Human UDP-glucuronosyltransferase 1A1 Research Articles

Collaborative modification strategy to develop a highly selective fluorescent probe for human UDP-glucuronosyltransferase 1A10

UDP-glucuronosyltransferase 1A10 (UGT1A10) is an important phase II human metabolic enzyme. However, few methods are available to facilely monitor the function of UGT1A10 in biological systems. Here we report the first sensitive and selective fluorescent “off–on” UGT1A10 probe substrate UPr6. Using a collaborative modification strategy, we introduced a rigid propyl morpholine group to the amide and a methoxy group next to 4′-phenoxy of the 4-phenyl-1,8-naphthalimide skeleton to obtain UPr6. Visualization methods were established to evaluate the function of UGT1A10 in living cells, rat tissues, and zebrafish. From a Chinese herbal medicine compound library, two natural products EUC and DAP from licorice were discovered as potent UGT1A10 inhibitors, which remarkably improved the sensitivity of CPT-11 against a resistant human cancer cell line. This work established a feasible strategy to rationally design isoform-specific UGT probe substrates.

Computational prediction for the metabolism of human UDP-glucuronosyltransferase 1A1 substrates

UDP-glucuronosyltransferase (UGT) 1A1, one of the most important isoforms in UGTs superfamily, has attracted increasing concerns for its special role in the clearance and detoxification of endogenous and exogenous substances. To avoid the clinical drug-drug interactions, it is of great importance to have the knowledge of the metabolic profile of UGT1A1 substrates early. Herein, we purposed to establish machine learning models to predict the metabolic propeties of UGT1A1 substrates. On the basis of the literature-derived substrates database of UGT1A1, automatic metabolism prediction models for the aromatic hydroxyl (ArOH) and carboxyl (COOH) groups were developed with eight machine learning methods, among which, three methods, i.e. Random Forest, Random Subspace and J48, illustrated the best performance either for the aromatic hydroxyl and the carboxyl model. The models illustrated good robustness when they were evaluated with functions like “Precision”, “Recall”, “F-Measure”, “AUC”, “MCC”, etc. Nice accuracy was observed for the aromatic hydroxyl and carboxyl model of these methods, whose AUCs ranged from 0.901 to 0.997. Additionally, the ArOH model was applied to predict the UGT1A1-mediated metabolism of an external set. Two new unknown substrates, cytochrome P450 (CYPs)-mediated metabolites of gefitinib, were predicted and identified, which were validated by in vitro assays. In summary, this study provides a reliable and robust strategy to predict UGT1A1 metabolites, which will be helpful either in rational-optimization of drug metabolism or in avoiding drug-drug interactions in clinic.

Discovery and Characterization of the Key Constituents in Ginkgo biloba Leaf Extract With Potent Inhibitory Effects on Human UDP-Glucuronosyltransferase 1A1.

Human UDP-glucuronosyltransferase 1A1 (hUGT1A1) is one of the most essential phase II enzymes in humans. Dysfunction or strong inhibition of hUGT1A1 may result in hyperbilirubinaemia and clinically relevant drug/herb-drug interactions (DDIs/HDIs). Recently, a high-throughput fluorescence-based assay was constructed by us to find the compounds/herbal extracts with strong inhibition against intracellular hUGT1A1. Following screening of over one hundred of herbal products, the extract of Ginkgo biloba leaves (GBL) displayed the most potent hUGT1A1 inhibition in HeLa-UGT1A1 cells (Hela cells overexpressed hUGT1A1). Further investigations demonstrated that four biflavones including bilobetin, isoginkgetin, sciadopitysin and ginkgetin, are key constituents responsible for hUGT1A1 inhibition in living cells. These biflavones potently inhibit hUGT1A1 in both human liver microsomes (HLM) and living cells, with the IC50 values ranging from 0.075 to 0.41 μM in living cells. Inhibition kinetic analyses and docking simulations suggested that four tested biflavones potently inhibit hUGT1A1-catalyzed NHPN-O-glucuronidation in HLM via a mixed inhibition manner, showing the K i values ranging from 0.07 to 0.74 μM. Collectively, our findings uncover the key constituents in GBL responsible for hUGT1A1 inhibition and decipher their inhibitory mechanisms against hUGT1A1, which will be very helpful for guiding the rational use of GBL-related herbal products in clinical settings.

Metabolism of 2,3-Dehydrosilybin A and 2,3-Dehydrosilybin B: A Study with Human Hepatocytes and Recombinant UDP-Glucuronosyltransferases and Sulfotransferases.

2,3-Dehydrosilybin A and 2,3-dehydrosilybin B are a pair of enantiomers formed by the oxidation of the natural flavonolignans silybin A and silybin B, respectively. However, the antioxidant activity of 2,3-dehydrosilybin molecules is much stronger than that of their precursors. Here, we investigated the biotransformation of pure 2,3-dehydrosilybin A and 2,3-dehydrosilybin B in isolated human hepatocytes, and we also aimed to identify human UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) with activity toward their respective enantiomers. After incubation with hepatocytes, both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B were converted to hydroxyl derivatives, methylated hydroxyl derivatives, methyl derivatives, sulfates, and glucuronides. The products of direct conjugations predominated over those of oxidative metabolism, and glucuronides were the most abundant metabolites. Furthermore, we found that recombinant human UGTs 1A1, 1A3, 1A7, 1A8, 1A9, and 1A10 were capable of catalyzing the glucuronidation of both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B. UGTs 1A1 and 1A7 showed the highest activity toward 2,3-dehydrosilybin A, and UGT1A9 showed the highest activity toward 2,3-dehydrosilybin B. The sulfation of 2,3-dehydrosilybin A and B was catalyzed by SULTs 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, and 1E1, of which SULT1A3 exhibited the highest activity toward both enantiomers. We conclude that 2,3-dehydrosilybin A and B are preferentially metabolized by conjugation reactions, and that several human UGT and SULT enzymes may play a role in these conjugations.

In vitro inhibition of human UDP-glucuronosyltransferase (UGT) 1A1 by osimertinib, and prediction of in vivo drug-drug interactions

Osimertinib is the only third-generation epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI) approved by Food and Drug Administration (FDA). This study aimed to know the inhibitory effect of osimertinib on human UDP-glucosyltransferases (UGTs) and human liver microsomes (HLMs), as well as to identify its potential to cause drug-drug interaction (DDI) arising from the modulation of UGT activity. High inhibitory effect of osimertinib was shown towards UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A10, 2B7 and 2B15. Especially, osimertinib exhibited competitive inhibition against UGT1A1 with a Ki,u of 0.87 ± 0.12 μM. It also noncompetitively inhibited SN-38 glucuronidation in pooled HLMs with a Ki,u of 3.32 ± 0.25 μM. Results from quantitative prediction study indicated that osimertinib administered at 80 mg/day may result in a 4.83 % increase in the AUC of drugs mainly metabolized by UGT1A1, implying low risk of DDI via liver metabolism. However, the ratios of [I]gut/Ki,u are much higher than 11 in HLMs and recombinant UGT1A1, indicating a risk for interaction in intestine. The effects of osimertinib on intestinal UGT should be paid more attention on to avoid unnecessary clinical DDI risks.

Metabolism of flutamide in diet control fischer 344 and brown Norway x F 344 rats, and its hydroxylation and conjugation by human CYP450s and UDP-glucuronosyltransferases

In our current report, flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl)-phenyl] propanamide) treated diet control and non limited-fed Fischer 344 (F344) and Brown Norway (BN) x F344 rats showed that diet control reduces spontaneous and flutamide-induced hyperplasia((1)). In this continued study, we found that serum concentrations of active metabolite of flutamide, 2-hydroxyflutamide (OH-flu), were 181 ± 26.6 ng/mL and 68 ± 8.0 ng/mL (p<0.05), in non-limited fed and diet control F344 rats. In BN x F344 rats, the serum concentrations of 2-OH-flutamide were 232 ± 57 ng/mL and 52 ± 6 ng/mL (p<0.05) in non-limited fed and diet control animals. In diet control groups, liver microsomes from flutamide-treated F344 rats showed high 7-ethoxyresorufin O-deethylase (EROD) activity, while 7-Benzoxyresorufin O-dealkylase (BROD) activity was not affected significantly. Both rat and human liver microsomes showed flutamide oxidation activity. Human liver microsomes showed 10 times higher activity than rat liver microsomes (0.673 ± 0.04 vs 0.063 ± 0.008 nmol OH-flu/min/mg protein). Microsomes from human tissues such as colon, colon cancer, kidney, bladder, pancreas, prostate, prostate cancer, or ovarian cancer, showed no or non-detectable activity for flutamide hydroxylation. CYP450 1A1, 1A2, 1B1 and 2C19 from human lymphoblastoid cell lines, oxidized flutamide to OH-flu in vitro with activities from 0.118 ± 0.005 to 0.275 ± 0.010 nmol/min/mg protein. Microsomes isolated from human kidney, colon, and liver showed UDP-glucuronosyltransferase (UGT) activity for glucuronidation of OH-flu. Human kidney showed the highest activity. Several human recombinant UGTs (1A1, 1A4, 1A6, 1A7, 1A9, and 1A10) also showed glucuronidation activity for OH-flu. It was found that UGT1A6 was more active than other human UGTs.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems

The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quantitative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Furthermore, the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small molecules on this conjugative enzyme.

Comparisons between Animal Models and Intestinal Crypt Cultures when Investigating the Impact of Fatty Acids on Gene Expression in Intestinal Epithelial Cells

Developmental delay of UDP‐glucuronosyltransferase 1A1 (UGT1A1) expression is central toward the development of neonatal hyperbilirubinemia, observed clinically as increased total serum bilirubin (TSB) in newborns. UGT1A1 is the sole enzyme responsible for the glucuronidation and detoxification of bilirubin. We have developed a murine model that resembles neonatal hyperbilirubinemia. In humanized UGT1 (hUGT1) mice, the expression of intestinal but not hepatic UGT1A1 is inversely associated with TSB levels. In addition, the expression of intestinal UGT1A1 is closely associated with proliferation and maturation of intestinal epithelial cells (IECs). It is known that fatty acids (FAs), the major component of breast milk, plays an important role in intestinal development. We have treated hUGT1 neonatal mice with oleic acid (OA, 16:1), a monounsaturated fatty acid. Following OA exposure, TSB levels in hUGT1 neonatal mice were reduced significantly, and RT‐qPCR analysis demonstrated that intestinal human UGT1A1 was induced, along with increased expression of Cyp4a10, a target gene of peroxisome proliferator‐activated receptor alpha (PPARα). In addition, IEC maturation markers including sucrase‐isomaltase (Sis) and alkaline phosphatase 3 (Akp3) are also induced. To examine the mechanism of OA induction, intestinal crypt cells were isolated from hUGT1 mice. The organoids were exposed to different fatty acids, including OA, palmitoleic acid (PA), arachidonic acid (AA), linoleic acid (LA), and docosahexaenoic acid (DHA). Gene expression parameters were monitored 24 hours after exposure. We found that OA‐ and PA‐treated organoids have similar survival rates in comparison to controls, but at similar concentrations of LA, DHA or AA cell death and decreased organoid viability was observed. At lower FA concentrations, RT‐qPCR analysis demonstrated that the UGT1A1 gene was not induced in any of the samples, including the IEC maturation markers Sis and Akp3. However, increased expression of the Cyp4a10 gene resulted in DHA, AA, and LA‐treated organoids. By comparing the in vivo and in vitro experiments, it suggests that unlike Cyp4a10, the regulation of UGT1A1 by fatty acids is associated with the development of the intestinal tract. Under current culture conditions, the application of intestinal organoid cultures in the study of developmental‐associated events is limited.Support or Funding InformationSupported in part by NIH grants GM126074 and ES010337

Inhibition of UGT1A1 by natural and synthetic flavonoids

Flavonoids are widely distributed phytochemicals in vegetables, fruits and medicinal plants. Recent studies demonstrate that some natural flavonoids are potent inhibitors of the human UDP-glucuronosyltransferase 1A1 (UGT1A1), a key enzyme in detoxification of endogenous harmful compounds such as bilirubin. In this study, the inhibitory effects of 56 natural and synthetic flavonoids on UGT1A1 were assayed, while the structure–inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated. The results demonstrated that the C-3 and C-7 hydroxyl groups on the flavone skeleton would enhance UGT1A1 inhibition, while flavonoid glycosides displayed weaker inhibitory effects than their corresponding aglycones. Further investigation on inhibition kinetics of two strong flavonoid-type UGT1A1 inhibitors, acacetin and kaempferol, yielded interesting results. Both flavonoids were competitive inhibitors against UGT1A1-mediated NHPN-O-glucuronidation, but were mixed and competitive inhibitors toward UGT1A1-mediated NCHN-O-glucuronidation, respectively. Furthermore, docking simulations showed that the binding areas of NHPN, kaempferol and acacetin on UGT1A1 were highly overlapping, and convergence with the binding area of bilirubin within UGT1A1. In summary, detailed structure-inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated carefully and the findings shed new light on the interactions between flavonoids and UGT1A1, and will contribute considerably to the development of flavonoid-type drugs without strong UGT1A1 inhibition.

Oligomerization and Catalytic Parameters of Human UDP-Glucuronosyltransferase 1A10: Expression and Characterization of the Recombinant Protein.

UDP-glucuronosyltransferase (UGT), as an integral membrane protein localized in the endoplasmic reticulum, has the ability to detoxify potentially hazardous xenobiotic substances. Most UGTs are expressed in liver, but UGT1A10 has proven to be an extrahepatic enzyme considerably expressed throughout the gastrointestinal tract. Earlier studies indicated that different UGT isoforms could exist in higher-order homo-oligomers or at least dimers within the membrane, but the formation of intermolecular disulfide bridges between UGT molecules was not often observed. In this study, we expressed recombinant human UGT1A10 in human embryonic kidney (HEK)293 and Chinese hamster ovary (CHO) cells to examine its oligomeric states and characterize its enzymatic activities against two therapeutically interesting substrates, morphine and entacapone, including determination of the catalytic rate constant (kcat) values for the first time. It was observed that a majority of the UGT1A10 protein expressed in HEK293 cells existed in covalently crosslinked higher-order oligomers via formation of intermolecular disulfide bonds, whereas formation of the intermolecular disulfide bonds was not observed in the UGT1A10 protein expressed in CHO cells. Owing to the formation of the covalently crosslinked higher-order oligomers, the UGT1A10 protein expressed in HEK293 cells had much lower catalytic activity (particularly the catalytic rate constant kcat) against both morphine and entacapone, compared with the UGT1A10 protein form expressed in CHO cells against the same substrates.

Humanized UGT1 Mice, Regulation of UGT1A1, and the Role of the Intestinal Tract in Neonatal Hyperbilirubinemia and Breast Milk-Induced Jaundice.

Neonatal hyperbilirubinemia and the onset of bilirubin encephalopathy and kernicterus result in part from delayed expression of UDP-glucuronosyltransferase 1A1 (UGT1A1) and the ability to metabolize bilirubin. It is generally believed that acute neonatal forms of hyperbilirubinemia develop due to an inability of hepatic UGT1A1 to metabolize efficiently bilirubin for clearance through the hepatobiliary tract. Newly developed mouse models designed to study bilirubin metabolism have led to new insight into the role of the intestinal tract in controlling neonatal hyperbilirubinemia. Humanization of mice with the UGT1 locus (hUGT1 mice) and the UGT1A1 gene provide a unique tool to study the onset of hyperbilirubinemia since the human UGT1A1 gene is developmentally regulated during the neonatal period in hUGT1 mice. A new mechanism outlying developmental expression of intestinal UGT1A1 is presented and its implications in the control of neonatal hyperbilirubinemia discussed. New findings linking breast milk protection against necrotizing enterocolitis and intestinal control of UGT1A1 may help explain the contribution of breast milk toward the development of neonatal hyperbilirubinemia. Our findings outline a new model that includes an active intestinal ROS /IκB kinase/nuclear receptor corepressor 1 loop that can be applied to an understanding of breast milk-induced jaundice.

Bacterial Outer Membrane Vesicles from Dextran Sulfate Sodium-Induced Colitis Differentially Regulate Intestinal UDP-Glucuronosyltransferase 1A1 Partially Through Toll-Like Receptor 4/Mitogen-Activated Protein Kinase/Phosphatidylinositol 3-Kinase Pathway.

UDP-glucuronosyltransferase 1A1 (UGT1A1) constitutes an important part of intestinal epithelial barrier and catalyzes glucuronidation of many endogenous compounds and drugs. Downregulation of UGT1A1 in inflammation has been reported, whereas the association with gut dysbiosis is poorly defined. This study verified the involvement of gut microbiota in intestinal UGT1A1 regulation using dextran sulfate sodium (DSS)-induced rat colitis model plus fecal microbiota transplantation (FMT). Generally, both DSS induction and colitis-to-normal FMT suppressed mRNA and protein expressions of UGT1A1 and nuclear xenobiotic receptors (NRs) in colon, but enhanced mRNA and decreased protein of rat UGT1A1/rat NRs in small intestine. Normal-to-colitis FMT alleviated DSS-induced changes. Bacterial outer membrane vesicles (OMVs) from colitis rats and rats receiving colitis feces reduced both mRNA and protein of human UGT1A1 (hUGT1A1)/human NRs (hNRs) in Caco-2 cells. Interestingly, using deoxycholate to reduce lipopolysaccharide, normal OMVs upregulated hUGT1A1/hNRs, whereas colitis OMVs decreased, indicating the involvement of other OMVs components in UGT1A1 regulation. The 10- to 50-kDa fractions from both normal and colitis OMVs downregulated hUGT1A1, human PXR, and human PPAR-γ, whereas >50-kDa fractions from normal rats upregulated hUGT1A1 and human CAR. Additionally, the conditioned medium from OMVs-stimulated rat primary macrophages also reduced hUGT1A1/hNRs expression. Both Toll-like receptor (TLR)2 and TLR4 were activated by DSS, colitis-to-normal FMT, and the opposite, whereas only TLR4 was increased in OMVs-treated cells. TLR4 small interfering RNA blocked hUGT1A1/hNRs downregulation and phosphatidylinositol 3-kinase/Akt, extracellular signal-regulated kinase, and nuclear factor κB phosphorylation evoked by bacterial OMVs. Taken together, this study demonstrated that gut microbiota regulate intestinal UGT1A1 partially through secreting OMVs, which interact with intestinal epithelial cells directly or via activating macrophage.

Histone Modifications Regulate the Developmental Expression of Human Hepatic UDP-Glucuronosyltransferase 1A1.

Human UDP-glucuronosyltransferase 1A1 (UGT1A1) is a unique enzyme involved in bilirubin conjugation. We previously characterized the hepatic expression of transcription factors affecting UGT1A1 expression during development. Accordingly, in this study, we characterized the ontogenetic expression of hepatic UGT1A1 from the perspective of epigenetic regulation. We observed significant histone-3-lysine-4 dimethylation (H3K4me2) enrichment in the adult liver and histone-3-lysine-27 trimethylation (H3K27me3) enrichment in the fetal liver, indicating that dynamic alterations of histone methylation were associated with ontogenetic UGT1A1 expression. We further showed that the transcription factor hepatocyte nuclear factor 1α (HNF1A) affects histone modifications around the UGT1A1 locus. In particular, we demonstrated that by recruiting HNF1A the cofactors mixed-lineage leukemia 1, the transcriptional coactivator p300, and nuclear receptor coactivator 6 aggregate at the UGT1A1 promoter, thereby regulating histone modifications and subsequent UGT1A1 expression. In this study, we proposed new ideas for the developmental regulation of metabolic enzymes via histone modifications, and our findings will potentially contribute to the development of age-specific therapies.

Comparison of the inhibition potentials of icotinib and erlotinib against human UDP-glucuronosyltransferase 1A1

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a key role in detoxification of many potentially harmful compounds and drugs. UGT1A1 inhibition may bring risks of drug–drug interactions (DDIs), hyperbilirubinemia and drug-induced liver injury. This study aimed to investigate and compare the inhibitory effects of icotinib and erlotinib against UGT1A1, as well as to evaluate their potential DDI risks via UGT1A1 inhibition. The results demonstrated that both icotinib and erlotinib are UGT1A1 inhibitors, but the inhibitory effect of icotinib on UGT1A1 is weaker than that of erlotinib. The IC50 values of icotinib and erlotinib against UGT1A1-mediated NCHN-O-glucuronidation in human liver microsomes (HLMs) were 5.15 and 0.68 μmol/L, respectively. Inhibition kinetic analyses demonstrated that both icotinib and erlotinib were non-competitive inhibitors against UGT1A1-mediated glucuronidation of NCHN in HLMs, with the Ki values of 8.55 and 1.23 μmol/L, respectively. Furthermore, their potential DDI risks via UGT1A1 inhibition were quantitatively predicted by the ratio of the areas under the concentration–time curve (AUC) of NCHN. These findings are helpful for the medicinal chemists to design and develop next generation tyrosine kinase inhibitors with improved safety, as well as to guide reasonable applications of icotinib and erlotinib in clinic, especially for avoiding their potential DDI risks via UGT1A1 inhibition.

A turn on fluorescent probe for human UDP-glucuronosyltransferase 1A1 and bilirubin metabolism evaluation

A turn on fluorescent probe for human UDP-glucuronosyltransferase 1A1 and bilirubin metabolism evaluation

Inter-isoform Hetero-dimerization of Human UDP-Glucuronosyltransferases (UGTs) 1A1, 1A9, and 2B7 and Impacts on Glucuronidation Activity.

Human UDP-glucuronosyltransferases (UGTs) play a pivotal role in phase II metabolism by catalyzing the glucuronidation of endobiotics and xenobiotics. The catalytic activities of UGTs are highly impacted by both genetic polymorphisms and oligomerization. The present study aimed to assess the inter-isoform hetero-dimerization of UGT1A1, 1A9, and 2B7, including the wild type (1A1*1, 1A9*1, and 2B7*1) and the naturally occurring (1A1*1b, 1A9*2/*3/*5, and 2B7*71S/*2/*5) variants. The related enzymes were double expressed in Bac-to-Bac systems. The fluorescence resonance energy transfer (FRET) technique and co-immunoprecipitation (Co-IP) revealed stable hetero-dimerization of UGT1A1, 1A9, and 2B7 allozymes. Variable FRET efficiencies and donor-acceptor distances suggested that genetic polymorphisms resulted in altered affinities to the target protein. In addition, the metabolic activities of UGTs were differentially altered upon hetero-dimerization via double expression systems. Moreover, protein interactions also changed the regioselectivity of UGT1A9 for querectin glucuronidation. These findings provide in-depth understanding of human UGT dimerization as well as clues for complicated UGT dependent metabolism in humans.

Three-dimensional polyacrylamide gel-based DNA microarray method effectively identifies UDP-glucuronosyltransferase 1A1 gene polymorphisms for the correct diagnosis of Gilbert's syndrome.

Gilbert's syndrome is a mild genetic liver disorder characterized by unconjugated hyperbilirubinemia due to defects in the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene. The T-3279G mutation in the phenobarbital responsive enhancer module (PBREM), the TA-insertion in the TATA box, creating the A(TA)7TAA motif instead of A(TA)6TAA and the G211A mutation in coding exon 1, particularly in Asian populations, of the human UGT1A1 gene are the three common genotypes found in patients with Gilbert's syndrome. Different approaches for detecting the T-3279G, A(TA)6/7TAA and G211A mutations of the UGT1A1 gene have been described. In this study, to the best of our knowledge, we established a three-dimensional polyacrylamide gel-based DNA microarray method for the first time, in order to study UGT1A1 gene polymorphisms. This method, based on a step-by-step three-dimensional polyacrylamide gel-based DNA microarray protocol, successfully identified all possible genotypes of T-3279G, A(TA)6/7TAA and G211A in 20 patients with hyperbilirubinemia. In addition, sequencing was performed to confirm these results. The data from the current study demonstrate that the three-dimensional polyacrylamide gel microarray method has the potential to be applied as a useful, reliable and cost-effective tool to detect the T-3279G, the A(TA)6/7TAA and the G211A mutations of the UGT1A1 gene in patients with hyperbilirubinemia and thereby aid in the diagnosis of Gilbert's syndrome.

Identification and characterization of naturally occurring inhibitors against UDP-glucuronosyltransferase 1A1 in Fructus Psoraleae (Bu-gu-zhi)

As an edible traditional Chinese herb, Fructus psoraleae (FP) has been widely used in Asia for the treatment of vitiligo, bone fracture and osteoporosis. Several cases on markedly elevated bilirubin and acute liver injury following administration of FP and its related proprietary medicine have been reported, but the mechanism in FP-associated toxicity has not been well investigated yet. This study aimed to investigate the inhibitory effects of FP extract and its major constituents against human UDP-glucuronosyltransferase 1A1 (UGT1A1), the key enzyme responsible for metabolic elimination of bilirubin. To this end, N-(3-carboxy propyl)-4-hydroxy-1,8-naphthalimide (NCHN), a newly developed specific fluorescent probe for UGT1A1, was used to evaluate the inhibitory effects of FP extract or its fractions in human liver microsomes (HLM), while LC-UV fingerprint and UGT1A1 inhibition profile were combined to identity and characterize the naturally occurring inhibitors of UGT1A1 in FP. Our results demonstrated that both the extract of FP and five major components of FP displayed evident inhibitory effects on UGT1A1 in HLM. Among these five identified naturally occurring inhibitors, bavachin and corylifol A were found to be strong inhibitors of UGT1A1 with the inhibition kinetic parameters (Ki) values lower than 1μM, while neobavaisoflavone, isobavachalcone, and bavachinin displayed moderate inhibitory effects against UGT1A1 in HLM, with the Ki values ranging from 1.61 to 9.86μM. These findings suggested that FP contains natural compounds with potent inhibitory effects against human UGT1A1, which may be one of the important reasons for triggering FP-associated toxicity, including elevated bilirubin levels and liver injury.

Molecular cloning and functional analysis of minipig UDP-glucuronosyltransferase 1A6

1. UDP-glucuronosyltransferase 1A6 (UGT1A6) plays important roles in the glucuronidation of numerous drugs, environmental pollutants, and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies because many of their physiological characteristics are similar to those of humans. The aim of the present study was to examine similarities and differences in the enzymatic properties of UGT1A6 between humans and minipigs.2. Minipig UGT1A6 (mpUGT1A6) cDNA was cloned by the RACE method, and the corresponding proteins were expressed in insect cells. The enzymatic function of mpUGT1A6 was analyzed by the kinetics of serotonin glucuronidation.3. Amino acid homology between human UGT1A6 (hUGT1A6) and mpUGT1A6 was 79.9%. The kinetics of serotonin glucuronidation by recombinant hUGT1A6 and mpUGT1A6 enzymes fit the Michaelis–Menten equation. The Km, Vmax, and CLint values of hUGT1A6 were 10.5 mM, 4.04 nmol/min/mg protein, and 0.39 µL/min/mg protein, respectively. The Km value of mpUGT1A6 was similar to that of hUGT1A6, whereas the Vmax and CLint values of mpUGT1A6 were approximately 2-fold higher than those of hUGT1A6.4. These results suggest that the enzymatic properties of UGT1A6 enzymes are moderately different between humans and minipigs.

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.