Publications by authors named "Moshe Finel"

114 Publications

A broad-spectrum substrate for the human UDP-glucuronosyltransferases and its use for investigating glucuronidation inhibitors.

Int J Biol Macromol 2021 Jun 16;180:252-261. Epub 2021 Mar 16.

Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Electronic address:

Strong inhibition of the human UDP-glucuronosyltransferase enzymes (UGTs) may lead to undesirable effects, including hyperbilirubinaemia and drug/herb-drug interactions. Currently, there is no good way to examine the inhibitory effects and specificities of compounds toward all the important human UGTs, side-by-side and under identical conditions. Herein, we report a new, broad-spectrum substrate for human UGTs and its uses in screening and characterizing of UGT inhibitors. Following screening a variety of phenolic compound(s), we have found that methylophiopogonanone A (MOA) can be readily O-glucuronidated by all tested human UGTs, including the typical N-glucuronidating enzymes UGT1A4 and UGT2B10. MOA-O-glucuronidation yielded a single mono-O-glucuronide that was biosynthesized and purified for structural characterization and for constructing an LC-UV based MOA-O-glucuronidation activity assay, which was then used for investigating MOA-O-glucuronidation kinetics in recombinant human UGTs. The derived K values were crucial for selecting the most suitable assay conditions for assessing inhibitory potentials and specificity of test compound(s). Furthermore, the inhibitory effects and specificities of four known UGT inhibitors were reinvestigated by using MOA as the substrate for all tested UGTs. Collectively, MOA is a broad-spectrum substrate for the human UGTs, which offers a new and practical tool for assessing inhibitory effects and specificities of UGT inhibitors.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.03.073DOI Listing
June 2021

A fluorescence-based microplate assay for high-throughput screening and evaluation of human UGT inhibitors.

Anal Chim Acta 2021 Apr 8;1153:338305. Epub 2021 Feb 8.

Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China. Electronic address:

Human UDP-glucuronosyltransferase enzymes (hUGTs), one of the most important classes of conjugative enzymes, are responsible for the glucuronidation and detoxification of a variety of endogenous substances and xenobiotics. Inhibition of hUGTs may cause undesirable effects or adverse drug-drug interactions (DDI) via modulating the glucuronidation rates of endogenous toxins or the drugs that are primarily conjugated by the inhibited hUGTs. Herein, to screen hUGTs inhibitors in a more efficient way, a novel fluorescence-based microplate assay has been developed by utilizing a fluorogenic substrate. Following screening of series of 4-hydroxy-1,8-naphthalimide derivatives, we found that 4-HN-335 is a particularly good substrate for a panel of hUGTs. Under physiological conditions, 4-HN-335 can be readily O-glucuronidated by ten hUGTs, such reactions generate a single O-glucuronide with a high quantum yield (Ф = 0.79) and bring remarkable changes in fluorescence emission. Subsequently, a fluorescence-based microplate assay is developed to simultaneously measure the inhibitory effects of selected compound(s) on ten hUGTs. The newly developed fluorescence-based microplate assay is time- and cost-saving, easy to manage and can be adapted for 96-well microplate format with the Z-factor of 0.92. We further demonstrate the utility of the fluorescence-based assay for high-throughput screening of two compound libraries, resulting in the identification of several potent UGT inhibitors, including natural products and FDA-approved drugs. Collectively, this study reports a novel fluorescence-based microplate assay for simultaneously sensing the residual activities of ten hUGTs, which strongly facilitates the identification and characterization of UGT inhibitors from drugs or herbal constituents and the investigations on UGT-mediated DDI.
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http://dx.doi.org/10.1016/j.aca.2021.338305DOI Listing
April 2021

Neobavaisoflavone Induces Bilirubin Metabolizing Enzyme UGT1A1 via PPARα and PPARγ.

Front Pharmacol 2020 8;11:628314. Epub 2021 Feb 8.

Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

UDP-glucuronosyltransferase 1A1 (UGT1A1) is an essential enzyme in mammals that is responsible for detoxification and metabolic clearance of the endogenous toxin bilirubin and a variety of xenobiotics, including some crucial therapeutic drugs. Discovery of potent and safe UGT1A1 inducers will provide an alternative therapy for ameliorating hyperbilirubinaemia and drug-induced hepatoxicity. This study aims to find efficacious UGT1A1 inducer(s) from natural flavonoids, and to reveal the mechanism involved in up-regulating of this key conjugative enzyme by the flavonoid(s) with strong UGT1A1 induction activity. Among all the tested flavonoids, neobavaisoflavone (NBIF) displayed the most potent UGT1A1 induction activity, while its inductive effects were confirmed by both western blot and glucuronidation activity assays. A panel of nuclear receptor reporter assays demonstrated that NBIF activated PPARα and PPARγ in a dose-dependent manner. Meanwhile, we also found that NBIF could up-regulate the expression of PPARα and PPARγ in hepatic cells, suggesting that the induction of UGT1A1 by NBIF was mainly mediated by PPARs. In silico simulations showed that NBIF could stably bind on pocket II of PPARα and PPARγ. Collectively, our results demonstrated that NBIF is a natural inducer of UGT1A1, while this agent induced UGT1A1 mainly via activating and up-regulating PPARα and PPARγ. These findings suggested that NBIF can be used as a promising lead compound for the development of more efficacious UGT1A1 inducers to treat hyperbilirubinaemia and UGT1A1-associated drug toxicities.
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http://dx.doi.org/10.3389/fphar.2020.628314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897654PMC
February 2021

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

J Pharm Anal 2020 Jun 23;10(3):263-270. Epub 2020 May 23.

Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.

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.
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http://dx.doi.org/10.1016/j.jpha.2020.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322753PMC
June 2020

Human efflux transport of testosterone, epitestosterone and other androgen glucuronides.

J Steroid Biochem Mol Biol 2020 03 6;197:105518. Epub 2019 Nov 6.

Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Finland.

Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen glucuronides investigated. In kinetic analyses, MRP3 transported glucuronides of androsterone, epitestosterone and etiocholanolone at low K values, between 0.4 and 4 μM, while the K values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 μM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by K values over 100 μM. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.
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http://dx.doi.org/10.1016/j.jsbmb.2019.105518DOI Listing
March 2020

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

Eur J Pharm Sci 2020 Jan 25;141:105118. Epub 2019 Oct 25.

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 University of Helsinki, Finland.

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' K 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.
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http://dx.doi.org/10.1016/j.ejps.2019.105118DOI Listing
January 2020

Characterization of human UGT2A3 expression using a prepared specific antibody against UGT2A3.

Drug Metab Pharmacokinet 2019 Aug 13;34(4):280-286. Epub 2019 May 13.

Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan; WPI Nano Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan. Electronic address:

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p < 0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues.
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http://dx.doi.org/10.1016/j.dmpk.2019.05.001DOI Listing
August 2019

Efflux transport of nicotine, cotinine and trans-3'-hydroxycotinine glucuronides by human hepatic transporters.

Basic Clin Pharmacol Toxicol 2019 Dec 19;125(6):490-498. Epub 2019 Jul 19.

Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.

Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans-3'-hydroxycotinine (OH-cotinine) glucuronides. To this end, we tested their transport by multidrug resistance-associated proteins 1 (MRP1, ABCC1) and MRP3-6 (ABCC3-6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P-gp, ABCB1) that are expressed in the apical membranes of these cells. ATP-dependent transport of these glucuronides was evaluated in inside-out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP-dependent transport was observed only for OH-cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated K value of 895 µmol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 µmol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH-cotinine inhibited MRP2-4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.
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http://dx.doi.org/10.1111/bcpt.13281DOI Listing
December 2019

Inhibition of human carboxylesterases by magnolol: Kinetic analyses and mechanism.

Chem Biol Interact 2019 Aug 4;308:339-349. Epub 2019 Jun 4.

Translational Medicine Center, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine & Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 200473, China. Electronic address:

Magnolol, the most abundant bioactive constituent of the Chinese herb Magnolia officinalis, has been found with multiple biological activities, including anti-oxidative, anti-inflammatory and enzyme-regulatory activities. In this study, the inhibitory effects and inhibition mechanism of magnolol on human carboxylesterases (hCEs), the key enzymes responsible for the hydrolytic metabolism of a variety of endogenous esters as well as ester-bearing drugs, have been well-investigated. The results demonstrate that magnolol strongly inhibits hCE1-mediated hydrolysis of various substrates, whereas the inhibition of hCE2 by magnolol is substrate-dependent, ranging from strong to moderate. Inhibition of intracellular hCE1 and hCE2 by magnolol was also investigated in living HepG2 cells, and the results showed that magnolol could strongly inhibit intracellular hCE1, while the inhibition of intracellular hCE2 was weak. Inhibition kinetic analyses and docking simulations revealed that magnolol inhibited both hCE1 and hCE2 in a mixed manner, which could be partially attributed to its binding at two distinct ligand-binding sites in each carboxylesterase, including the catalytic cavity and the regulatory domain. In addition, the potential risk of the metabolic interactions of magnolol via hCE1 inhibition was predicted on the basis of a series of available pharmacokinetic data and the inhibition constants. All these findings are very helpful in deciphering the metabolic interactions between magnolol and hCEs, and also very useful for avoiding deleterious interactions via inhibition of hCEs.
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http://dx.doi.org/10.1016/j.cbi.2019.06.003DOI Listing
August 2019

Recent progress and challenges in screening and characterization of UGT1A1 inhibitors.

Acta Pharm Sin B 2019 Mar 14;9(2):258-278. Epub 2018 Sep 14.

Institute of Interdisciplinary Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.

Uridine-diphosphate glucuronosyltransferase 1A1 (UGT1A1) is an important conjugative enzyme in mammals that is responsible for the conjugation and detoxification of both endogenous and xenobiotic compounds. Strong inhibition of UGT1A1 may trigger adverse drug/herb-drug interactions, or result in metabolic disorders of endobiotic metabolism. Therefore, both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have recommended assaying the inhibitory potential of drugs under development on the human UGT1A1 prior to approval. This review focuses on the significance, progress and challenges in discovery and characterization of UGT1A1 inhibitors. Recent advances in the development of UGT1A1 probes and their application for screening UGT1A1 inhibitors are summarized and discussed in this review for the first time. Furthermore, a long list of UGT1A1 inhibitors, including information on their inhibition potency, inhibition mode, and affinity, has been prepared and analyzed. Challenges and future directions in this field are highlighted in the final section. The information and knowledge that are presented in this review provide guidance for rational use of drugs/herbs in order to avoid the occurrence of adverse effects UGT1A1 inhibition, as well as presenting methods for rapid screening and characterization of UGT1A1 inhibitors and for facilitating investigations on UGT1A1-ligand interactions.
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http://dx.doi.org/10.1016/j.apsb.2018.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437557PMC
March 2019

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

Data Brief 2019 Feb 17;22:977-986. Epub 2018 Dec 17.

Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia.

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 v 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).
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http://dx.doi.org/10.1016/j.dib.2018.12.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356001PMC
February 2019

Case report by Toce and co-authors: Have all the reasons for poor morphine glucuronidation been addressed?

Br J Clin Pharmacol 2019 04 8;85(4):859-860. Epub 2019 Feb 8.

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.

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http://dx.doi.org/10.1111/bcp.13868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422727PMC
April 2019

Metabolism of Scoparone in Experimental Animals and Humans.

Planta Med 2019 Apr 8;85(6):453-464. Epub 2019 Feb 8.

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland.

Scoparone, a major constituent of the Chinese herbal medicine Yin Chen Hao, expresses beneficial effects in experimental models of various diseases. The intrinsic doses and effects of scoparone are dependent on its metabolism, both in humans and animals. We evaluated in detail the metabolism of scoparone in human, mouse, rat, pig, dog, and rabbit liver microsomes and in humans . Oxidation of scoparone to isoscopoletin via 6-O-demethylation was the major metabolic pathway in liver microsomes from humans, mouse, rat, pig and dog, whereas 7-O-demethylation to scopoletin was the main reaction in rabbit. The scoparone oxidation rates in liver microsomes were 0.8 - 1.2 µmol/(min*g protein) in mouse, pig, and rabbit, 0.2 - 0.4 µmol/(min*g protein) in man and dog, and less than 0.1 µmol/(min*g) in rat. In liver microsomes of all species, isoscopoletin was oxidized to 3-[4-methoxy-ρ-(3, 6)-benzoquinone]-2-propenoate and esculetin, which was formed also in the oxidation of scopoletin. Human CYP2A13 exhibited the highest rate of isoscopoletin and scopoletin oxidation, followed by CYP1A1 and CYP1A2. Glucuronidation of isoscopoletin and scopoletin was catalyzed by the human UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, and UGT2B17. Dog was most similar to man in scoparone metabolism. Isoscopoletin glucuronide and sulfate conjugates were the major scoparone metabolites in humans, and they were completely excreted within 24 h in urine. Scoparone and its metabolites did not activate key nuclear receptors regulating CYP and UGT enzymes. These results outline comprehensively the metabolic pathways of scoparone in man and key preclinical animal species.
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http://dx.doi.org/10.1055/a-0835-2301DOI Listing
April 2019

Inhibition of UGT1A1 by natural and synthetic flavonoids.

Int J Biol Macromol 2019 Apr 28;126:653-661. Epub 2018 Dec 28.

Translational Medicine Center, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, & Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 200473, China. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.12.171DOI Listing
April 2019

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

Chemosphere 2019 Jan 18;215:870-880. Epub 2018 Oct 18.

Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chemosphere.2018.10.129DOI Listing
January 2019

A Novel Pathogenic Variant in a Sudanese Child with Type 1 Crigler-Najjar Syndrome.

Drug Metab Dispos 2019 01 1;47(1):45-48. Epub 2018 Nov 1.

Department of Pediatrics, Milton S. Hershey Penn State Medical Center, Hershey, Pennsylvania (W.E.); Division of Pharmaceutical Chemistry and Technology, University of Helsinki, Helsinki, Finland (E.J., J.M., M.F.); Pediatric Genomics Discovery Program, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut (W.J., A.G.S., M.K., S.A.L.); Departments of Pathology and Cell Biology (A.C.I.) and Surgery (S.J.L.), Columbia University Medical Center, New York, New York; and Department of Pediatrics, University of Rochester Medical Center, Rochester, New York (A.C.)

Uridine diphosphate glucuronosyltransferases (UGTs) are key enzymes responsible for the body's ability to process a variety of endogenous and exogenous compounds. Significant gains in understanding UGT function have come from the analysis of variants seen in patients. We cared for a Sudanese child who showed clinical features of type 1 Crigler-Najjar syndrome (CN-1), namely severe unconjugated hyperbilirubinemia leading to liver transplantation. CN-1 is an autosomal recessive disorder caused by damaging mutations in the gene for UGT1A1, the hepatic enzyme responsible for bilirubin conjugation in humans. Clinical genetic testing was unable to identify a known pathogenic mutation in this child. Instead, a novel homozygous variant resulting in an in-frame deletion, p.Val275del, was noted. Sanger sequencing demonstrated that this variant segregated with the disease phenotype in this family. We further performed functional testing using recombinantly expressed UGT1A1 with and without the patient variant, demonstrating that p.Val275del results in a complete lack of glucuronidation activity, a hallmark of CN-1. Sequence analysis of this region shows a high degree of conservation across all known catalytically active human UGTs, further suggesting that it plays a key role in the enzymatic function of UGTs. Finally, we note that the patient's ethnicity likely played a role in his variant being previously undescribed and advocate for greater diversity and inclusion in genomic medicine.
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http://dx.doi.org/10.1124/dmd.118.084368DOI Listing
January 2019

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

Mol Pharm 2018 03 15;15(3):923-933. Epub 2018 Feb 15.

Institute of Biomedicine, Faculty of Medicine , University of Turku , FI-20014 Turku , Finland.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150735PMC
March 2018

Efflux transport of estrogen glucuronides by human MRP2, MRP3, MRP4 and BCRP.

J Steroid Biochem Mol Biol 2018 04 22;178:99-107. Epub 2017 Nov 22.

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Finland. Electronic address:

Estrone, estradiol and estriol are endogenous human estrogens that are rapidly conjugated with glucuronic acid in both intestinal and hepatic epithelial cells. The resulting glucuronides, estrone-3-glucuronide (E-G), estradiol-3- and 17-glucuronides (E-3G and E-17G), as well as estriol-3- and 16-glucuronides (E-3G and E-16G) are found in human plasma and urine. Unlike E-17G, the efflux transport of other estrogen glucuronides by human transporters has not yet been investigated comprehensively. We have studied the transport of E-G, E-3G, E-3G, E-16G and estrone-3-sulfate (E-S), another important estrogen conjugate, using the vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP that were expressed in insect cells. The transport screening assays revealed that whereas E-S was a good and specific substrate for BCRP, the less transporter-specific conjugates, E-G and E-3G, were still transported by BCRP at 10-fold higher rates than E-S. BCRP also transported E-16G at higher rates than the studied MRPs, while it transported E-3G at lower rates than MRP3. MRP2 exhibited lower or equal transport rates of E-G, E-3G, E-3G and E-16G in comparison to MRP3 and BCRP in the screening assays, mainly due to its high K values, between 180 and 790 μM. MRP3 transported all the tested glucuronides at rather similar rates, at K values below 20 μM, but lower V values than other transporters. In the case of E-3G, MRP3 was the most active transporter in the screening assay. MRP4 transported only E-16G at considerable rates, while none of the tested estrogen conjugates was transported by MDR1 at higher rates than control vesicles. These new results, in combination with previously reported in vivo human data, stimulate our understanding on the substrate specificity and role of efflux transporters in disposition of estrogen glucuronides in humans.
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http://dx.doi.org/10.1016/j.jsbmb.2017.11.007DOI Listing
April 2018

Clopidogrel Carboxylic Acid Glucuronidation is Mediated Mainly by UGT2B7, UGT2B4, and UGT2B17: Implications for Pharmacogenetics and Drug-Drug Interactions.

Drug Metab Dispos 2018 02 14;46(2):141-150. Epub 2017 Nov 14.

Department of Clinical Pharmacology, Faculty of Medicine, University of Helsinki, and Helsinki University Hospital (H.K., A.M.F., M.Ne., E.K.T., A.T., M.T.H., M.K.I., P.J.N., M.Ni., J.T.B.) and Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki (M.F.), Helsinki, Finland

The antiplatelet drug clopidogrel is metabolized to an acyl--d-glucuronide, which causes time-dependent inactivation of CYP2C8. Our aim was to characterize the UDP-glucuronosyltransferase (UGT) enzymes that are responsible for the formation of clopidogrel acyl--d-glucuronide. Kinetic analyses and targeted inhibition experiments were performed using pooled human liver and intestine microsomes (HLMs and HIMs, respectively) and selected human recombinant UGTs based on preliminary screening. The effects of relevant polymorphisms on the pharmacokinetics of clopidogrel were evaluated in 106 healthy volunteers. UGT2B7 and UGT2B17 exhibited the greatest level of clopidogrel carboxylic acid glucuronidation activities, with a CL of 2.42 and 2.82 l⋅min⋅mg, respectively. Of other enzymes displaying activity (UGT1A3, UGT1A9, UGT1A10-H, and UGT2B4), UGT2B4 (CL 0.51 l⋅min⋅mg) was estimated to contribute significantly to the hepatic clearance. Nonselective UGT2B inhibitors strongly inhibited clopidogrel acyl--d-glucuronide formation in HLMs and HIMs. The UGT2B17 inhibitor imatinib and the UGT2B7 and UGT1A9 inhibitor mefenamic acid inhibited clopidogrel carboxylic acid glucuronidation in HIMs and HLMs, respectively. Incubation of clopidogrel carboxylic acid in HLMs with UDPGA and NADPH resulted in strong inhibition of CYP2C8 activity. In healthy volunteers, the deletion allele was associated with a 10% decrease per copy in the plasma clopidogrel acyl--d-glucuronide to clopidogrel carboxylic acid area under the plasma concentration-time curve from 0 to 4 hours (AUC) ratio ( < 0.05). To conclude, clopidogrel carboxylic acid is metabolized mainly by UGT2B7 and UGT2B4 in the liver and by UGT2B17 in the small intestinal wall. The formation of clopidogrel acyl--d-glucuronide is impaired in carriers of the deletion. These findings may have implications regarding the intracellular mechanisms leading to CYP2C8 inactivation by clopidogrel.
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http://dx.doi.org/10.1124/dmd.117.078162DOI Listing
February 2018

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.

Mol Pharm 2017 10 13;14(10):3299-3311. Epub 2017 Sep 13.

Division of Pharmaceutical Chemistry and Technology, and ‡Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki , 00014 University of Helsinki, Finland.

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-17β-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 (K values of 168, 13, and 3 μM, respectively) in comparison to MRP3 (K 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 (K 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 (V and K 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-17β-glucuronide, at a high rate (V = 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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00366DOI Listing
October 2017

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

Xenobiotica 2018 Feb 25;48(2):109-116. Epub 2017 Jul 25.

a Laboratory of Pharmaceutical Resource Discovery , Biotechnology Department, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian , China.

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 V and mild substrate inhibition, whereas the kinetics in HLM was biphasic. UGT1A1 (high K value), UGT1A10-H (low K 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.
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http://dx.doi.org/10.1080/00498254.2017.1283719DOI Listing
February 2018

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

Mol Pharm 2017 09 14;14(9):2875-2883. Epub 2016 Dec 14.

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki , Finland.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.6b00852DOI Listing
September 2017

Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae.

Mol Pharm 2016 07 8;13(7):2274-82. Epub 2016 Jun 8.

Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.

Glucuronidation is one of the most common pathways in mammals for detoxification and elimination of hydrophobic xenobiotic compounds, including many drugs. Metabolites, however, can form active or toxic compounds, such as acyl glucuronides, and their safety assessment is often needed. The absence of efficient means for in vitro synthesis of correct glucuronide metabolites frequently limits such toxicological analyses. To overcome this hurdle we have developed a new approach, the essence of which is a coexpression system containing a human, or another mammalian UDP-glucuronosyltransferases (UGTs), as well as UDP-glucose-6-dehydrogenase (UGDH), within the budding yeast, Saccharomyces cerevisiae. The system was first tested using resting yeast cells coexpressing UGDH and human UGT1A6, 7-hydroxycoumarin as the substrate, in a reaction medium containing 8% glucose, serving as a source of UDP-glucuronic acid. Glucuronides were readily formed and recovered from the medium. Subsequently, by selecting suitable mammalian UGT enzyme for the coexpression system we could obtain the desired glucuronides of various compounds, including molecules with multiple conjugation sites and acyl glucuronides of several carboxylic acid containing drugs, namely, mefenamic acid, flufenamic acid, and zomepirac. In conclusion, a new and flexible yeast system with mammalian UGTs has been developed that exhibits a capacity for efficient production of various glucuronides, including acyl glucuronides.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00954DOI Listing
July 2016

Influence of metabolism on endocrine activities of bisphenol S.

Chemosphere 2016 Aug 21;157:152-9. Epub 2016 May 21.

Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia. Electronic address:

Bisphenol S (BPS; bis[4-hydroxyphenyl]sulfone) is commonly used as a replacement for bisphenol A in numerous consumer products. The main goal of this study was to examine the influence of different metabolic reactions that BPS undergoes on the endocrine activity. We demonstrate that hydroxylation of the aromatic ring of BPS, catalyzed mainly by the cytochrome P450 enzymes CYP3A4 and CYP2C9, is its major in-vitro phase I biotransformation. Nevertheless, coupled oxidative-conjugative reactions analyses revealed that glucuronidation and formation of BPS glucuronide is the predominant BPS metabolic pathway. BPS reactive metabolites that can be tracked as glutathione conjugates were not detected in the present study. Two in-vitro systems were used to evaluate the endocrine activity of BPS and its two main metabolites, BPS glucuronide and hydroxylated BPS 4-(4-hydroxy-benzenesulfonyl)-benzene-1,2-diol (BPSM1). In addition, we have tested two structural analogs of BPS, bis[4-(2-hydroxyetoxy)phenyl]sulfone (BHEPS) and 4,4-sulfonylbis(2-methylphenol) (dBPS). The test systems were yeast cells, for evaluating estrogenic and androgenic activities, and the GH3.TRE-Luc reporter cell line for measuring thyroid hormone activity. BPS and BPSM1 were weak agonists of the estrogen receptor, EC50 values of 8.4 × 10(-5) M and 6.7 × 10(-4) M, respectively. Additionally, BPSM1 exhibited weak antagonistic activity toward the thyroid hormone receptor, with an IC50 of 4.3 × 10(-5) M. In contrast to BPSM1, BPS glucuronide was inactive in these assays, inhibiting neither the estrogen nor the thyroid hormone receptors. Hence, glucuronidation appears to be the most important pathway for both BPS metabolism and detoxification.
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http://dx.doi.org/10.1016/j.chemosphere.2016.05.027DOI Listing
August 2016

Bisphenol-A glucuronidation in human liver and breast: identification of UDP-glucuronosyltransferases (UGTs) and influence of genetic polymorphisms.

Xenobiotica 2017 Jan 21;47(1):1-10. Epub 2016 Mar 21.

a Department of Veterinary Clinical Sciences , Pharmacogenomics Laboratory, Program in Individualized Medicine (PrIMe), College of Veterinary Medicine, Washington State University , Pullman , WA , USA and.

1. Bisphenol-A is a ubiquitous environmental contaminant that is primarily metabolized by glucuronidation and associated with various human diseases including breast cancer. Here we identified UDP-glucuronosyltransferases (UGTs) and genetic polymorphisms responsible for interindividual variability in bisphenol-A glucuronidation in human liver and breast. 2. Hepatic UGTs showing the highest bisphenol-A glucuronidation activity included UGT2B15 and UGT1A9. Relative activity factor normalization indicated that UGT2B15 contributes >80% of activity at bisphenol-A concentrations under 5 μM, while UGT1A9 contributes up to 50% of activity at higher concentrations. 3. Bisphenol-A glucuronidation by liver microsomes (46 donors) ranged from 0.25 to 4.3 nmoles/min/mg protein. Two-fold higher glucuronidation (p = 0.018) was observed in UGT1A9 *22/*22 livers compared with *1/*1 and *1/*22 livers. However, no associations were observed for UGT2B15*2 or UGT1A1*28 genotypes. 4. Bisphenol-A glucuronidation by breast microsomes (15 donors) ranged from <0.2 to 56 fmoles/min/mg protein. Breast mRNA expression of UGTs capable of glucuronidating bisphenol-A was highest for UGT1A1, followed by UGT2B4, UGT1A9, UGT1A10, UGT2B7 and UGT2B15. Bisphenol-A glucuronidation was over 10-fold lower in breast tissues with the UGT1A1*28 allele compared with tissues without this allele (p = 0.006). 5. UGT2B15 and UGT1A9 contribute to glucuronidation variability in liver, while UGT1A1 is important in breast.
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http://dx.doi.org/10.3109/00498254.2016.1156784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531270PMC
January 2017

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

Drug Metab Dispos 2015 Nov 1;43(11):1769-72. Epub 2015 Sep 1.

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland

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.
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http://dx.doi.org/10.1124/dmd.115.065680DOI Listing
November 2015

Glucuronidation of estrone and 16α-hydroxyestrone by human UGT enzymes: The key roles of UGT1A10 and UGT2B7.

J Steroid Biochem Mol Biol 2015 Nov 26;154:104-11. Epub 2015 Jul 26.

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Finland. Electronic address:

The glucuronidation of estrone and 16α-hydroxyestrone by recombinant human UDP-glucuronosyltransferase enzymes (UGTs) of subfamilies 1A, 2A and 2B was studied. Microsomes from human liver and small intestine were also tested for the glucuronidation of these two estrogens. The results revealed that UGT1A10 is by far the most active enzyme in estrone glucuronidation. UGT1A10 also exhibited high rate of 16α-hydroxyestrone conjugation at the 3-OH, whereas UGT2B7 catalyzed its glucuronidation at high rates at the 16-OH. Human liver microsomes exhibited high rates of 16α-hydroxyestrone-16-glucuronide formation, but very low formation rates of either 16α-hydroxyestrone-3-glucuronide or estrone glucuronide. On the other hand, human intestine microsomes catalyzed the formation of all these 3 different glucuronides at high rates. Kinetic analyses revealed very low Km value for 16α-hydroxyestrone glucuronidation by UGT2B7, below 4 μM, suggesting higher affinity than commonly found among UGTs and their substrates. In further studies with UGT1A10, mutant F93G exhibited increased glucuronidation rates of 16α-hydroxyestrone, but not estrone, whereas mutations in F90 did not reveal any activity with either estrogen. Taken together, the results of this study significantly expand our understanding on the metabolism of estrogens and their interactions with the human UGTs.
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http://dx.doi.org/10.1016/j.jsbmb.2015.07.013DOI Listing
November 2015

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

Biosens Bioelectron 2015 Oct 8;72:261-7. Epub 2015 May 8.

Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bios.2015.05.003DOI Listing
October 2015

Exploring the structure-activity relationships of ABCC2 modulators using a screening approach.

Bioorg Med Chem 2015 Jul 17;23(13):3513-25. Epub 2015 Apr 17.

Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00790 Helsinki, Finland; Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.

ABCC2 is a transporter with key influence on liver and kidney pharmacokinetics. In order to explore the structure-activity relationships of compounds that modulate ABCC2, and by doing so gain insights into drug-drug interactions, we screened a library of 432 compounds for modulators of radiolabeled β-estradiol 17-(β-d-glucuronide) (EG) and fluorescent 5(6)-carboxy-2',7'-dichlorofluorescein transport (CDCF) in membrane vesicles. Following the primary screen at 80μM, dose-response curves were used to investigate in detail 86 compounds, identifying 16 low μM inhibitors and providing data about the structure-activity relationships in four series containing 19, 24, 10, and eight analogues. Measurements with the CDCF probe were consistently more robust than for the EG probe. Only one compound was clearly probe-selective with a 50-fold difference in the IC50s obtained by the two assays. We built 24 classification models using the SVM and fused-XY Kohonen methods, revealing molecular descriptors related to number of rings, solubility and lipophilicity as important to distinguish inhibitors from inactive compounds. This study is to the best of our knowledge the first to provide details about structure-activity relationships in ABCC2 modulation.
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http://dx.doi.org/10.1016/j.bmc.2015.04.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461526PMC
July 2015

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

Xenobiotica 2015 30;45(6):511-9. Epub 2014 Dec 30.

Faculty of Pharmacy, University of Ljubljana , Ljubljana , Slovenia and.

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.
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http://dx.doi.org/10.3109/00498254.2014.999140DOI Listing
March 2016