitable inhibitors, nilotinib or atractylenolide I, it was demonstrated that UGT1A10-H, but not the commercial UGT1A10, provides a good tool to study the role of native UGT1A10 in the human intestine. The results also suggest that much of the data in the literature on UGT1A10 activity may have to be re-evaluated.

The Polymorphic Variant P24T of UDP-Glucuronosyltransferase 1A4 and Its Unusual Consequences.

The P24T polymorphic variant of the human UDP-glucuronosyltransferase 1A4 (UGT1A4*2, 70C>A) occurs within the signal peptide, five amino acids upstream of the cleavage site and the start of the mature protein. Bioinformatic analysis of the variant suggested that the signal peptide of part of the translated protein is cleaved two residues upstream of the regular site, whereas the rest is cleaved as usual. To test this, recombinant UGT1A4-P24T, with a C-terminal His-tag, was expressed in sf9 insect cells and affinity-purified for N-terminal protein sequencing. The results were in agreement with the in silico prediction. About half of the mutant protein was cleaved at the regular site, between S28 and G29, whereas the other half was cleaved two amino acids upstream, between A26 and E27. The glucuronidation of two substrates, dexmedetomidine and trifluoperazine, was assayed using membrane-enriched UGT1A4-P24T and wild-type UGT1A4. The variant exhibited much lower glucuronidation rates, but kinetic analyses revealed large differences between them only in the Vmax values. The Km values for both substrates were not affected by the mutation and its consequences. This might suggest that the unusual signal peptide cleavage in UGT1A4-P24T somehow disturbs protein folding. Moreover, it raises the possibility that the effect of UGT1A4-P24T on the glucuronidation rate in mammalian expression systems would be mild since they contain more effective post-translation protein control systems in the endoplasmic reticulum. In summary, our results reveal the effect of a polymorphic mutation on the signal sequence cleavage and thereby also the mature UGT.

An optimized ratiometric fluorescent probe for sensing human UDP-glucuronosyltransferase 1A1 and its biological applications.

This study aimed to develop a practical ratiometric fluorescent probe for highly selective and sensitive detection of human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most important phase II enzymes. 4-Hydroxy-1,8-naphthalimide (HN) was selected as the fluorophore for this study because it possesses intramolecular charge transfer (ICT) feature and displays outstanding optical properties. A series of N-substituted derivatives with various hydrophobic, acidic and basic groups were designed and synthesized to evaluate the selectivity of HN derivatives toward UGT1A1. Our results demonstrated that the introduction of an acidic group to HN could significantly improve the selectivity of UGT1A1. Among the synthesized fluorescent probes, NCHN (N-3-carboxy propyl-4-hydroxy-1,8-naphthalimide) displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following UGT1A1-catalyzed glucuronidation. UGT1A1-catalyzed NCHN-4-O-glucuronidation generated a single fluorescent product with a high quantum yield (Φ=0.688) and brought remarkable changes in both color and fluorescence in comparison with the parental substrate. The newly developed probe has been successfully applied for sensitive measurements of UGT1A1 activities in human liver preparations, as well as for rapid screening of UGT1A1 modulators, using variable enzyme sources. Furthermore, its potential applications for live imaging of endogenous UGT1A1in cells have also been demonstrated.

Dog UDP-glucuronosyltransferase enzymes of subfamily 1A: cloning, expression, and activity.

Understanding drug glucuronidation in the dog, a preclinical animal, is important but currently poorly characterized at the level of individual enzymes. We have constructed cDNAs for the 10 dog UDP-glucuronosyltransferases of subfamily 1A (dUGT1As), expressed them in insect cells, and assayed their activity as well as the activity of the nine human UGT1As, toward 14 compounds. The goal was to find out whether individual dUGT1As and individual human UGT1As have similar substrate specificities. The results revealed similarities but also many differences. For example, similarly to the human UGT1A10, dUGT1A11 exhibited high glucuronidation activity toward the 3-OH of 17-ß-estradiol, 17-α-estradiol, and ethinylestradiol, and also conjugated the drug entacapone. Unlike the human UGT1A10, however, it failed to catalyze considerable rates of R-propranolol, diclofenac, and indomethacin glucuronidation. The estrogen glucuronidation assays revealed that dUGT1A8 and dUGT1A10 have a capacity to catalyze the formation of (linked) diglucuronides, an activity no human UGT1A exhibited. dUGT1A2-dUGT1A4 are homologs of the human UGT1A4, but none of them catalyzed N-glucuronidation of dexmedetomidine. Contrary to the human UGT1A4, however, dUGT1A2-dUGT1A4 catalyzed indomethacin and diclofenac glucuronidation. It may be concluded that, perhaps with the exception of UGT1A6, high similarities in substrate specificity between individual dog and human UGTs of subfamily 1A are rare or partial. Activity assays with liver and intestine microsomes of both dog and human further revealed interspecies differences, particularly in glucuronidation rates. In the dog, the microsomes assays also strongly suggested important roles for dUGTs of other subfamilies, mainly in the liver.

Differences in the glucuronidation of bisphenols F and S between two homologous human UGT enzymes, 1A9 and 1A10.

1. Bisphenol S (BPS) and bisphenol F (BPF) are bisphenol A (BPA) analogues commonly used in the manufacturing of industrial and consumer products. 2. Bisphenols are often detoxified through conjugation with glucuronic acid or sulfate. In this work, we have examined the glucuronidation of BPS and BPF by recombinant human UDP-glucuronosyltransferase (UGT) enzymes. In addition, we have reexamined BPA glucuronidation, using extra-hepatic UGTs that were not tested previously. 3. The results revealed that UGT1A9, primarily a hepatic enzyme, is mainly responsible for BPS glucuronidation, whereas UGT1A10, an intestine enzyme that is highly homologous to UGT1A9 at the protein level, is by far the most active UGT in BPF glucuronidation. In contrast to the latter two UGTs that display significant specificity in the glucuronidation of BPS and BPF, UGT2A1 that is mainly expressed in the airways, exhibited high activity toward all the tested bisphenols, BPS, BPF and BPA. UGT1A10 exhibited somewhat higher BPA glucuronidation activity than UGT1A9, but it was lower than UGT2A1 and UGT2B15. 4. The new findings demonstrate interesting differences in the glucuronidation patterns of bisphenols and provide new insights into the role of extra-hepatic tissues in their detoxification.

Albumin stimulates the activity of the human UDP-glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the effects are enzyme and substrate dependent.

Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro-in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2-4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction's K(m), increasing its V(max), or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions' K(m) are concerned. In the cases of V(max) values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V(max) increases. Additionally, the BSA effects may be UGT subfamily dependent since K(m) decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V(max) increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs.